CN118510747A - Nanomaterials comprising tetravalent lipid compounds - Google Patents

Abstract

The present disclosure describes compositions, formulations, nanoparticles (such as lipid nanoparticles) and/or nanomaterials, and methods of their use.

Description

Translated fromChinese

包含四价脂质化合物的纳米材料Nanomaterials containing quadrivalent lipid compounds

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本申请要求2021年12月20日提交的第63/291,593号和2022年6月22日提交的第63/354,640号美国临时申请的优先权，这些申请中的每个申请的全部内容通过引用并入本文中。This application claims priority to U.S. Provisional Application Nos. 63/291,593, filed on December 20, 2021, and 63/354,640, filed on June 22, 2022, the entire contents of each of which are incorporated herein by reference.

背景技术Background Art

药物递送系统的递送在化学、生物学和医学领域带来了挑战。例如，由于对于系统的分子特性如何控制向组织的递送和赋予药物功效的理解不足，药物递送系统受到阻碍。The delivery of drug delivery systems presents challenges in chemistry, biology, and medicine. For example, drug delivery systems are hampered by a poor understanding of how the molecular properties of the system control delivery to tissues and confer drug efficacy.

发明内容Summary of the invention

本发明认识到需要组合物、制剂、纳米粒子和/或纳米材料及其使用方法。此外，本公开认识到组合物、制剂、纳米粒子和/或纳米材料的结构特征影响体内、体外和离体功能反应。例如，本公开尤其描述了本文所述的一种或多种组分的选择和组合影响脂质纳米粒子的功能活性。在一些实施例中，例如，功能活性可指期望的向性、稳定性和/或药物递送功效。在一些实施例中，本公开尤其描述了一种或多种组分的不同比率影响本文所述的组合物、制剂、纳米粒子和/或纳米材料的一种或多种功能活性。The present invention recognizes the need for compositions, preparations, nanoparticles and/or nanomaterials and methods of use thereof. In addition, the disclosure recognizes that the structural features of compositions, preparations, nanoparticles and/or nanomaterials affect in vivo, in vitro and ex vivo functional responses. For example, the disclosure particularly describes that the selection and combination of one or more components described herein affect the functional activity of lipid nanoparticles. In certain embodiments, for example, functional activity may refer to desired tropism, stability and/or drug delivery efficacy. In certain embodiments, the disclosure particularly describes that the different ratios of one or more components affect one or more functional activities of compositions, preparations, nanoparticles and/or nanomaterials described herein.

此外，本公开尤其认识到，与参考脂质结构相比，脂质的化学结构赋予了改进的特性。例如，在一些实施例中，本公开描述了式I化合物：In addition, the present disclosure particularly recognizes that the chemical structure of the lipid confers improved properties compared to the reference lipid structure. For example, in some embodiments, the present disclosure describes a compound of formula I:

或其N-氧化物、或其药学上可接受的盐，其中L¹、L^1'、L^1”、L²、L^2'、L^2”、L³、L^3'、L^3”、R¹、R^1'、R^1”、X¹、X²和X³中的每一者如本文所定义。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L¹ , L^1′ , L^1″ , L 2 , L^2′ , L^2″ , L³ , L^3′ , L^3″ , R¹ , R^1′ , R^1″ , X¹ , X² and X³ is as defined herein.

此外，如本文所述，本公开展示了包括四价核心(例如，来源于2,2-双(羟甲基)丙烷-1,3-二醇的核心)特征的可电离脂质的惊人特性(例如，货物(诸如治疗剂或预防剂)的出乎意料的向性、稳定性和递送功效)以及该可电离脂质的组合物、制剂、纳米粒子和/或纳米材料(例如，LNP和/或含有LNP的组合物、制剂、纳米粒子和/或纳米材料)以及它们的使用的方法。In addition, as described herein, the present disclosure demonstrates surprising properties (e.g., unexpected tropism, stability, and delivery efficacy of cargo (such as therapeutic or prophylactic agents)) of ionizable lipids comprising a tetravalent core (e.g., a core derived from 2,2-bis(hydroxymethyl)propane-1,3-diol) feature, as well as compositions, formulations, nanoparticles and/or nanomaterials (e.g., LNPs and/or compositions, formulations, nanoparticles and/or nanomaterials containing LNPs), and methods of their use.

此外，本公开认识到包含一种或多种可电离脂质的脂质纳米粒子(LNP)组合物。例如，本公开提供，包含一种或多种所公开的可电离脂质的LNP组合物和/或制剂赋予出人意料的向性。In addition, the present disclosure recognizes lipid nanoparticle (LNP) compositions comprising one or more ionizable lipids. For example, the present disclosure provides that LNP compositions and/or formulations comprising one or more disclosed ionizable lipids confer unexpected tropism.

在一些实施例中，所提供的组合物、制剂、纳米粒子和/或纳米材料适用于治疗方法、递送方法、产生多肽的方法或延迟/遏制疾病或病症进展的方法。In some embodiments, provided compositions, formulations, nanoparticles and/or nanomaterials are useful in therapeutic methods, delivery methods, methods of producing polypeptides, or methods of delaying/restraining the progression of a disease or condition.

在一些实施例中，所提供的组合物、制剂、纳米粒子和/或纳米材料适用于制造方法。In some embodiments, provided compositions, formulations, nanoparticles, and/or nanomaterials are suitable for use in methods of manufacturing.

在一些实施例中，所提供的组合物、制剂、纳米粒子和/或纳米材料适用于表征方法。In some embodiments, provided compositions, formulations, nanoparticles, and/or nanomaterials are suitable for use in characterization methods.

涉及本发明的一个方面(例如方法)的实施例的要素可应用于涉及本发明的其它方面的实施例中，且反之亦然。Elements of embodiments relating to one aspect of the invention (eg, a method) may be applied in embodiments relating to other aspects of the invention, and vice versa.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1描绘根据本公开的实施例的LNP制剂的示范性mRNA筛选系统。FIG. 1 depicts an exemplary mRNA screening system for LNP formulations according to embodiments of the present disclosure.

图2描绘根据本公开的实施例的LNP制剂的示范性siRNA筛选系统。FIG. 2 depicts an exemplary siRNA screening system for LNP formulations according to embodiments of the present disclosure.

定义definition

约：如本文中所使用，术语“约”或“大约”当在本文中用于指代值时，是指在上下文中与所参考的值类似的值。一般来说，熟悉上下文的本领域技术人员应当理解在该上下文中“约”或“大约”所涵盖的相关偏差度。例如，在一些实施例中，术语“约”可以涵盖在参考值的任一方向(大于或小于)的25％、20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％的值的范围，除非另有说明或从上下文明显可见(除了此类数字将会超过可能值的100％的情况)。About: As used herein, the term "about" or "approximately" when used in this article to refer to a value refers to a value similar to the referenced value in the context. In general, those skilled in the art who are familiar with the context should understand the relevant degree of deviation covered by "about" or "approximately" in this context. For example, in some embodiments, the term "about" can cover a range of values of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater than or less than) of the reference value, unless otherwise stated or obvious from the context (except for the case where such numbers will exceed 100% of the possible value).

施用：如本文中所使用，术语“施用”通常是指向受试者或系统施用组合物。本领域的普通技术人员将清楚可以在适当的情况下用于向受试者(例如人类)施用的各种途径。例如，在一些实施例中，施用可以是经眼的、口服的、肠胃外的、局部的等。在一些特定实施例中，施用可以是支气管的(例如，通过支气管滴注)、颊的、真皮的(其可以是或包括，例如，局部施用(真皮、真皮内、真皮间、透皮等)中的一者或多者)、肠内的、动脉内的、真皮内的、胃内的、髓内的、肌内的、鼻内的、腹膜内的、鞘内的、静脉内的、心室内的、特定器官内(例如肝内)的、粘膜的、鼻的、口腔的、直肠的、皮下的、舌下的、局部的、气管的(例如，通过气管内滴注)、阴道的、玻璃体的等。在一些实施例中，施用可以涉及间歇给药(例如，时间上间隔的多个剂量)和/或定期(例如，由共同时间段间隔的单独剂量)给药。在一些实施例中，施用可能涉及至少选定的一段时间内的持续给药(例如，灌注)。在一些实施例中，包括脂质纳米粒子的药物组合物可调配成通过肠胃外(肌肉内、腹膜内、静脉内(IV)或皮下注射)、经皮(被动地或使用离子电渗疗法或电穿孔)或经粘膜(经鼻、经阴道、经直肠或舌下)施用途径或使用生物溶蚀性插入物施用，且可以按适于每一种施用途径的剂型调配。Administration: As used herein, the term "administering" generally refers to administering a composition to a subject or system. A person of ordinary skill in the art will appreciate the various routes that can be used for administration to a subject (e.g., a human being) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or include, for example, one or more of topical administration (dermal, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., within the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may involve intermittent administration (e.g., multiple doses spaced apart in time) and/or regular administration (e.g., separate doses spaced apart by a common time period). In some embodiments, administration may involve continuous administration (e.g., perfusion) over at least a selected period of time. In some embodiments, pharmaceutical compositions comprising lipid nanoparticles may be formulated to be administered by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration or using bioerodible inserts, and may be formulated in dosage forms suitable for each route of administration.

脂肪族：如本文中所使用，术语“脂肪族”或“脂肪族基团(aliphatic group)”意指完全饱和或含有一个或多个不饱和单元的直链(即无支链)或支链型被取代或未被取代的烃链，或完全饱和或含有一个或多个不饱和单元但不是芳香族的单环烃或双环烃(在本文中也被称作“碳环(carbocycle)”、“碳环(carbocyclic)”或“环脂肪族(cycloaliphatic)”或环烷基(cycloalkyl)”)，其具有与分子的其余部分的单个连接点。除非另有规定，否则脂肪族基团含有1-6个脂肪族碳原子。在一些实施例中，脂肪族基团含有1-5个碳原子。在一些实施例中，脂肪族基团含有1-4个碳原子。在一些实施例中，脂肪族基团含有1-3个碳原子，且在一些实施例中，脂肪族基团含有1-2个碳原子。在一些实施例中，“碳环(carbocyclic)”(或“环脂肪族”或“碳环(carbocycle)”或“环烷基”)是指完全饱和或含有一个或多个不饱和单元但不是芳香族的任选地被取代的单环C₃-C₈烃或任选地被取代的C₆-C₁₂双环烃，其具有与分子的其余部分的单个连接点。合适的脂肪族基团包含但不限于直链或支链型被取代或未被取代的烷基、烯基、炔基基团及其混合物，如(环烷基)烷基、(环烯基)烷基或(环烷基)烯基。Aliphatic: As used herein, the term "aliphatic" or "aliphatic group""Carbocyclic" (or "cycloaliphatic" or "cycloalkyl") refers to a monocyclic C 3 alkyl group that is fully saturated or contains one or more unsaturated units, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic (also referred to herein as "carbocycle", "carbocyclic" or "cycloaliphatic" or "cycloalkyl"), which has a single point of attachment to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 carbon atoms. In some embodiments, an aliphatic group contains 1-4 carbon atoms. In some embodiments, an aliphatic group contains 1-3 carbon atoms, and in some embodiments, an aliphatic group contains 1-2 carbon atoms. In some embodiments, "carbocyclic" (or "cycloaliphatic" or "carbocycle" or "cycloalkyl") refers to an optionally substituted monocyclic C₃ alkyl group that is fully saturated or contains one or more unsaturated units but is not aromatic._-C8 hydrocarbon or an optionally substituted_C6 -_C12 bicyclic hydrocarbon having a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched substituted or unsubstituted alkyl, alkenyl, alkynyl groups and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

烯基：如本文中所使用，术语“烯基”是指具有一个或多个双键的如本文所定义的烷基基团。在一些实施例中，单独使用或作为较大部分的一部分使用的术语“烯基”是指任选地被取代的直链或支链烃链，其具有至少一个双键并且具有(除非另有规定)2-20、2-18、2-16、2-14、2-12、2-10、2-8、2-6、2-4或2-3个碳原子(例如，C_2-20、C_2-18、C_2-16、C_2-14、C_2-12、C_2-10、C_2-8、C_2-6、C_2-4或C_2-3)。示范性烯基基团包含乙烯基、丙烯基、丁烯基、戊烯基、己烯基以及庚烯基。Alkenyl: As used herein, the term "alkenyl" refers to an alkyl group as defined herein having one or more double bonds. In some embodiments, the term "alkenyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched hydrocarbon chain having at least one double bond and having (unless otherwise specified) 2-20,_2-18 , 2-16, 2-14, 2-12, 2-10,_2-8 , 2-6, 2-4 or 2-3 carbon atoms (e.g., C_2-20 , C 2-18, C_2-16 , C_2-14 , C 2-12, C_2-10 , C_2-8 , C_2-6 , C_2-4 or C_2-3 ). Exemplary alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl and heptenyl.

亚烯基：术语“亚烯基”是指二价烯基基团。经取代的亚烯基链是含有至少一个双键的聚亚甲基基团，其中一个或多个氢原子被取代基置换。合适的取代基包含下文关于经取代的脂肪族基团所描述的取代基。Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for substituted aliphatic groups.

烷基：如本文中所使用，术语“烷基”具有其在本领域中的普通含义，并且可以包括饱和脂肪族基团，包括直链烷基基团、支链烷基基团、环烷基(脂环族)基团、被烷基取代的环烷基基团以及被环烷基取代的烷基基团。在一些实施例中，烷基具有1-100个碳原子。在某些实施例中，直链或支链烷基在其主链中具有约1-20个碳原子(例如，对于直链为C₁-C₂₀；对于支链为C₂-C₂₀)，并另选地约1-10个。在一些实施例中，环烷基环在其环结构中具有约3-10个碳原子，其中此类环为单环或双环，并且另选地在环结构中具有约5个、6个或7个碳原子。在一些实施例中，烷基基团可以是低级烷基基团，其中低级烷基基团包括1-4个碳原子(例如，对于直链低级烷基为C₁-C₄)。Alkyl: As used herein, the term "alkyl" has its ordinary meaning in the art, and may include saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, cycloalkyl groups substituted by alkyl groups, and alkyl groups substituted by cycloalkyl groups. In some embodiments, the alkyl group has 1-100 carbon atoms. In certain embodiments, the straight chain or branched chain alkyl has about 1-20 carbon atoms in its main chain (e.g., C₁ -C₂₀ for straight chain; C₂ -C₂₀ for branched chain), and alternatively about 1-10. In some embodiments, the cycloalkyl ring has about 3-10 carbon atoms in its ring structure, wherein such ring is monocyclic or bicyclic, and alternatively has about 5, 6 or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group includes 1-4 carbon atoms (e.g., C₁ -C₄ for straight chain lower alkyl).

亚烷基：术语“亚烷基(alkylene)”或“亚烷基(alkylenyl)”是指直链(即无支链)或支链型被取代或未被取代的二价烷基基团(即二价饱和烃链)。以上提到的单价烷基中的任何单价烷基可以是通过从烷基中提取第二氢原子的亚烷基。在一些实施例中，“亚烷基”是聚亚甲基基团，即-(CH₂)_n-，其中n为正整数，优选地为1至10、1至9、1至8、1至7、1至6、1至5、1至4、1至3、1至2、2至5或4至8。被取代的亚烷基为其中一个或多个亚甲基氢原子被取代基置换的聚亚甲基基团。合适的取代基包含下文关于经取代的脂肪族基团所描述的取代基。Alkylene: The term "alkylene" or "alkylenyl" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted divalent alkyl group (i.e., a divalent saturated hydrocarbon chain). Any of the monovalent alkyl groups mentioned above may be an alkylene group obtained by abstracting the second hydrogen atom from the alkyl group. In some embodiments, "alkylene" is a polymethylene group, i.e., -(CH₂ )_n -, wherein n is a positive integer, preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, or 4 to 8. Substituted alkylene is a polymethylene group in which one or more methylene hydrogen atoms are replaced by a substituent. Suitable substituents include the substituents described below for substituted aliphatic groups.

炔基：如本文中所使用，术语“炔基”是指具有一个或多个三键的如本文所定义的烷基基团。在一些实施例中，单独使用或作为较大部分的一部分使用的术语“炔基”是指任选地被取代的直链或支链烃基团，其具有至少一个三键并且具有(除非另有规定)2-20、2-18、2-16、2-14、2-12、2-10、2-8、2-6、2-4或2-3个碳原子(例如，C_2-20、C_2-18、C_2-16、C_2-14、C_2-12、C_2-10、C_2-8、C_2-6、C_2-4或C_2-3)。示范性炔基基团包含乙炔基、丙炔基、丁炔基、戊炔基、己炔基以及庚炔基。Alkynyl: As used herein, the term "alkynyl" refers to an alkyl group as defined herein having one or more triple bonds. In some embodiments, the term "alkynyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12,_2-10 , 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C_2-20 , C_2-18 , C_2-16 , C 2-14, C_2-12 , C_2-10 , C_2-8 , C_2-6 , C_2-4 , or C_2-3 ). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

氨基酸：如本文所使用，在其最广泛意义上是指可以例如通过一个或多个肽键的形成而被并入多肽链中的任何化合物和/或物质。在一些实施例中，氨基酸具有通用结构H₂N-C(H)(R)-COOH。在一些实施例中，氨基酸是天然存在的氨基酸。在一些实施例中，氨基酸为天然氨基酸；在一些实施例中，氨基酸为D-氨基酸；在一些实施例中，氨基酸为L-氨基酸。“标准氨基酸”是指天然存在的肽中常见的二十种标准L-氨基酸中的任一种。“非标准氨基酸”是指除标准氨基酸以外的任何氨基酸，不管其是以合成方式制备的还是从天然来源获得的。在一些实施例中，氨基酸，包含多肽中的羧基端和/或氨基端氨基酸，与上述通用结构相比，可以含有结构修饰。例如，在一些实施例中，与通用结构相比，氨基酸可以通过甲基化、酰胺化、乙酰化、聚乙二醇化、糖基化、磷酸化和/或取代(例如氨基基团、羧酸基、一个或多个质子和/或羟基基团的取代)来修饰。在一些实施例中，与含有其它方面相同的未修饰氨基酸的多肽相比，此类修饰可以例如改变含有经修饰氨基酸的多肽的循环半衰期。在一些实施例中，与含有其它方面相同的未修饰氨基酸的多肽相比，此类修饰不会显著改变含有经修饰氨基酸的多肽的相关活性。如从上下文中将清楚的，在一些实施例中，术语“氨基酸”可以用于指游离氨基酸；在一些实施例中，它可以用来指多肽的氨基酸残基。Amino Acid: As used herein, in its broadest sense refers to any compound and/or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has a general structure H₂ NC(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. In some embodiments, an amino acid, including a carboxyl-terminal and/or amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the general structure described above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., substitution of an amino group, a carboxylic acid group, one or more protons, and/or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing a modified amino acid compared to a polypeptide containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the associated activity of a polypeptide containing a modified amino acid compared to a polypeptide containing an otherwise identical unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

动物：如本文中所使用，是指动物界的任何成员。在一些实施例中，“动物”是指任何性别和处于任何发育阶段的人类。在一些实施例中，“动物”是指处于任何发育阶段的非人类动物。在某些实施例中，非人类动物为哺乳动物(例如啮齿动物、小鼠、大鼠、兔、猴、狗、猫、绵羊、牛、灵长类动物和/或猪)。在一些实施例中，动物包括但不限于哺乳动物、鸟类、爬行动物、两栖动物、鱼类、昆虫和/或蠕虫。在一些实施例中，动物可以是转基因动物、基因工程动物和/或克隆。Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans of any sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and/or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and/or clones.

适体：如本文中所使用，术语“适体”是指由与特定分子标靶(例如，伞形拓扑聚糖)紧密结合的核酸(例如，RNA、DNA)组成的大分子。特定适体可用直链核苷酸序列来描述并且长度通常为约15至60个核苷酸。以不受任何理论约束为前提，预期适体中的核苷酸链形成分子内相互作用，使分子折叠成复杂的三维形状，并且此三维形状使适体紧密结合于其靶分子的表面。鉴于存在于所有可能的核苷酸序列范围内的分子形状的非凡多样性，可以获得用于大量分子标靶(包含蛋白质和小分子)的适体。除高特异性以外，适体通常对其标靶具有极高亲和力(例如，对于蛋白质的亲和力在皮摩尔到低纳摩尔范围内)。在许多实施例中，适体具有化学稳定性并且可以煮沸或冷冻而不损失活性。因为适体是合成分子，所以可对适体进行多种修饰，从而优化其关于特定应用的功能。例如，可以修饰适体以显著降低其用于体内应用时在血液中被酶降解的灵敏度。另外，可以修饰适体以改变其生物分布或血浆滞留时间。Aptamer: As used herein, the term "aptamer" refers to a macromolecule composed of nucleic acids (e.g., RNA, DNA) that tightly bind to a specific molecular target (e.g., an umbrella-topology glycan). Specific aptamers can be described by linear nucleotide sequences and are typically about 15 to 60 nucleotides in length. Without being bound by any theory, it is expected that the nucleotide chains in the aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to tightly bind to the surface of its target molecule. In view of the extraordinary diversity of molecular shapes present in all possible nucleotide sequences, aptamers for a large number of molecular targets (including proteins and small molecules) can be obtained. In addition to high specificity, aptamers generally have extremely high affinity for their targets (e.g., affinity for proteins in the picomolar to low nanomolar range). In many embodiments, aptamers are chemically stable and can be boiled or frozen without loss of activity. Because aptamers are synthetic molecules, aptamers can be modified in a variety of ways to optimize their function with respect to specific applications. For example, aptamers can be modified to significantly reduce their sensitivity to enzymatic degradation in blood when used for in vivo applications. Additionally, aptamers can be modified to alter their biodistribution or plasma residence time.

芳基：术语“芳基”是指具有总共六个到十四个环成员(例如，C_6-14)的单环及双环系统，其中该系统中的至少一个环是芳香族的并且其中该系统中的每个环含有三个到七个环成员。术语“芳基”可以与术语“芳基环”互换使用。在一些实施例中，“芳基”是指包含但不限于苯基、萘基、蒽基等的芳香族环系统，其可以带有一个或多个取代基。除非另有规定，否则“芳基”基团为烃。Aryl: The term "aryl" refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C_6-14 ), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" can be used interchangeably with the term "aryl ring". In some embodiments, "aryl" refers to an aromatic ring system including but not limited to phenyl, naphthyl, anthracenyl, etc., which may bear one or more substituents. Unless otherwise specified, an "aryl" group is a hydrocarbon.

关联：如本文中所使用的该术语，如果一个事件或实体的存在、水平、程度、类型和/或形式与另一个事件或实体相关，那么两个事件或实体彼此“关联”。例如，如果特定实体(例如，多肽、遗传特征(genetic signature)、代谢物、微生物等)的存在、水平和/或形式与特定疾病、病症或病状的发病率和/或对特定疾病、病症或病状的易感性相关(例如，在相关群体内)，那么认为所述特定实体与所述疾病、病症或病状相关。在一些实施例中，如果两个或更多个实体直接或间接相互作用，使得其彼此具有和/或保持物理接近性，那么其在物理上彼此“相关”。在一些实施例中，彼此物理关联的两个或更多个实体彼此共价连接；在一些实施例中，在彼此物理关联的两个或更多个实体彼此不共价连接，而是非共价关联，例如，借助于氢键、范德华力相互相用(van der Waals interaction)、疏水性相互作用、磁性和它们的组合。Association: As used herein, two events or entities are "associated" with each other if the presence, level, degree, type, and/or form of one event or entity is associated with another event or entity. For example, if the presence, level, and/or form of a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is associated with the incidence of a particular disease, disorder, or condition and/or susceptibility to a particular disease, disorder, or condition (e.g., within a related population), then the particular entity is considered to be associated with the disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly so that they have and/or maintain physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

生物相容的：如本文中所使用，术语“生物相容的”是指材料在放置成与活组织接触(例如，体内)时不会对此类组织造成显著伤害。在某些实施例中，如果材料对细胞无毒，那么其具有“生物相容性”。在某些实施例中，如果材料体外添加到细胞导致低于或等于20％的细胞死亡，且/或体内施用材料未诱发显著发炎或其它此类不良作用，那么该材料具有“生物相容性”。Biocompatible: As used herein, the term "biocompatible" refers to a material that does not cause significant damage to living tissue when placed in contact with such tissue (e.g., in vivo). In certain embodiments, a material is "biocompatible" if it is not toxic to cells. In certain embodiments, a material is "biocompatible" if addition of the material to cells in vitro results in less than or equal to 20% cell death, and/or administration of the material in vivo does not induce significant inflammation or other such adverse effects.

可生物降解的：如本文中所使用，术语“可生物降解的”是指当材料被引入细胞中时，材料被分解(例如，通过细胞机制，诸如通过酶促降解、通过水解和/或通过它们的组合分解)成细胞可以重新使用或处理而不会对细胞产生显著毒性作用的组分。在某些实施例中，通过可生物降解的材料的分解产生的组分是生物相容的，并且因此不会在体内诱发显著发炎和/或其它不良作用。在一些实施例中，可生物降解的聚合物材料分解成其组成性单体。在一些实施例中，可生物降解的材料(包含例如可生物降解的聚合物材料)的分解涉及酯键的水解。或者或另外，在一些实施例中，可生物降解的材料(包含例如可生物降解的聚合物材料)的分解涉及氨基甲酸酯键的裂解。示范性可生物降解的聚合物包含例如羟基酸(如乳酸和乙醇酸)的聚合物，包含但不限于聚(羟基酸)、聚(乳酸)(poly(lactic acid)，PLA)、聚(乙醇酸)(poly(glycolic acid)，PGA)、聚(乳酸-共-乙醇酸)(poly(lactic-co-glycolic acid)，PLGA)和与PEG的共聚物、聚酸酐、聚(原酸)酯、聚酯、聚氨基甲酸酯、聚(丁酸)、聚(戊酸)、聚(己内酯)、聚(羟基烷酸酯)、聚(丙交酯-共-己内酯)、其共混物和共聚物。许多天然存在的聚合物也是可生物降解的，包含例如：蛋白质，如白蛋白、胶原蛋白、明胶和谷醇溶蛋白(例如玉米蛋白)；及多糖，如海藻酸酯、纤维素衍生物和聚羟基烷酸酯(例如聚羟基丁酸酯)、其共混物和共聚物。本领域的技术人员将了解或能够确定这类聚合物何时是其生物相容的和/或可生物降解的衍生物(例如，与母体聚合物有关，其结构大体上相同，仅在如本领域中已知的特定化学基团的取代或添加方面不同)。Biodegradable: As used herein, the term "biodegradable" means that when a material is introduced into a cell, the material is broken down (e.g., by cellular mechanisms, such as by enzymatic degradation, by hydrolysis, and/or by a combination thereof) into components that the cell can reuse or process without causing significant toxic effects to the cell. In certain embodiments, the components produced by the decomposition of the biodegradable material are biocompatible and therefore do not induce significant inflammation and/or other adverse effects in vivo. In some embodiments, the biodegradable polymeric material decomposes into its constituent monomers. In some embodiments, the decomposition of the biodegradable material (including, for example, a biodegradable polymeric material) involves the hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the decomposition of the biodegradable material (including, for example, a biodegradable polymeric material) involves the cleavage of carbamate bonds. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including but not limited to poly(hydroxy acids), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA) and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example: proteins such as albumin, collagen, gelatin, and prolamins (e.g., zein); and polysaccharides such as alginates, cellulose derivatives, and polyhydroxyalkanoates (e.g., polyhydroxybutyrate), blends and copolymers thereof. One skilled in the art will understand or be able to determine when such polymers are biocompatible and/or biodegradable derivatives thereof (e.g., related to the parent polymer, having substantially the same structure, differing only in the substitution or addition of specific chemical groups as known in the art).

生物活性：如本文中所使用，是指通过所关注药剂或实体实现的可观测的生物效应或结果。例如，在一些实施例中，特异性结合相互作用是一种生物活性。在一些实施例中，生物路径或事件的调节(例如诱导、增强或抑制)是一种生物活性。在一些实施例中，生物活性的存在或程度通过检测由所关注的生物路径或事件产生的直接或间接产物评估。Biological activity: As used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or extent of a biological activity is assessed by detecting a direct or indirect product produced by a biological pathway or event of interest.

生物样品：如本文中所使用，术语“生物样品”通常是指获自或来源于如本文所述的所关注生物来源(例如，组织或生物体或细胞培养物)的样品。在一些实施例中，所关注的来源包括生物体，如动物或人类。在一些实施例中，生物样品是或包括生物组织或流体。在一些实施例中，生物样品可以是或包括骨髓；血液；血液细胞；腹水；组织或细针活检样品；含有细胞的体液；自由浮动的核酸；痰；唾液；尿；脑脊液、腹膜液；胸膜液；粪便；淋巴；妇科液；皮肤拭子；阴道拭子；口腔拭子；鼻拭子；冲洗或灌洗，诸如导管灌洗或支气管肺泡灌洗；抽吸物；刮屑；骨髓样本；组织活检样本；手术样本；粪便、其他体液、分泌物和/或排泄物；和/或来自其中的细胞等。在一些实施例中，生物样品为或包括从受试者获得的细胞。在一些实施例中，获得的细胞是或包含从其获得样品的个体的细胞。在一些实施例中，样品是通过任何适当的方式直接从所关注的来源获得的“初级样品”。例如，在一些实施例中，初级生物样品通过选自由活检(例如，细针抽吸或组织活检)、手术、体液(例如，血液、淋巴液、粪便等)等的收集组成的组的方法来获得。在一些实施例中，如从上下文将清楚的，术语“样品”是指通过处理(例如，通过除去初级样品的一种或多种组分和/或通过向该初级样品添加一种或多种试剂)初级样品来获得的制剂。例如，使用半渗透膜进行过滤。此类“经处理的样品”可以包括例如从样品中提取的或通过对初级样品进行诸如mRNA的扩增或逆转录、某些组分的分离和/或纯化等技术而获得的核酸或蛋白质。Biological sample: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a biological source of interest as described herein (e.g., a tissue or organism or cell culture). In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; body fluids containing cells; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washing or lavage, such as catheter lavage or bronchoalveolar lavage; aspirate; scrapings; bone marrow sample; tissue biopsy sample; surgical sample; feces, other body fluids, secretions and/or excretions; and/or cells therefrom, etc. In some embodiments, the biological sample is or includes cells obtained from a subject. In some embodiments, the obtained cells are or include cells of the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of a biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing (e.g., by removing one or more components of the primary sample and/or by adding one or more reagents to the primary sample) a primary sample. For example, filtering is performed using a semi-permeable membrane. Such "processed samples" may include, for example, nucleic acids or proteins extracted from a sample or obtained by performing techniques such as amplification or reverse transcription of mRNA, separation and/or purification of certain components on the primary sample.

二价：如本文中所使用，术语“二价”是指具有两个连接点的化学部分。例如，“二价C_1-8(或C_1-6)饱和或不饱和的直链或支链烃链”是指如本文所定义的直链或支链二价亚烷基链、亚烯基链和亚炔基链。Divalent: As used herein, the term "divalent" refers to a chemical moiety having two points of attachment. For example, "divalent C_1-8 (or C_1-6 ) saturated or unsaturated straight or branched hydrocarbon chain" refers to a straight or branched divalent alkylene chain, alkenylene chain, and alkynylene chain as defined herein.

桥接双环基：如本文中所使用，术语“桥接双环基”、“桥接双环(bridgedbicycle)”、“桥接双环(bridged bicyclic)”和“桥接双环(bridged bicyclic ring)”是指任何双环系统，即碳环或杂环，饱和或部分不饱和，具有至少一个桥。如IUPAC所定义，“桥”是无支链原子链或连接两个桥头的原子或价键，其中“桥头”是环系统的与三个或更多个骨架原子(不包括氢)结合的任何骨架原子。在一些实施例中，桥接双环基团具有7-12个环成员和0-4个独立地选自氮、氧或硫的杂原子。此类桥接双环基团是本领域所熟知的，并且包括下文所示的那些基团，其中每个基团在任何可取代的碳或氮原子处连接至分子的其余部分。除非另有规定，桥连双环基团任选地被一个或多个针对脂肪族基团所示的取代基取代。另外或另选地，桥连双环基团的任何可取代的氮任选地被取代。示例性桥接双环包含但不限于：Bridged bicyclic group: As used herein, the terms "bridged bicyclic group", "bridged bicycle", "bridged bicyclic" and "bridged bicyclic ring" refer to any bicyclic system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, with at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, wherein a "bridgehead" is any backbone atom of a ring system that is bound to three or more backbone atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Such bridged bicyclic groups are well known in the art and include those groups shown below, wherein each group is connected to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, the bridged bicyclic group is optionally substituted by one or more substituents shown for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of the bridged bicyclic group is optionally substituted. Exemplary bridged double rings include, but are not limited to:

癌症：术语“癌症”、“恶性肿瘤”、“赘瘤”、“肿瘤””及“癌瘤”在本文中用以指展现相对较异常、不受控制和/或自主性生长，使其展现以显著失去控制的细胞增殖为特征的异常生长表型的细胞。在一些实施例中，肿瘤可以是或包括癌前(例如良性)、恶性、转移前、转移性和/或非转移性的细胞。本公开明确地鉴别其教示内容可能尤其与其相关的某些癌症。在一些实施例中，相关癌症可以通过实体肿瘤表征。在一些实施例中，相关癌症可以通过血液系统肿瘤表征。一般来说，本领域已知的不同类型的癌症的实例包括：例如，造血系统癌症，包括白血病、淋巴瘤(霍奇金病和非霍奇金病)、骨髓瘤和骨髓增殖性疾病；肉瘤；黑色素瘤；腺瘤；实体组织癌瘤；口腔、咽、喉和肺的鳞状细胞癌；肝癌；泌尿生殖道癌，诸如前列腺癌、子宫颈癌、膀胱癌、子宫癌和子宫内膜癌以及肾细胞癌；骨癌、胰腺癌、皮肤癌、皮肤或眼内黑色素瘤、内分泌系统癌、甲状腺癌、甲状旁腺癌、头颈癌、乳腺癌、胃肠道癌和神经系统癌、良性病变，诸如乳头状瘤等。Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by significantly uncontrolled cell proliferation. In some embodiments, a tumor can be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic cells. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancers may be characterized by solid tumors. In some embodiments, the relevant cancers may be characterized by hematological tumors. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin's disease and non-Hodgkin's disease), myelomas and myeloproliferative disorders; sarcomas; melanomas; adenomas; solid tissue carcinomas; squamous cell carcinomas of the oral cavity, pharynx, larynx and lung; liver cancer; genitourinary tract cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer, and renal cell carcinoma; bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and nervous system cancer, benign lesions, such as papilloma, etc.

载剂：如本文中所使用，是指与组合物一起施用的稀释剂、佐剂、赋形剂或媒剂。在一些示范性实施例中，载剂可以包含无菌液体，例如水和油，包含石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。在一些实施例中，载剂是或包含一种或多种固体组分。Carrier: As used herein, refers to a diluent, adjuvant, excipient or vehicle with which the composition is administered. In some exemplary embodiments, the carrier may comprise a sterile liquid, such as water and oil, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

碳环基：如本文中所使用，术语“碳环基”、“碳环(carbocycle)”和“碳环(carbocyclic ring)”是指如本文所述的具有3至14个成员的饱和或部分不饱和环状脂肪族单环、双环或多环环系统，其中该脂肪族环系统如本文所述任选地被取代。碳环基团包含但不限于环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环庚基、环庚烯基、环辛基、环辛烯基、降冰片烷基、金刚烷基和环辛二烯基。在一些实施例中，“碳环基”(或“环脂肪族”)是指完全饱和或含有一个或多个不饱和单元但不是芳香族的任选地被取代的单环C₃-C₈烃或任选地被取代的C₆-C₁₂双环烃，其具有与分子的其余部分的单个连接点。术语“环烷基”是指具有约3至约10个环碳原子的任选地被取代的饱和环系统。在一些实施例中，环烷基基团具有3-6个碳。示范性单环环烷基环包含环丙基、环丁基、环戊基、环己基和环庚基。术语“环烯基”是指含有至少一个碳-碳双键并具有约3至约10个碳原子的任选地被取代的非芳香族单环或多环环系统。示范性单环环烯基包含环戊烯基、环己烯基和环庚烯基。Carbocyclyl: As used herein, the terms "carbocyclyl,""carbocycle," and "carbocyclic ring" refer to a saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring system having 3 to 14 members as described herein, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, "carbocyclyl" (or "cycloaliphatic") refers to an optionally substituted monocyclic_C3 -_C8 hydrocarbon or an optionally substituted_C6 -_C12 bicyclic hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single point of attachment to the rest of the molecule. The term "cycloalkyl" refers to an optionally substituted saturated ring system having from about 3 to about 10 ring carbon atoms. In certain embodiments, the cycloalkyl group has 3-6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl and cycloheptenyl.

类似的：如本文中所使用，术语“类似的”是指两种或更多种试剂、实体、情形、条件集等，其可能彼此不相同，但是足够相似以允许能够在其间进行比较，使得本领域技术人员将了解，可以基于所观测到的差异或相似性，合理地得出结论。在一些实施例中，通过多个大体上一致的特征和一种或少量变化特征来表征类似的条件、情形、个体或群体的集合。本领域的技术人员将结合上下文了解，在待视为类似的两种或两种此类试剂、实体、情形、条件集的任何既定情况中需要何种程度的一致性。例如，本领域的普通技术人员将了解，情形、个体或群体的集合当特征在于足够数量和类型的大体上相同的特征以确保以下合理结论时是彼此类似的：在情形、个体或群体的不同集合下或使用情形、个体或群体的不同集合获得的结果或观测到的现象的差异是由有所变化的那些特征的变化引起的或指示有所变化的那些特征的变化。Similar: As used herein, the term "similar" refers to two or more reagents, entities, situations, condition sets, etc., which may be different from each other, but are similar enough to allow comparisons to be made therebetween, so that those skilled in the art will understand that, based on the observed differences or similarities, a reasonable conclusion can be drawn. In certain embodiments, a similar condition, situation, individual or group set is characterized by a plurality of substantially consistent features and one or a small amount of variable features. Those skilled in the art will understand in context what degree of consistency is required in any given situation of two or two such reagents, entities, situations, condition sets to be considered similar. For example, those of ordinary skill in the art will understand that the set of situations, individuals or groups is similar to each other when characterized by the substantially identical features of sufficient quantity and type to ensure the following reasonable conclusions: the difference in the results or observed phenomena obtained under different sets of situations, individuals or groups or using different sets of situations, individuals or groups is caused by the changes in those features that have changed or indicates the changes in those features that have changed.

组合物：本领域技术人员将了解，术语“组合物”可以用于指包括一种或多种指定组分的离散物理实体。一般来说，除非另有规定，否则组合物可以为任何形式，例如，气体、凝胶、液体、固体等。Composition: Those skilled in the art will appreciate that the term "composition" can be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition can be in any form, for example, gas, gel, liquid, solid, etc.

包括：本文中被描述为“包括”一个或多个指定的要素或步骤的组合物或方法是开放式的，意指指定的要素或步骤是必需的，但是可以在组合物或方法的范围内加入其它要素或步骤。为了避免冗长，还应理解，任何被描述为“包括”(或其“包括(comprises)”)一个或多个指定的要素或步骤的组合物或方法也描述了“基本上由(consisting essentiallyof)”相同的指定的要素或步骤“组成”(或其“基本上由(consists essentially of)”相同的指定的要素或步骤“组成”)的相应更有限的组合物或方法，意指所述组合物或方法包含指定的基本要素或步骤，并且还可以包含不会实质上影响所述组合物或方法的基本和新颖特征的额外要素或步骤。还应理解，任何在本文中被描述为“包括”一个或多个指定的要素或步骤或“基本上由”一个或多个的指定要素或步骤“组成”的组合物或方法也描述了“由”指定的要素或步骤“组成”(consisting of/consists of)但不包括任何其它未指定要素或步骤的相应更有限的和封闭式的组合物或方法。在本文中所公开的任何组合物或方法中，可以用任何指定的基本要素或步骤的已知或所公开的等效物取代所述要素或步骤。Comprising: A composition or method described herein as "comprising" one or more specified elements or steps is open-ended, meaning that the specified elements or steps are required, but other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is also understood that any composition or method described as "comprising" (or "comprises") one or more specified elements or steps also describes a corresponding more limited composition or method "consisting essentially of" the same specified elements or steps (or "consists essentially of" the same specified elements or steps), meaning that the composition or method includes the specified basic elements or steps, and may also include additional elements or steps that do not substantially affect the basic and novel features of the composition or method. It is also understood that any composition or method described herein as "comprising" one or more specified elements or steps or "consisting essentially of" one or more specified elements or steps also describes a corresponding more limited and closed composition or method "consisting of/consists of" the specified elements or steps but does not include any other unspecified elements or steps. In any composition or method disclosed herein, any specified essential element or step may be substituted with known or disclosed equivalents to that element or step.

“改善”、“增加”、“抑制”或“减少”：如本文中所使用，术语“改善”、“增加”、“抑制”、“减少”或其语法同等成分指示相对于基线或其它参考测量的值。在一些实施例中，适当的参考测量可以是或包括在不存在(例如，之前和/或之后)特定药剂或治疗的其它方面类似的条件下或在存在适当的类似参考药剂的情况下在特定系统中(例如，在单一个体中)的测量。在一些实施例中，适当的参考测量可以是或包括在已知或预期以特定方式在相关药剂或治疗存在下作出反应的类似系统中的测量。"Improve," "increase," "inhibit," or "decrease": As used herein, the terms "improve," "increase," "inhibit," "decrease," or their grammatical equivalents indicate values relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or include a measurement in a particular system (e.g., in a single individual) in the absence (e.g., before and/or after) of a particular agent or treatment under otherwise similar conditions or in the presence of an appropriate, similar reference agent. In some embodiments, an appropriate reference measurement may be or include a measurement in a similar system that is known or expected to respond in a particular manner in the presence of the relevant agent or treatment.

测定：本文所述的许多方法包含“测定”步骤。本领域技术人员在阅读本说明书后将了解，这种“测定”可以通过使用本领域技术人员可用的各种技术中的任一种来利用或实现，包含例如本文中明确提到的特定技术。在一些实施例中，测定涉及对物理样品的操作。在一些实施例中，测定涉及对数据或信息的考量和/或操作，例如利用计算机或其它适合执行相关分析的处理单元。在一些实施例中，测定涉及从来源接收相关信息和/或材料。在一些实施例中，测定涉及比较样品或实体与类似参考物的一个或多个特征。Determination: Many of the methods described herein include a "determination" step. Those skilled in the art will appreciate after reading this specification that such "determination" can be utilized or implemented using any of a variety of techniques available to those skilled in the art, including, for example, specific techniques explicitly mentioned herein. In some embodiments, determination involves manipulation of a physical sample. In some embodiments, determination involves consideration and/or manipulation of data or information, such as using a computer or other processing unit suitable for performing the relevant analysis. In some embodiments, determination involves receiving relevant information and/or material from a source. In some embodiments, determination involves comparing one or more features of a sample or entity to a similar reference.

包裹：术语“包裹”在本文中用以指物质完全被另一种材料包围。Encapsulated: The term "encapsulated" is used herein to mean that a substance is completely surrounded by another material.

赋形剂：如本文中所使用，是指可以被包含在药物组合物中，例如用以提供或促成所期望的稠度或稳定作用的非治疗剂。合适的药物赋形剂包含例如淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙稀、乙二醇、水、乙醇等。Excipient: As used herein, refers to a non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc.

表达：如本文中所使用，术语核酸序列的“表达”是指从核酸序列产生任何基因产物。在一些实施例中，基因产物可以是转录物。在一些实施例中，基因产物可以是多肽。在一些实施例中，核酸序列的表达涉及以下中的一者或多者：(1)从DNA序列产生RNA模板(例如，通过转录)；(2)处理RNA转录本(例如，通过剪接、编辑、5'帽形成和/或3'末端形成)；(3)将RNA翻译成多肽或蛋白质；且/或(4)翻译后修饰多肽或蛋白质。Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing the RNA transcript (e.g., by splicing, editing, 5' cap formation and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translationally modifying the polypeptide or protein.

卤代脂肪族：术语“卤代脂肪族”是指被一个或多个卤素原子(例如，一个、二个、三个、四个、五个、六个或七个卤素，诸如氟、碘、溴或氯)取代的脂肪族基团。在一些实施例中，脂肪族基团含有1-7个卤素原子。在一些实施例中，脂肪族基团含有1-5个卤素原子。在一些实施例中，脂肪族基团含有1-3个卤素原子。Halogenated aliphatic: The term "halogenated aliphatic" refers to an aliphatic group substituted with one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halogens, such as fluorine, iodine, bromine, or chlorine). In some embodiments, the aliphatic group contains 1-7 halogen atoms. In some embodiments, the aliphatic group contains 1-5 halogen atoms. In some embodiments, the aliphatic group contains 1-3 halogen atoms.

卤代烷基：术语“卤代烷基”是指被一个或多个卤素原子(例如，一个、二个、三个、四个、五个、六个或七个卤素，诸如氟、碘、溴或氯)取代的烷基基团。在一些实施例中，卤代烷基基团含有1-7个碳原子。在一些实施例中，卤代烷基基团含有1-5个碳原子。在一些实施例中，卤代烷基基团含有1-3个碳原子。Haloalkyl: The term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms (e.g., one, two, three, four, five, six, or seven halogens, such as fluorine, iodine, bromine, or chlorine). In some embodiments, the haloalkyl group contains 1-7 carbon atoms. In some embodiments, the haloalkyl group contains 1-5 carbon atoms. In some embodiments, the haloalkyl group contains 1-3 carbon atoms.

杂亚烷基：如本文中所使用，术语“杂亚烷基(heteroalkylene)”或“杂亚烷基(heteroalkylenyl)”表示任选地被取代的直链(即无支链)或支链二价烷基(即二价饱和烃链)，除碳原子外还具有一个到五个杂原子。术语“杂原子”如下所述。在一些实施例中，杂亚烷基基团含有2-10个碳原子，其中1-3个碳原子任选地且独立地被选自氧、氮和硫的杂原子置换。在一些实施例中，杂亚烷基基团含有2-8个碳原子，其中1-3个碳原子任选地且独立地被选自氧、氮和硫的杂原子置换。在一些实施例中，杂亚烷基基团含有4-8个碳原子，其中1-3个碳原子任选地且独立地被选自氧、氮和硫的杂原子置换。在一些实施例中，杂亚烷基基团含有2-5个碳原子，其中1-2个碳原子任选地且独立地被选自氧、氮和硫的杂原子置换。在其他实施例中，杂亚烷基基团含有1-3个碳原子，其中1个碳原子任选地且独立地被选自氧、氮和硫的杂原子置换。合适的杂亚烷基基团包括但不限于-CH₂O-、-(CH₂)₂O-、-CH₂OCH₂-、-O(CH₂)₂-、-(CH₂)₃O-、-(CH₂)₂OCH₂-、-CH₂O(CH₂)₂-、-O(CH₂)₃-、-(CH₂)₄O-、-(CH₂)₃OCH₂-、-CH₂O(CH₂)₃-、-(CH₂)₂O(CH₂)₂-、-O(CH₂)₄-。除非另有说明，C_x杂亚烷基是指在被杂原子置换之前具有x个碳原子的杂亚烷基。Heteroalkylene: As used herein, the term "heteroalkylene" or "heteroalkylenyl" means an optionally substituted straight chain (i.e., unbranched) or branched divalent alkyl (i.e., a divalent saturated hydrocarbon chain), having one to five heteroatoms in addition to carbon atoms. The term "heteroatom" is described below. In some embodiments, the heteroalkylene group contains 2-10 carbon atoms, wherein 1-3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, the heteroalkylene group contains 2-8 carbon atoms, wherein 1-3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, the heteroalkylene group contains 4-8 carbon atoms, wherein 1-3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, the heteroalkylene group contains 2-5 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In other embodiments, the heteroalkylene group contains 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen and sulfur. Suitable heteroalkylene groups include, but are not limited to,_-CH2O- ,_- (_CH2 )2O-,_-CH2OCH2- , -O(_CH2 )_2- , -₍ CH2)_3O- , -(CH2)2OCH2-_, -CH2O(_CH2 )_2-, -O(_CH2 )_3- , -(CH2)_4O- , -(_{CH2)3OCH2-, -CH2O(CH2)3-, -(CH2)2O(CH2)2-,-O(CH2)4-.Unless} otherwise specified,_Cx heteroalkylene refers to a heteroalkylene group having x carbon atoms before being replaced_bya heteroatom.

杂芳基：单独使用或作为较大部分的一部分使用的术语“杂芳烷基”或“杂芳-(heteroar-)”(例如，“杂芳基”和“杂芳烷氧基”)是指以下单环或双环环基团：具有5至10个环原子(例如，5至6元单环杂芳基或9至10元双环杂芳基)；在环阵列中具有6个、10个或14个π电子；以及除碳原子外还具有一至五个杂原子。示范性杂芳基基团包含但不限于噻吩基、呋喃基、吡咯基、咪唑基、吡唑基、三唑基、四唑基、噁唑基、异噁唑基、噁二唑基、噻唑基、异噻唑基、噻二唑基、吡啶基、吡啶酮基、哒嗪基、嘧啶基、吡嗪基、吲哚嗪基、嘌呤基、萘啶基、蝶啶基、咪唑并[1,2-a]嘧啶基、咪唑并[1,2-a]吡啶基、噻吩并嘧啶基、三唑并吡啶基及苯并异噁唑基。如本文中所使用，术语“杂芳基”和“杂芳-”还包含其中杂芳族环与一个或多个芳基、环脂肪族或杂环基环稠合的基团，其中连接基团或连接点位于杂芳族环(即，具有1至3个杂原子的双环杂芳基环)上。非限制性实例包含吲哚基、异吲哚基、苯并噻吩基、苯并呋喃基、二苯并呋喃基、吲唑基、苯并咪唑基、苯并噻唑基、苯并噻二唑基、苯并噁唑基、喹啉基、异喹啉基、噌啉基、酞嗪基、喹唑啉基、喹喔啉基、4H-喹嗪基、咔唑基、吖啶基、吩嗪基、吩噻嗪基、吩噁嗪基、四氢喹啉基、四氢异喹啉基、吡啶并[2,3-b]-1,4-噁嗪-3(4H)-酮及苯并异噁唑基。术语“杂芳基”与术语“杂芳基环”、“杂芳基基团”或“杂芳香族基”可以互换使用，这些术语中的任一个都包含任选地被取代的环。Heteroaryl: The terms "heteroaralkyl" or "heteroar-" (e.g., "heteroaryl" and "heteroaralkoxy"), used alone or as part of a larger moiety, refer to monocyclic or bicyclic ring radicals having: 5 to 10 ring atoms (e.g., 5 to 6 membered monocyclic heteroaryl or 9 to 10 membered bicyclic heteroaryl); 6, 10 or 14 pi electrons in the ring array; and one to five heteroatoms in addition to carbon atoms. Exemplary heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and benzisoxazolyl. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which includes rings that are optionally substituted.

杂原子：术语“杂原子”是指氧、硫、氮、磷或硅中的一者或多者(包括氮、硫、磷或硅的任何氧化形式；任何碱性氮的季铵化形式或；杂环的可取代的氮，例如，N(如在3,4-二氢-2H-吡咯基中)、NH(如在吡咯烷基中)或NR⁺(如在N-取代的吡咯烷基中))。Heteroatom: The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; the quaternized form of any basic nitrogen; a substitutable nitrogen of a heterocycle, for example, N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

杂环：术语“杂环(heterocycle)”、“杂环基(heterocyclyl)”、“杂环基团(heterocyclic radica)”和“杂环(heterocyclic ring)”在本文中可互换地使用，并且是指稳定的3至8元单环、7至12元双环或10至16元多环杂环部分，其为饱和或部分不饱和的并且除碳原子外还具有一个或多个(诸如一至四个)杂原子，如上文所定义。当关于杂环的环原子使用时，术语“氮”包含经取代的氮。例如，在具有0-3个选自氧、硫或氮的杂原子的饱和或部分不饱和的环中，氮可为N(如3,4-二氢-2H-吡咯基中)、NH(如吡咯烷基中)或NR⁺(如N取代的吡咯烷基中)。杂环可以在任何杂原子或碳原子处连接到其侧基，从而产生稳定结构，并且任何环原子可以任选地经取代。此类饱和或部分不饱和的杂环基基团的实例包含但不限于氮杂环丁烷基、氧杂环丁烷基、四氢呋喃基、四氢噻吩基、吡咯烷基、哌啶基、十氢喹啉基、噁唑烷基、哌嗪基、四氢吡喃基、二氧杂环己烷基、二氧戊环基、二氮呯基、噁氮呯基、噻氮呯基、吗啉基、硫吗啉基和/或杂环基基团可以是单环、双环、三环或多环的，优选地是单环、双环或三环的，更优选地是单环或双环的。术语“杂环烷基”是指被杂环基取代的烷基，其中烷基和杂环基部分独立地为任选地经取代的。双环杂环还包含其中杂环与一个或多个芳基、杂芳基或环脂肪族环稠合的基团。示范性双环杂环基团包含吲哚啉基、异吲哚啉基、苯并间二氧杂环戊烯基、1,3-二氢异苯并呋喃基、2,3-二氢苯并呋喃基和四氢喹啉基。双环杂环还可以是螺环系统(例如，除碳原子以外还具有一个或多个如上文所定义的杂原子(例如一个、两个、三个或四个杂原子)的7至11元螺环稠合杂环)。双环杂环还可以示桥环系统(例如，具有一个、两个或三个桥接原子的7至11元桥接杂环)。Heterocycle: The terms "heterocycle", "heterocyclyl", "heterocyclic radica" and "heterocyclic ring" are used interchangeably herein and refer to a stable 3-8 membered monocyclic, 7-12 membered bicyclic or 10-16 membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (such as one to four) heteroatoms in addition to carbon atoms, as defined above. When used with respect to the ring atoms of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl). The heterocycle may be attached to its side group at any heteroatom or carbon atom to produce a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclyl groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepine, oxazepine, thiazepine, morpholinyl, thiomorpholinyl and/or The heterocyclyl group can be monocyclic, bicyclic, tricyclic or polycyclic, preferably monocyclic, bicyclic or tricyclic, more preferably monocyclic or bicyclic. The term "heterocycloalkyl" refers to an alkyl substituted by a heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aryl, heteroaryl or cycloaliphatic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl and tetrahydroquinolinyl. The bicyclic heterocycle can also be a spirocyclic system (e.g., a 7- to 11-membered spirocyclic fused heterocycle having one or more heteroatoms (e.g., one, two, three or four heteroatoms) as defined above in addition to carbon atoms). The bicyclic heterocycle can also represent a bridged ring system (eg, a 7- to 11-membered bridged heterocycle having one, two, or three bridging atoms).

抑制剂：如本文中所使用，术语“抑制剂”是指其存在、水平或程度与标靶的水平或活性降低相关的实体、条件或事件)。在一些实施例中，抑制剂可以直接作用(在这种情况下，其直接对其靶标施加其影响，例如通过与该靶标结合)；在一些实施例中，抑制剂可以间接起作用(在这种情况下，其通过与靶标的调节剂相互作用且/或以其他方式改变该靶标的调节剂来施加其影响，使得该靶标的水平和/或活性降低)。在一些实施例中，抑制剂是其存在或水平与相对于特定参考水平或活性(例如，在适当的参考条件，如存在已知的抑制剂，或不存在所讨论的抑制剂等条件下观测到的水平或活性)降低的标靶水平或活性相关的抑制剂。Inhibitor: As used herein, the term "inhibitor" refers to an entity, condition, or event whose presence, level, or extent is associated with a decreased level or activity of a target. In some embodiments, an inhibitor may act directly (in which case it exerts its effect directly on its target, such as by binding to the target); in some embodiments, an inhibitor may act indirectly (in which case it exerts its effect by interacting with and/or otherwise altering a modulator of the target, such that the level and/or activity of the target is decreased). In some embodiments, an inhibitor is an inhibitor whose presence or level is associated with a decreased level or activity of a target relative to a particular reference level or activity (e.g., a level or activity observed under appropriate reference conditions, such as the presence of a known inhibitor, or the absence of the inhibitor in question).

体外：如本文中所使用，术语“体外”是指在人造环境中(例如，在试管或反应器中、在细胞培养物中等)而非在多细胞生物体内发生的事件。In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reactor, in cell culture, etc.) rather than in a multicellular organism.

经分离：如本文中所使用，是指物质和/或实体(1)已与最初产生时与其相关的至少一些组分分离(无论在自然界中和/或在实验环境中)，和/或(2)通过人力设计、产生、制备和/或制造。经分离的物质和/或实体可与约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或超过约99％的与其最初缔合的其它组分分离。在一些实施例中，经分离的试剂的纯度为约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或超过约99％。如本文中所使用，如果物质基本上不含其它组分，则其为“纯的”。在一些实施例中，如本领域技术人员将理解，一种物质在已经与某些其它组分(诸如例如一种或多种载剂或赋形剂(例如，缓冲液、溶剂、水等))合并之后仍然可以被视为“经分离的”或甚至“纯的”；在此类实施例中，物质的分离或纯度百分比的计算不包含此类载剂或赋形剂。仅举一个例子，在一些实施例中，当以下情况时，自然界中存在的生物聚合物(例如多肽或多核苷酸)被认为是“经分离的”：a)由于其起源或衍生来源不与在自然界中的天然状态下伴随生物聚合物的组分中的一些或所有组分相关联；b)它基本上不含来自自然界中产生生物聚合物的物种的相同物种的其他多肽或核酸；c)由来自不属于自然界中产生生物聚合物的物种的细胞或其他表达系统的组分表达或以其他方式与其相关。因此，举例来说，在一些实施例中，经化学合成或在不同于在自然界中产生多肽的细胞系统中合成的多肽被视为“经分离”多肽。另选地或附加地，在一些实施例中，已经经受一种或多种纯化技术的多肽就其已经与a)在自然界中相关；和/或b)在起初产生时相关的其它组分分离来说可以被视为“经分离”多肽。Isolated: as used herein, refers to a substance and/or entity that (1) has been separated from at least some of the components with which it was originally associated (whether in nature and/or in an experimental setting), and/or (2) has been designed, produced, prepared, and/or manufactured by human effort. An isolated substance and/or entity may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which it was originally associated. In some embodiments, the purity of the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after it has been combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the calculation of the separation or purity percentage of the substance does not include such carriers or excipients. As just one example, in some embodiments, a biopolymer (e.g., a polypeptide or polynucleotide) occurring in nature is considered "isolated" when: a) it is not associated with some or all of the components that accompany the biopolymer in its native state in nature by virtue of its origin or source of derivation; b) it is substantially free of other polypeptides or nucleic acids from the same species as the species in which the biopolymer is produced in nature; c) it is expressed by or otherwise associated with components of a cell or other expression system that is not of the species in which the biopolymer is produced in nature. Thus, for example, in some embodiments, a polypeptide that has been chemically synthesized or synthesized in a cellular system other than that in which the polypeptide is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide insofar as it has been separated from other components with which it is a) associated in nature; and/or b) associated when originally produced.

体内：如本文中所使用，是指在多细胞生物体，如人类和非人类动物内发生的事件。在基于细胞的系统的情况下，该术语可用于指代在活细胞(与例如体外系统相反)内发生的事件。In vivo: as used herein refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

连接子：如本文中所使用，用以指代将不同元件彼此连接的多元件药剂的部分。例如，本领域的普通技术人员将理解，其结构包含两个或更多个功能性结构域或组织结构域的多肽通常包含此类结构域之间的将其彼此连接的一段氨基酸。在一些实施例中，多肽包括具有一般形式S1-L'-S2的总体结构的连接子元件“L”，其中S1和S2可相同或不同且表示两个通过连接子彼此缔合的结构域。在一些实施例中，多肽连接子的长度为至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100个或更多个氨基酸。在一些实施例中，连接子的特征在于，其不采用刚性三维结构，而是采用能为多肽提供柔性的三维结构。当工程化多肽(例如，融合多肽)时可以适当地使用本领域已知的多种不同的连接子元件(参见，例如，Holliger,P.等人，(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人，(1994)Structure 2:1 121-1123)。Connector: As used herein, it refers to the part of a multi-element medicament that connects different elements to each other. For example, it will be understood by those of ordinary skill in the art that a polypeptide whose structure includes two or more functional domains or organizational domains generally includes a segment of amino acids between such domains that connect them to each other. In some embodiments, the polypeptide includes a connector element "L" with a general structure of the general form S1-L'-S2, wherein S1 and S2 may be the same or different and represent two domains that are associated with each other by a connector. In some embodiments, the length of the polypeptide connector is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized in that it does not adopt a rigid three-dimensional structure, but rather adopts a three-dimensional structure that provides flexibility to the polypeptide. When engineering polypeptides (e.g., fusion polypeptides), a variety of different linker elements known in the art can be appropriately used (see, e.g., Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J. et al., (1994) Structure 2: 1 121-1123).

纳米粒子：如本文中所使用，术语“纳米粒子”是指直径小于1000纳米(nm)的粒子。在一些实施例中，如由美国国家科学基金会(National Science Foundation)定义，纳米粒子的直径小于300nm。在一些实施例中，如由美国国家卫生研究院(National Institutesof Health)定义，纳米粒子的直径小于100nm。在一些实施例中，纳米粒子为微胞，这在于其包括通过微胞膜自本体溶液分离的闭合区室，所述膜通常包括包围且围封一空间或区室(例如，以界定内腔)的两亲实体。在一些实施例中，微胞膜包括至少一种聚合物，例如可生物相容的和/或可生物降解的聚合物。在一些实施例中，本文所述的脂质纳米粒子的平均流体动力学直径可以为约30至约170nm。在一些实施例中，本文所述的脂质纳米粒子平均流体动力学直径可为约30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm、150nm、155nm、160nm、165nm、170nm或具有由前述各值中的任何两个限定的端点的任何范围。例如，在一些实施例中，本文所述的脂质纳米粒子的平均流体动力学直径在50nm与100nm之间。Nanoparticle: As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, as defined by the National Science Foundation, the diameter of the nanoparticle is less than 300 nm. In some embodiments, as defined by the National Institutes of Health, the diameter of the nanoparticle is less than 100 nm. In some embodiments, the nanoparticle is a micelle, in that it includes a closed compartment separated from a bulk solution by a micelle membrane, which typically includes an amphiphilic entity that surrounds and encloses a space or compartment (e.g., to define an inner cavity). In some embodiments, the micelle membrane includes at least one polymer, such as a biocompatible and/or biodegradable polymer. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein can be from about 30 to about 170 nm. In some embodiments, the lipid nanoparticle average hydrodynamic diameter described herein can be about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm or any scope with the endpoint limited by any two of the aforementioned values. For example, in some embodiments, the lipid nanoparticle average hydrodynamic diameter described herein is between 50nm and 100nm.

纳米粒子组合物：如本文中所使用，术语“纳米粒子组合物”是指含有至少一种纳米粒子和至少一种额外试剂或成分的组合物。在一些实施例中，纳米粒子组合物含有如本文所述的纳米粒子的大体上均一集合。Nanoparticle composition: As used herein, the term "nanoparticle composition" refers to a composition containing at least one nanoparticle and at least one additional agent or ingredient. In some embodiments, a nanoparticle composition contains a substantially homogeneous collection of nanoparticles as described herein.

核酸：如本文中所使用，在其最广泛意义上是指并入或可以并入寡核苷酸链中的任何化合物和/或物质。在一些实施例中，核酸是通过磷酸二酯键并入或可以通过磷酸二酯键并入寡核苷酸链中的化合物和/或物质。如从上下文将清楚的，在一些实施例中，“核酸”是指单个核酸残基(例如，核苷酸和/或核苷)；在一些实施例中，“核酸”是指包含单个核酸残基的寡核苷酸链。在一些实施例中，“核酸”为或包含RNA；在一些实施例中，“核酸”为或包含DNA。在一些实施例中，核酸是一个或多个天然核酸残基、包括一个或多个天然核酸残基或由一个或多个天然核酸残基组成。在一些实施例中，核酸是一个或多个核酸类似物、包括一个或多个核酸类似物或由一个或多个核酸类似物组成。在一些实施例中，核酸类似物与核酸的不同之处在于其不利用磷酸二酯主链。例如，在一些实施例中，核酸是一个或多个“肽核酸”、包括一个或多个“肽核酸”或由一个或多个“肽核酸”组成，其在本领域中是已知的并且在主链中具有代替磷酸二酯键的肽键，被视为处于本发明的范围内。或者或另外，在一些实施例中，核酸具有一个或多个硫代磷酸酯和/或5'-N-氨基亚磷酸酯键而非磷酸二酯键。在一些实施例中，核酸为以下、包括以下或由以下组成：一个或多个天然核苷(例如腺苷、胸苷、鸟苷、胞苷、尿苷、脱氧腺苷、脱氧胸苷、脱氧鸟苷和脱氧胞苷)。在一些实施例中，“核酸”为以下、包括以下或由以下组成：一个或多个核苷类似物(例如2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤、2-硫代胞苷、甲基化碱基、嵌入的碱基及其组合)。在一些实施例中，与天然核酸中的糖类相比，核酸包括一种或多种经修饰的糖(例如2'-氟核糖、核糖、2'-脱氧核苷、阿拉伯糖和己糖)。在一些实施例中，核酸具有对如RNA或蛋白质等功能性基因产物进行编码的核苷酸序列。在一些实施例中，核酸包含一个或多个内含子。在一些实施例中，通过以下中的一种或多种制备核酸：从天然来源分离、基于互补模板的聚合进行酶促合成(体内或体外)、在重组细胞或系统中繁殖和化学合成。在一些实施例中，核酸的长度为至少3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、20、225、250、275、300、325、350、375、400、425、450、475、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000个或更多个残基。在一些实施例中，核酸是部分或全部单链的；在一些实施例中，核酸是部分或全部双链的。在一些实施例中，核酸具有包括对多肽进行编码的至少一个元件的核苷酸序列，或者是对多肽进行编码的互补序列。在一些实施例中，核酸具有酶活性。Nucleic acid: As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and/or substances that are or can be incorporated into an oligonucleotide chain through phosphodiester bonds. As will be clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, nucleic acid is, includes, or consists of one or more natural nucleic acid residues. In some embodiments, nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, nucleic acid is, includes, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoamidate bonds rather than phosphodiester bonds. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is, includes, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, embedded bases, and combinations thereof). In some embodiments, the nucleic acid includes one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxynucleosides, arabinose, and hexose) compared to sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of: separation from a natural source, enzymatic synthesis (in vivo or in vitro) based on polymerization of a complementary template, propagation in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide, or is a complementary sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

可操作地连接：如本文中所使用，并置是指所描述的组分处于准许其以其预期方式作用的关系中的并接。“可操作地连接”于功能元件的控制元件以使得功能元件的表达和/或活动在与控制元件相容的条件下实现的方式相关联。在一些实施例中，“可操作地连接的”控制元件与感兴趣的编码元件邻接(例如，共价连接)；在一些实施例中，控制元件反作用于所关注功能元件或以其他方式作用于所关注功能元件。Operably linked: As used herein, juxtaposition refers to the joining of the described components in a relationship that permits them to function in their intended manner. A control element "operably linked" to a functional element is associated in a manner such that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is adjacent to (e.g., covalently linked to) the coding element of interest; in some embodiments, the control element reacts to or otherwise acts on the functional element of interest.

为了本发明的目的，根据元素周期表(CAS版本，Handbook of Chemistry andPhysics，第75版)来识别化学元素。另外，有机化学的一般原理在“Organic Chemistry”,Thomas Sorrell,University Science Books,Sausalito:1999以及“March's AdvancedOrganic Chemistry”，第5版：Smith,M.B.和March,J.,John Wiley&Sons,New York:2001，其全部内容通过引用并入本文。For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th edition). In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th edition: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

肠胃外：如本文中所使用的短语“肠胃外施用(parenteral administration)”和“经肠胃外施用(administered parenterally)”具有其在本领域中所理解的含义，是指除肠内和局部施用以外的施用模式，通常是通过注射，并且包含但不限于静脉内、肌肉内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内以及胸骨内注射和输注。Parenteral: As used herein, the phrases "parenteral administration" and "administered parenterally" have their art-understood meanings and refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

患者：如本文中所使用,术语“患者”是指例如出于实验、诊断、预防、美容和/或治疗目的而施用或可以施用所提供的组合物的任何生物体。典型的患者包含动物(例如,哺乳动物，诸如小鼠、大鼠、兔、非人灵长类动物和/或人类)。在一些实施例中，患者是人类。在一些实施例中，患者患有或易患一种或多种病症或病状。在一些实施例中，患者表现出病症或病状的一种或多种症状。在一些实施例中，患者已被诊断为患有一种或多种病症或病状。在一些实施例中，病症或病状是或包含癌症，或存在一种或多种肿瘤。在一些实施例中，患者正在接受或已经接受某些疗法以诊断和/或治疗疾病、病症或病状。Patient: As used herein, the term "patient" refers to any organism to which a provided composition is or can be administered, e.g., for experimental, diagnostic, preventive, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. In some embodiments, the patient suffers from or is susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or comprises cancer, or one or more tumors are present. In some embodiments, the patient is receiving or has received certain therapies to diagnose and/or treat a disease, disorder, or condition.

药物组合物：如本文中所使用，术语“药物组合物”是指与一种或多种药学上可接受的载剂一起调配的活性剂。在一些实施例中，活性剂以适于在治疗方案中施用的单位剂量的量存在，当向相关群体施用时，所述治疗方案显示出达到预定治疗作用的统计学上显著的概率。在一些实施例中，药物组合物可以专门配制用于以固体或液体形式施用，包含适合于以下的那些：口服施用，例如灌服剂(水性或非水性溶液或悬浮液)、片剂(例如针对以下的那些：口腔、舌下和全身吸收，用于施用于舌部的丸剂、粉剂、颗粒剂、糊剂)；肠胃外施用，例如，通过皮下、肌内、静脉内或硬膜外注射，例如，作为无菌溶液或悬浮液或缓释调配物；局部施用，例如，作为乳膏、软膏或缓释贴或喷雾施用于皮肤、肺或口腔；阴道内或直肠内，例如，作为子宫托、乳膏或泡沫；舌下含服；经眼施用；透皮使用；或经鼻、肺和其他粘膜表面的施用。Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a therapeutic regimen that, when administered to a relevant population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for: oral, sublingual and systemic absorption, pills, powders, granules, pastes for application to the tongue); parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, for example, as a sterile solution or suspension or sustained release formulation; topical administration, for example, as a cream, ointment or sustained release patch or spray to the skin, lungs or mouth; intravaginal or intrarectal, for example, as a pessary, cream or foam; sublingual administration; ocular administration; transdermal use; or administration to the nose, lungs and other mucosal surfaces.

药学上可接受的：如本文中所使用，短语“药学上可接受的”是指在合理的医学判断范围内，适用于与人类和动物的组织接触而无过度毒性、刺激、过敏反应或其它问题或并发症，与合理的益处/风险比相匹配的那些化合物、材料、组合物和/或剂型。Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

药学上可接受的载剂：如本文中所使用，术语“药学上可接受的载剂”意指药学上可接受的材料、组合物或媒剂，如液体或固体填充剂、稀释剂、赋形剂或溶剂包裹材料，其参与将本发明化合物从身体的一个器官或部分携带或运输到身体的另一个器官或部分。在与调配物的其它成分相容并且对患者无害的意义上来讲，每种载剂必须是“可接受的”。可以用作药学上可接受的载剂的材料的一些实例包含：糖，诸如乳糖、葡萄糖和蔗糖；淀粉，诸如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，诸如羧甲基纤维素钠、乙基纤维素、醋酸纤维素；黄芪粉；麦芽；明胶；滑石；赋形剂，诸如可可脂和栓剂蜡；油类，诸如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；二醇，诸如丙二醇；多元醇，诸如甘油、山梨醇、甘露醇和聚乙二醇；酯类，诸如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，诸如氢氧化镁和氢氧化铝；海藻酸；无热原水；等渗盐水；林格氏溶液；乙醇；pH缓冲溶液；聚酯、聚碳酸酯和/或聚酸酐；以及药物调配物中使用的其他无毒相容物质。Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, which participates in carrying or transporting the compounds of the invention from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; astragalus powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffer solutions; polyesters, polycarbonates and/or polyanhydrides; and other nontoxic compatible substances used in pharmaceutical formulations.

药学上可接受的盐：如本文中所使用，术语“药学上可接受的盐”是指适合于在药学背景中使用的这类化合物的盐，即在合理的医学判断范围内，适合与人类和低等动物的组织接触而无过度毒性、刺激、过敏反应等，并且与合理的益处/风险比相匹配的盐。药学上可接受的盐是本领域中熟知的。例如，S.M.Berge等人在J.Pharmaceutical Sciences,66:1-19(1977)中详细描述了药学上可接受的盐。在一些实施例中，药学上可接受的盐包含但不限于无毒的酸加成盐，其是氨基与无机酸或与有机酸形成的盐，所述无机酸如盐酸、氢溴酸、磷酸、硫酸以及高氯酸，所述有机酸如乙酸、马来酸、酒石酸、柠檬酸、琥珀酸或丙二酸，或通过使用本领域中所用的其它方法，如离子交换法形成的盐。在一些实施例中，药学上可接受的盐包含但不限于：己二酸盐、藻酸盐、抗坏血酸盐、天冬氨酸盐、苯磺酸盐、苯甲酸盐、硫酸氢盐、硼酸盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、柠檬酸盐、环戊烷丙酸盐、二葡糖酸盐、十二烷基硫酸盐、乙磺酸盐、甲酸盐、富马酸盐、葡庚糖酸盐、甘油磷酸盐、葡糖酸盐、半硫酸盐、庚酸盐、己酸盐、氢碘化物、2-羟基-乙磺酸盐、乳糖酸盐(lactobionate)、乳酸盐、月桂酸盐、月桂基硫酸盐、苹果酸盐、马来酸盐、丙二酸盐、甲磺酸盐、2-萘磺酸盐、烟酸盐、硝酸盐、油酸盐、草酸盐、棕榈酸盐、扑酸盐、果胶酸盐、过硫酸盐、3-苯基丙酸盐、磷酸盐、苦味酸盐、新戊酸盐、丙酸盐、硬脂酸盐、琥珀酸盐、硫酸盐、酒石酸盐、硫氰酸盐、对甲苯磺酸盐、十一烷酸盐、戊酸盐等。代表性碱金属盐或碱土金属盐包含钠盐、锂盐、钾盐、钙盐、镁盐等。在一些实施例中，适当时，药学上可接受的盐包含使用抗衡离子形成的无毒的铵、季铵以及胺阳离子，抗衡离子例如是卤离子、氢氧根、羧酸根、硫酸根、磷酸根、硝酸根、具有1至6个碳原子的烷基、磺酸根以及芳基磺酸根。Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds suitable for use in a pharmaceutical context, i.e., within the scope of reasonable medical judgment, suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and salts that match a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M.Berge et al. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include but are not limited to non-toxic acid addition salts, which are salts formed of amino groups with inorganic acids or with organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art, such as salts formed by ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates and arylsulfonates, as appropriate.

预防(prevent/prevention)：如本文所使用，当结合疾病、病症和/或病状的存在使用时，是指降低患上疾病、病症和/或病状的风险和/或延迟疾病、病症或病状的一个或多个特征或症状的发作。当疾病、病症或病状的发作已被延迟预定一段时间时，可以认为预防完成。Prevent (prevent): as used herein, when used in conjunction with the presence of a disease, disorder and / or condition, refers to reducing the risk of developing a disease, disorder and / or condition and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder or condition. Prevention can be considered to be achieved when the onset of a disease, disorder or condition has been delayed for a predetermined period of time.

蛋白质：如本文中所使用，术语“蛋白质”是指多肽(即，由至少两个氨基酸通过肽键相互连接而成的串)。蛋白质可以包含氨基酸以外的部分(例如，可以是糖蛋白、蛋白聚糖等)和/或可以以其他方式加工或修饰。本领域普通技术人员将理解，“蛋白质”可以是如由细胞产生的完整多肽链(具有或不具有信号序列)或者可以是其特征部分。普通技术人员将理解，蛋白质有时可以包含多于一条多肽链，例如，通过一个或多个二硫键连接或通过其他方式相关联。多肽可含有L-氨基酸、D-氨基酸或其两者，且可含有本领域中已知的多种氨基酸修饰或类似物中的任一种。有用的修饰包含例如末端乙酰化、酰胺化、甲基化等。在一些实施例中，蛋白质可以包括天然氨基酸、非天然氨基酸、合成氨基酸及它们的组合。术语“肽”一般用于指长度小于约100个氨基酸、小于约50个氨基酸、小于20个氨基酸或小于10个氨基酸的多肽。在一些实施例中，蛋白质是抗体、抗体片段、其生物活性部分和/或其特征部分。Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids connected to each other by peptide bonds). Proteins may contain parts other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be processed or modified in other ways. It will be understood by those of ordinary skill in the art that a "protein" may be a complete polypeptide chain (with or without a signal sequence) such as produced by a cell or may be a characteristic part thereof. It will be understood by those of ordinary skill that a protein may sometimes contain more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. A polypeptide may contain L-amino acids, D-amino acids, or both thereof, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, a protein may include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

多肽：如本文所使用的术语“多肽”通常具有作为至少三个氨基酸的聚合物的本领域公认的含义。本领域的普通技术人员将理解，术语“多肽”旨在足够一般，以便涵盖具有本文所列举的完整序列的多肽，同时涵盖表示此类完整多肽的功能性片段(例如，保留至少一种活性的片段)的多肽。此外，本领域的普通技术人员将理解，蛋白质序列通常可以在不破坏活性的情况下进行一些取代。因此，如本文所使用的相关术语“多肽”涵盖保留活性并与同一类别的另一种多肽共享至少约30至40％(通常大于约50％、60％、70％或80％)的总体序列一致性，并且进一步通常包含至少一个具有更高一致性的区(在一个或多个高度保守区中，所述一致性通常大于90％或甚至95％、96％、97％、98％或99％)，一般涵盖至少3-4个以及通常高达20个或更多个氨基酸的任何多肽。多肽可含有L-氨基酸、D-氨基酸或其两者，且可含有本领域中已知的多种氨基酸修饰或类似物中的任一种。有用的修饰包含例如末端乙酰化、酰胺化、甲基化等。在一些实施例中，蛋白质可以包括天然氨基酸、非天然氨基酸、合成氨基酸及它们的组合。术语“肽”一般用于指长度小于约100个氨基酸、小于约50个氨基酸、小于20个氨基酸或小于10个氨基酸的多肽。在一些实施例中，蛋白质是抗体、抗体片段、其生物活性部分和/或其特征部分。Polypeptide: The term "polypeptide" as used herein generally has the art-recognized meaning of a polymer of at least three amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be general enough to cover polypeptides having the complete sequences listed herein, while covering polypeptides representing functional fragments of such complete polypeptides (e.g., fragments retaining at least one activity). In addition, it will be understood by those of ordinary skill in the art that protein sequences can generally be substituted without destroying activity. Therefore, the related term "polypeptide" as used herein covers an overall sequence identity of at least about 30 to 40% (usually greater than about 50%, 60%, 70% or 80%) that retains activity and shares with another polypeptide of the same class, and further generally includes at least one region with higher identity (in one or more highly conserved regions, the identity is generally greater than 90% or even 95%, 96%, 97%, 98% or 99%), generally covering any polypeptide of at least 3-4 and generally up to 20 or more amino acids. The polypeptide may contain L-amino acids, D-amino acids, or both thereof, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the protein may include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

预防：如本文中所使用，术语“预防”是指延迟特定疾病、病症或病状的一种或多种症状的发作和/或降低其频率和/或严重程度。在一些实施例中，在群体基础上评估预防，使得如果在易患疾病、病症或病状的群体中观测到疾病、病症或病状的一种或多种症状的发展、频率和/或强度的统计学上显著降低，那么认为药剂会“预防”特定疾病、病症或病状。当疾病、病症或病状的发作已被延迟预定一段时间时，可以认为预防完成。Prevention: As used herein, the term "prevention" refers to delaying the onset of and/or reducing the frequency and/or severity of one or more symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered accomplished when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

保护基：如本文中所使用，短语“保护基”是指避免潜在反应性官能团发生不希望的化学转化的临时取代基。这类保护基的实例包含羧酸酯、醇的硅醚以及醛和酮各自的缩醛和缩酮。“Si保护基”是包括Si原子的保护基，如Si-三烷基(例如，三甲基硅基、三丁基硅基、叔丁基二甲基硅基)、Si-三芳基、Si-烷基-二苯基(例如，叔丁基二苯基硅基)或Si-芳基-二烷基(例如，Si-苯基二烷基)。一般来说，Si保护基与氧原子连接。已经对保护基化学领域进行了回顾(Greene,T.W.；Wuts,P.G.M.Protective Groups in Organic Synthesis，第2版；Wiley:New York,1991)。下文详细描述了这类保护基(和相关的保护部分)。Protecting group: As used herein, the phrase "protecting group" refers to a temporary substituent that avoids undesirable chemical transformation of a potential reactive functional group. Examples of such protecting groups include carboxylic acid esters, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones. "Si protecting group" is a protecting group including Si atoms, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl, tert-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g., tert-butyldiphenylsilyl) or Si-aryl-dialkyl (e.g., Si-phenyldialkyl). In general, Si protecting groups are connected to oxygen atoms. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd edition; Wiley: New York, 1991). Such protecting groups (and associated protection moieties) are described in detail below.

羟基保护基是本领域中众所周知的，并且包含Protecting Groups in OrganicSynthesis,T.W.Greene和P.G.M.Wuts，第3版，John Wiley&Sons,1999中详细描述的羟基保护基，所述文献的全部内容以引用的方式并入本文中。适当保护的羟基的实例进一步包含但不限于酯、碳酸酯、磺酸酯、烯丙基醚、醚、硅醚、烷基醚、芳基烷基醚和烷氧基烷基醚。合适的酯的实例包含甲酸酯、乙酸酯、丙酸酯、戊酸酯、巴豆酸酯和苯甲酸酯。合适的酯的具体实例包含甲酸酯、苯甲酰甲酸酯、氯乙酸酯、三氟乙酸酯、甲氧基乙酸酯、三苯基甲氧基乙酸酯、对氯苯氧基乙酸酯、3-苯基丙酸酯、4-氧代戊酸酯、4,4-(亚乙基二硫基)戊酸酯、新戊酸酯(三甲基乙酸酯)、巴豆酸酯、4-甲氧基-巴豆酸酯、苯甲酸酯、对苯甲基苯甲酸酯、2,4,6-三甲基苯甲酸酯。合适的碳酸酯的实例包含碳酸9-芴基甲酯、碳酸乙酯、碳酸2,2,2-三氯乙酯、碳酸2-(三甲基硅基)乙酯、碳酸2-(苯磺酰基)乙酯、碳酸乙烯酯、碳酸烯丙酯以及碳酸对硝基苯甲酯。合适的硅醚的实例包含三甲基硅醚、三乙基硅醚、叔丁基二甲基硅醚、叔丁基二苯基硅醚、三异丙基硅醚和其它三烷基硅醚。合适的烷基醚的实例包括甲基醚、苯甲基醚、对甲氧基苯甲基醚、3,4-二甲氧基苯甲基醚、三苯甲基醚、叔丁基醚以及烯丙醚或其衍生物。烷氧基烷基醚包含缩醛，如甲氧基甲醚、甲硫基甲醚、(2-甲氧基乙氧基)甲醚、苯甲氧基甲醚、β-(三甲基硅基)乙氧基甲醚和四氢吡喃-2-基醚。合适的芳基烷基醚的实例包含苯甲醚、对甲氧基苯甲基(MPM)醚、3,4-二甲氧基苯甲醚、邻硝基苯甲醚、对硝基苯甲醚、对卤基苯甲醚、2,6-二氯苯甲醚、对氰基苯甲醚、2-吡啶甲基醚和4-吡啶甲基醚。Hydroxyl protecting groups are well known in the art, and include Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, 3rd edition, John Wiley & Sons, 1999, the full content of which is incorporated herein by reference. Examples of suitably protected hydroxyls further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silicon ethers, alkyl ethers, aryl alkyl ethers and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, propionates, valerates, crotonates and benzoates. Specific examples of suitable esters include formates, benzoyl formates, chloroacetates, trifluoroacetates, methoxyacetates, triphenylmethoxyacetates, p-chlorophenoxyacetates, 3-phenylpropionates, 4-oxopentanoates, 4,4-(ethylenedisulfide)pentanoates, pivalates (trimethylacetates), crotonates, 4-methoxy-crotonates, benzoates, p-phenylmethylbenzoates, 2,4,6-trimethylbenzoates. Examples of suitable carbonates include 9-fluorenylmethyl carbonate, ethyl carbonate, 2,2,2-trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, 2-(phenylsulfonyl)ethyl carbonate, ethylene carbonate, allyl carbonate, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, triisopropylsilyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trityl ether, tert-butyl ether and allyl ether or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl ether, methylthiomethyl ether, (2-methoxyethoxy)methyl ether, benzyloxymethyl ether, β-(trimethylsilyl)ethoxymethyl ether and tetrahydropyran-2-yl ether. Examples of suitable aryl alkyl ethers include anisole, p-methoxybenzyl (MPM) ether, 3,4-dimethoxyanisole, o-nitroanisole, p-nitroanisole, p-haloanisole, 2,6-dichloroanisole, p-cyanoanisole, 2-picolyl ether and 4-picolyl ether.

被保护的胺是本领域中众所周知的并且包含Greene(1999)中详细描述的被保护的胺。合适的单保护胺进一步包含但不限于芳烷基胺、氨基甲酸酯、烯丙基胺、酰胺等。合适的单保护氨基部分的实例包含叔丁氧基羰基氨基(-NHBOC)、乙氧基羰基氨基、甲氧基羰基氨基、三氯乙氧基羰基氨基、烯丙氧基羰基氨基(-NHAlloc)、苯甲氧基羰基氨基(-NHCBZ)、烯丙基氨基、苯甲基氨基(-NHBn)、芴基甲基羰基(-NHFmoc)、甲酰胺基、乙酰胺基、氯乙酰胺基、二氯乙酰胺基、三氯乙酰胺基、苯基乙酰胺基、三氟乙酰胺基、苯甲酰胺基、叔丁基二苯基硅基等。合适的双保护胺包含被两个独立地选自上文描述为单保护胺的取代基的取代基取代的胺，并进一步包含环酰亚胺，例如邻苯二甲酰亚胺、马来酰亚胺、琥珀酰亚胺等。合适的双保护胺还包含吡咯等、2,2,5,5-四甲基-[1,2,5]氮杂二硅杂环戊烷等、以及叠氮化合物。Protected amines are well known in the art and include those described in detail in Greene (1999). Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected amino moieties include tert-butyloxycarbonylamino (-NHBOC), ethoxycarbonylamino, methoxycarbonylamino, trichloroethoxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxycarbonylamino (-NHCBZ), allylamino, benzylamino (-NHBn), fluorenylmethylcarbonyl (-NHFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, tert-butyldiphenylsilyl, and the like. Suitable double protected amines include amines substituted with two substituents independently selected from the substituents described above as mono-protected amines, and further include cyclic imides such as phthalimide, maleimide, succinimide, etc. Suitable double protected amines also include pyrrole, etc., 2,2,5,5-tetramethyl-[1,2,5]azadisilacyclopentane, etc., and azide compounds.

被保护的醛是本领域中众所周知的并且包含Greene(1999)中详细描述的被保护的醛。合适的被保护的醛进一步包含但不限于非环缩醛、环缩醛、腙、亚胺等。这类基团的实例包含二甲基缩醛、二乙基缩醛、二异丙基缩醛、二苯甲基缩醛、双(2-硝基苯甲基)缩醛、1,3-二噁烷、1,3-二氧杂环戊烷、缩氨基脲和其衍生物。Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzhydryl acetal, bis(2-nitrobenzyl)acetal, 1,3-dioxane, 1,3-dioxolane, semicarbazones, and derivatives thereof.

被保护的羧酸是本领域中众所周知的并且包括Greene(1999)中详细描述的被保护的羧酸。合适的被保护羧酸进一步包含但不限于任选地被取代的C_1-6脂肪族酯、任选地被取代的芳基酯、硅基酯、活化酯、酰胺、酰肼等。这类酯基的实例包含甲酯、乙酯、丙酯、异丙酯、丁酯、异丁酯、苯甲酯及苯基酯，其中每个基团任选地被取代。其它合适的被保护的羧酸包括噁唑啉和原酸酯。Protected carboxylic acids are well known in the art and include protected carboxylic acids described in detail in Greene (1999). Suitable protected carboxylic acids further include but are not limited to optionally substituted C_1-6 aliphatic esters, optionally substituted aryl esters, silicon-based esters, activated esters, amides, hydrazides, etc. Examples of this type of ester group include methyl esters, ethyl esters, propyl esters, isopropyl esters, butyl esters, isobutyl esters, benzyl esters and phenyl esters, wherein each group is optionally substituted. Other suitable protected carboxylic acids include oxazolines and orthoesters.

被保护的硫醇是本领域中众所周知的并且包含Greene(1999)中详细描述的被保护的硫醇。合适的被保护的硫醇进一步包含但不限于二硫化物、硫醚、硅基硫醚、硫酯、硫代碳酸酯及硫代氨基甲酸酯等。这类基团的实例包含但不限于烷基硫醚、苯甲基硫醚和经取代的苯甲基硫醚、三苯甲基硫醚和三氯乙氧基羰基硫酯等等。Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl thioethers and substituted benzyl thioethers, trityl thioethers, and trichloroethoxycarbonyl thioesters, and the like.

蛋白质：如本文所使用的术语“蛋白质”是指一种或多种作为离散单元发挥作用的多肽。如果单个多肽为离散功能单元且不需要与其它多肽进行永久或暂时的物理缔合以形成离散功能单元，那么术语“多肽”和“蛋白质”可以互换地使用。如果离散功能性单元包括彼此物理缔合的超过一个多肽，那么术语“蛋白质”可以用于指物理缔合且作为离散单元共同发挥作用的多个多肽。在一些实施例中，蛋白质可包啊很难除氨基酸之外的部分(例如，可以为糖蛋白、蛋白多糖等)和/或可以其它方式处理或修饰。本领域的普通技术人员将了解，在一些实施例中，术语“蛋白质”可以指由细胞产生的完整多肽链(例如，具有或不具有信号序列)，和/或指在细胞内具有活性的形式(例如，截短或复合形式)。在蛋白质包括多个多肽链的一些实施例中，这类链可以例如通过一个或多个二硫键彼此共价结合，或可以通过其它方式缔合。Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. If a single polypeptide is a discrete functional unit and does not need to be physically associated with other polypeptides permanently or temporarily to form a discrete functional unit, the terms "polypeptide" and "protein" can be used interchangeably. If the discrete functional unit includes more than one polypeptide physically associated with each other, the term "protein" can be used to refer to multiple polypeptides that are physically associated and function together as discrete units. In some embodiments, the protein may include parts other than amino acids (for example, it may be a glycoprotein, proteoglycan, etc.) and/or may be processed or modified in other ways. One of ordinary skill in the art will appreciate that in some embodiments, the term "protein" may refer to a complete polypeptide chain produced by a cell (for example, with or without a signal sequence), and/or to a form that is active in a cell (for example, a truncated or complex form). In some embodiments where the protein includes multiple polypeptide chains, such chains may be covalently bound to each other, for example, by one or more disulfide bonds, or may be associated in other ways.

纯：如本文中所使用，如果试剂或实体大体上不含其它组分，那么其为“纯”的。例如，含有超过约90％特定试剂或实体的制剂通常被认为是纯的制剂。在一些实施例中，试剂或实体是至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％纯的。Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or entity is generally considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

参考：如本文中所使用，描述了一种标准或对照，以此为基础进行比较。例如，在一些实施例中，将所关注药剂、动物、个体、群体、样品、序列或值与参考或对照药剂、动物、个体、群体、样品、序列或值进行比较。在一些实施例中，基本上在进行所关注的测试或确定的同时测试和/或确定参考或对照。在一些实施例中，参考或对照是任选地体现在有形介质中的历史参考或对照。通常，如本领域技术人员所理解，在与评估中的条件或环境类似的条件或环境下确定或表征参考或对照。本领域的技术人员将理解何时存在足够的相似性来证明对特定可能的参考或对照的依赖和/或比较。Reference: As used herein, describes a standard or control on which comparison is based. For example, in some embodiments, the agent, animal, individual, population, sample, sequence or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, the reference or control is tested and/or determined substantially at the same time as the test or determination of interest is performed. In some embodiments, the reference or control is a historical reference or control optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or environments similar to those under evaluation. Those skilled in the art will understand when there is sufficient similarity to justify reliance on and/or comparison of a particular possible reference or control.

样品：如本文中所使用，术语“样品”通常是指从所关注来源获得或源自所述来源的材料等分试样，如本文所述。在一些实施例中，所关注的来源为生物或环境来源。在一些实施例中，所关注的来源可以是或包括细胞或生物体，例如微生物、植物或动物(例如人类)。在一些实施例中，所关注的来源为或包括生物组织或流体。在一些实施例中，生物组织或流体可以是或包括羊水、房水、腹水、胆汁、骨髓、血液、母乳、脑脊髓液、耳垢、乳糜、食糜、射出的精液(ejaculate)、内淋巴液、渗出液、粪便、胃酸、胃液、淋巴液、粘液、心包液、外淋巴液、腹膜液、胸膜液、脓汁、发炎性分泌物(rheum)、唾液、皮脂、精液、血清、包皮垢、痰液、滑液、汗液、泪液、尿液、阴道分泌物、玻璃体液、呕吐物、和/或其组合或组分。在一些实施例中，生物流体可以是或包括胞内液、胞外液、血管内液(血浆)、间质液、淋巴液和/或跨细胞液。在一些实施例中，生物流体可以是或包括植物渗出液。在一些实施例中，生物组织或样品可以例如通过抽吸、活检(例如，细针或组织活检)、拭子(例如，口拭子、鼻拭子、皮肤拭子或阴道拭子)、刮擦、手术、洗涤或灌洗(例如，支气管肺泡、导管、鼻、眼部、口腔、子宫、阴道或其它洗涤或灌洗)获得。在一些实施例中，生物样品是或包括从个体获得的细胞。在一些实施例中，样品是通过任何适当的方式直接从所关注的来源获得的“初级样品”。在一些实施例中，如将从上下文显而易见，术语“样品”是指通过处理初级样品(例如，通过去除初级样品中的一种或多种组分和/或通过向初级样品中添加一种或多种试剂)而获得的制剂。例如，使用半渗透膜进行过滤。此“经处理的样品”可以包括例如从样品中提取的或通过对初级样品进行一种或多种诸如核酸的扩增或逆转录、某些组分的分离和/或纯化等技术而获得的核酸或蛋白质。Sample: As used herein, the term "sample" generally refers to an aliquot of material obtained from or derived from a source of interest, as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chyme, ejaculated semen (ejaculate), endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, inflammatory secretions (rheum), saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomitus, and/or combinations or components thereof. In certain embodiments, biological fluid can be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph and/or transcellular fluid. In certain embodiments, biological fluid can be or include plant exudate. In certain embodiments, biological tissue or sample can be obtained, for example, by suction, biopsy (for example, fine needle or tissue biopsy), swab (for example, oral swab, nasal swab, skin swab or vaginal swab), scraping, surgery, washing or lavage (for example, bronchoalveolar, duct, nose, eye, oral cavity, uterus, vagina or other washing or lavage). In certain embodiments, biological sample is or includes the cell obtained from individual. In certain embodiments, sample is "primary sample" obtained directly from the source of interest by any appropriate means. In certain embodiments, as will be apparent from the context, term "sample" refers to the preparation obtained by processing primary sample (for example, by removing one or more components in primary sample and/or by adding one or more reagents to primary sample). For example, filtering is performed using a semi-permeable membrane. This "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acids, separation and/or purification of certain components.

稳定纳米粒子组合物：当应用于本文中的组合物时，术语“稳定”意指组合物在一段时间内维持其物理结构(例如，粒子的大小范围和/或分布)的一个或多个方面。在一些实施例中，稳定的纳米粒子组合物是其平均粒度、最大粒度、粒度范围和/或粒度分布(即，高于指定尺寸和/或在指定尺寸范围外的粒子百分比)在指定条件下维持一段时间的组合物。在一些实施例中，所提供的稳定组合物是生物学相关活性维持一段时间的组合物。在一些实施例中，时间段为至少约一小时；在一些实施例中，时间段为约5小时、约10小时、约一(1)天、约一(1)周、约两(2)周、约一(1)个月、约两(2)个月、约三(3)个月、约四(4)个月、约五(5)个月、约六(6)个月、约八(8)个月、约十(10)个月、约十二(12)个月、约二十四(24)个月、约三十六(36)个月或更久。在一些实施例中，时间段在约一(1)天至约二十四(24)个月、约两(2)周至约十二(12)个月、约两(2)个月至约五(5)个月等的范围内。例如，如果纳米粒子群体经历长期储存、温度变化和/或pH变化并且组合物中的大多数纳米粒子保持规定范围内的直径，则纳米粒子组合物是稳定的。在一些实施例中，稳定组合物在环境条件下是稳定的。在一些实施例中，稳定组合物在生物条件(即37℃，在磷酸盐缓冲盐水中)下是稳定的。Stable nanoparticle compositions: When applied to the compositions herein, the term "stable" means that the composition maintains one or more aspects of its physical structure (e.g., size range and/or distribution of particles) over a period of time. In some embodiments, a stable nanoparticle composition is a composition whose average particle size, maximum particle size, size range, and/or size distribution (i.e., the percentage of particles above a specified size and/or outside a specified size range) is maintained under specified conditions for a period of time. In some embodiments, provided stable compositions are compositions whose biologically relevant activity is maintained for a period of time. In some embodiments, the period of time is at least about one hour; in some embodiments, the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer. In some embodiments, the time period is in the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. For example, a nanoparticle composition is stable if a population of nanoparticles is subjected to long-term storage, temperature changes, and/or pH changes and a majority of the nanoparticles in the composition maintain a diameter within a specified range. In some embodiments, a stable composition is stable under ambient conditions. In some embodiments, a stable composition is stable under biological conditions (i.e., 37° C. in phosphate buffered saline).

甾醇基：如本文中所使用，术语“甾醇基”是指饱和或部分不饱和并且被至少一个羟基取代的17元稠合多环部分，并且具有与任何可取代的碳或氧原子处的分子的其余部分的单个连接点。在一些实施例中，甾醇基是胆甾醇基或其变体或衍生物。在一些实施例中，胆甾醇基经修饰。在一些实施例中，胆甾醇基是氧化的胆甾醇基(例如，在β环结构上或烃尾结构上氧化)。在一些实施例中，胆甾醇基是酯化的胆甾醇基。在一些实施例中，甾醇基是植物甾醇基。示范性甾醇基基团包含但不限于25-羟基胆甾醇基(25-OH)、20α-羟基胆甾醇基(20α-OH)、27-羟基胆甾醇基、6-酮基-5α-羟基胆甾醇基、7-酮基胆甾醇基、7β-羟基胆甾醇基、7α-羟基胆甾醇基、7β-25-二羟基胆甾醇基、β-谷甾醇基、豆甾醇基、菜籽甾醇基和菜油甾醇基。Sterol group: As used herein, the term "sterol group" refers to a 17-membered fused polycyclic moiety that is saturated or partially unsaturated and substituted with at least one hydroxyl group, and has a single point of attachment to the rest of the molecule at any substitutable carbon or oxygen atom. In some embodiments, the sterol group is a cholesterol group or a variant or derivative thereof. In some embodiments, the cholesterol group is modified. In some embodiments, the cholesterol group is an oxidized cholesterol group (e.g., oxidized on the beta ring structure or on the hydrocarbon tail structure). In some embodiments, the cholesterol group is an esterified cholesterol group. In some embodiments, the sterol group is a phytosterol group. Exemplary steryl groups include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, β-sitosterol, stigmasterol, brassicasterol, and campesterol.

受试者：如本文中所使用，术语“受试者”是指生物体，通常是指哺乳动物(例如人类，在一些实施例中包含出生前的人类形式)。在一些实施例中，受试者患有相关疾病、病症、或病状。在一些实施例中，受试者易患疾病、病症或病状。在一些实施例中，受试者显示出疾病、病症或病状的一种或多种症状或特征。在一些实施例中，受试者未显示出疾病、病症或病状的任何症状或特征。在一些实施例中，受试者是指具有以下一个或多个特征的受试者，其特征在于易患或有风险患疾病、病症或病状。在一些实施例中，受试者是患者。在一些实施例中，受试者是正在和/或已经对其进行诊断和/或治疗的受试者。Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a disease, disorder, or condition of interest. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not display any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject refers to a subject having one or more of the following characteristics, characterized by being susceptible to or at risk for a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is a subject who is being and/or has been diagnosed and/or treated.

基本上：如本文中所使用，术语“基本上”是指展现全部或接近全部范围或程度的所关注特征或性质的定性条件。生物学领域的普通技术人员应理解，生物学和化学现象很少(如果有的话)达到完全和/或进行到完全或者实现或避免绝对结果。因此，术语“大体上”在本文中用于捕获许多生物学和化学现象中所固有的潜在完全性缺乏。Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting full or nearly full range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, reach completeness and/or proceed to perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

被取代或任选地被取代：如本文所述，本公开的化合物可以含有任选地被取代的和/或被取代的部分。一般来说，术语“经取代”无论前面有还是没有术语“任选地”，都意指指定部分的一个或多个氢经合适的取代基置换。“被取代”适用于一个或多个从结构中明确或暗示的氢(例如，至少是指并且至少是指)。除非另外指示，否则“任选地被取代的”基团可以在基团的每个可取代位置处具有合适的取代基，并且当任何既定结构中的超过一个位置可以经一个以上选自指定基团的取代基取代时，在每一位置处取代基可以是相同或不同的。本公开所设想的取代基组合优选是能使稳定的或化学上可行的化合物形成的取代基组合。如本文所使用的术语“稳定”是指化合物在经历允许其产生、检测和在某些实施例中其回收、纯化和用于本文中所公开的一种或多种目的条件时大体上不发生改变。被描述为“经取代的”基团优选地具有介于1至4个之间的取代基，更优选地1或2个取代基。被描述为“任选地被取代的”基团可以是未被取代的或“经取代的”，如上文所述。Substituted or optionally substituted: As described herein, the compounds of the present disclosure may contain optionally substituted and/or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens explicitly or implicitly removed from the structure (e.g., At least and At least ). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be the same or different at each position. The substituent combinations contemplated by the present disclosure are preferably substituent combinations that enable stable or chemically feasible compounds to be formed. The term "stable" as used herein refers to a compound that is substantially unchanged when subjected to conditions that allow its production, detection, and in certain embodiments, its recovery, purification, and use for one or more purposes disclosed herein. A group described as "substituted" preferably has between 1 and 4 substituents, more preferably 1 or 2 substituents. A group described as "optionally substituted" may be unsubstituted or "substituted", as described above.

合适的单价取代基包含卤素；-(CH₂)_0-4R^o；-(CH₂)_0-4OR^o；-O(CH₂)_0-4R^o、-O-(CH₂)_0-4C(O)OR^o；-(CH₂)_0-4CH(OR^o)₂；-(CH₂)_0-4Ph,其可以被R^o取代；-(CH₂)_0-4O(CH₂)_0-1Ph，其可以被R^o取代；-CH＝CHPh，其可以被R^o取代；-(CH₂)_0-4O(CH₂)_0-1-吡啶基，其可以被R^o取代；-NO₂；-CN；-N₃；-(CH₂)_0-4N(R^o)₂；-(CH₂)_0-4N(R^o)C(O)R^o；-N(R^o)C(S)R^o；-(CH₂)_0-4N(R^o)C(O)NR^o₂；-N(R^o)C(S)NR^o₂；-(CH₂)_0-4N(R^o)C(O)OR^o；-N(R^o)N(R^o)C(O)R^o；-N(R^o)N(R^o)C(O)NR^o₂；-N(R^o)N(R^o)C(O)OR^o；-(CH₂)_0-4C(O)R^o；-C(S)R^o；-(CH₂)_0-4C(O)OR^o；-(CH₂)_0-4C(O)SR^o；-(CH₂)_0-4C(O)OSiR^o₃；-(CH₂)_0-4OC(O)R^o；-OC(O)(CH₂)_0-4SR^o-、-SC(S)SR^o；-(CH₂)_0-4SC(O)R^o；-(CH₂)_0-4C(O)NR^o₂；-C(S)NR^o₂；-C(S)SR^o；-SC(S)SR^o、-(CH₂)_0-4OC(O)NR^o₂；-C(O)N(OR^o)R^o；-C(O)C(O)R^o；-C(O)CH₂C(O)R^o；-C(NOR^o)R^o；-(CH₂)_0-4SSR^o；-(CH₂)_0-4S(O)₂R^o；-(CH₂)_0-4S(O)₂OR^o；-(CH₂)_0-4OS(O)₂R^o；-S(O)₂NR^o₂；-(CH₂)_0-4S(O)R^o；-N(R^o)S(O)₂NR^o₂；-N(R^o)S(O)₂R^o；-N(OR^o)R^o；-C(NH)NR^o₂；-P(O)₂R^o；-P(O)R^o₂；-OP(O)R^o₂；-OP(O)(OR^o)₂；-SiR^o₃；-OSiR^o₃；-(C_1-4直链或支链亚烷基)O-N(R^o)₂；或-(C_1-4直链或支链亚烷基)C(O)O-N(R^o)₂，其中每个R^o可以如下文所定义被取代，并且独立地为氢、C_1-6脂肪族、-CH₂Ph、-O(CH₂)_0-1Ph、-CH₂-(5至6元杂芳基环)或具有0-4个独立地选自氮、氧或硫的杂原子的5至6元饱和、部分不饱和或芳基环；或除上述定义以外，两个独立出现的R^o与其中间原子一起形成具有0-4个独立地选自氮、氧或硫的杂原子的3至12元饱和、部分饱和或芳基单环或双环，其可以如下文所定义被取代。Suitable monovalent substituents include halogen; -(CH₂ )_0-4 R^o ; -(CH₂ )_0-4 OR^o ; -O(CH₂ )_0-4 R^o , -O-(CH₂ )_0-4 C(O)OR^o ; -(CH₂ )_0-4 CH(OR^o )₂ ; -(CH₂ )_0-4 Ph, which may be substituted with R^o ; -(CH₂ )_0-4 O(CH₂ )_0-1 Ph, which may be substituted with R^o ; -CH═CHPh, which may be substituted with R^o ; -(CH₂ )_0-4 O(CH₂ )_0-1 -pyridyl, which may be substituted with R^o ; -NO₂ ; -CN; -N₃ ; -(CH₂ )_0-4 N(R^o )₂ ; -(CH₂ )_0-4 N(R^o ) 2 ; )C(O)R^o ;-N(R^o )C(S)R^o ;-(CH₂ )_0-4 N(R^o )C(O)NR^o₂ ;-N(R^o )C(S)NR^o₂ ;-(CH₂ )_0-4 N(R^o )C(O)OR^o ;-N(R^o )N(R^o )C(O)R^o ;-N(R^o )N(R^o )C(O)NR^o₂ ;-N(R^o )N(R^o )C(O)OR^o ;-(CH₂ )_0-4 C(O)R^o ;-C(S)R^o ;-(CH₂ )_0-4 C(O)OR^o ;-(CH₂ )_0-4 C(O)SR^o ;-(CH₂ )_0-4 C(O)OSiR^o₃ ;-(CH₂ )_0-4 OC(O)R^o ;-OC(O)(CH₂ )_0-4 SR^o -,-SC(S)SR^o ;-(CH₂ )_0-4 SC(O)R^o ;-(CH₂ )_0-4 C(O)NR^o₂ ;-C(S)NR^o₂ ;-C(S)SR^o ;-SC(S)SR^o ,-(CH₂ )_0-4 OC(O)NR^o₂ ;-C(O)N(OR^o )R^o ;-C(O)C(O)R^o ;-C(O)CH₂ C(O)R^o ;-C(NOR^o )R^o ;-(CH₂ )_0-4 SSR^o ;-(CH₂ )_0-4 S(O)₂ R^o ;-(CH₂ )_0-4 S(O)₂ OR^o ; -(CH₂ )_0-4 OS(O)₂ R^o ; -S(O)₂ NR^o₂ ; -(CH₂ )_0-4 S(O)R^o ; -N(R^o )S(O)₂ NR^o₂ ; -N(R^o )S(O)₂ R^o ; -N(OR^o )R^o ; -C(NH)NR^o₂ ; -P(O)₂ R^o ; -P(O)R^o₂ ; -OP(O)R^o₂ ; -OP(O)(OR^o )₂ ; -SiR^o₃ ; -OSiR^o₃ ; -(C_1-4 straight or branched alkylene)ON(R^o )₂ ; or -(C_1-4 straight or branched alkylene)C(O)ON(R^o )₂ , wherein each R^o may be substituted as defined below and is independently hydrogen, C_1-6 aliphatic, -CH₂ Ph, -O(CH₂ )_0-1 Ph, -CH₂ -(5- to 6-membered heteroaryl ring) or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; or in addition to the above definition, two independent occurrences of R^o together with their middle atom form a 3- to 12-membered saturated, partially saturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

R^o上合适的单价取代基(或通过两个独立出现的R^o与其中间原子一起形成的环)独立地为卤素、-(CH₂)_0-2R^·、-(卤代R^·)、-(CH₂)_0-2OH、-(CH₂)_0-2OR^·、-(CH₂)_0-2CH(OR^·)₂；-O(卤代R^·)、-CN、-N₃、-(CH₂)_0-2C(O)R^·、-(CH₂)_0-2C(O)OH、-(CH₂)_0-2C(O)OR^·、-(CH₂)_0-2C(O)NH₂、-(CH₂)_0-2C(O)NHR^·、-(CH₂)_0-2C(O)NR^·₂、-(CH₂)_0-2SR^·、-(CH₂)_0-2SH、-(CH₂)_0-2NH₂、-(CH₂)_0-2NHR^·、-(CH₂)_0-2NR^·₂、-NO₂、-SiR^·₃、-OSiR^·₃、-C(O)SR^·、-(C_1-4直链或支链亚烷基)C(O)OR^·或-SSR^·，其中每个R^·未被取代或在前缀前面有“卤素”的情况下仅被一个或多个卤素取代，并且独立地选自C_1-4脂肪族、-CH₂Ph、-O(CH₂)_0-1Ph、或具有0-4个独立地选自氮、氧或硫的杂原子的5至6元饱和、部分不饱和或芳基环。R^o的饱和碳原子上的合适的二价取代基包含＝O和＝S。Suitable monovalent substituents on R^o (or the ring formed by two independent occurrences of R^o together with their middle atom) are independently halogen, -(CH₂ )_0-2 R^· , -(haloR^· ), -(CH₂ )_0-2 OH, -(CH₂ )_0-2 OR^· , -(CH₂ )_0-2 CH(OR^· )₂ ; -O(haloR^· ), -CN, -N₃ , -(CH₂ )_0-2 C(O)R^· , -(CH₂ )_0-2 C(O)OH, -(CH₂ )_0-2 C(O)OR^· , -(CH₂ )_0-2 C(O)NH₂ , -(CH₂ )_0-2 C(O)NHR^· , -(CH₂ )_0-2 C(O)NR^·₂ , -(CH₂ )_0-2 SR^· , -(CH_2)0-2 SH, -(CH₂ )_0-2 NH₂ , -(CH₂ )_0-2 NHR^· , -(CH₂ )_0-2 NR^· ,_{-NO 2,-SiR}^· , -OSiR^·_, -C(O)SR^· , -(C_1-4 straight or branched alkylene)C(O)OR^· , or -SSR^· , wherein each R^· is unsubstituted or, when the prefix is preceded by "halogen", is substituted only by one or more halogens, and is independently selected from C_1-4 aliphatic, -CH₂ Ph, -O(CH₂ )_0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on a saturated carbon atom of R^o include =0 and =S.

合适的二价取代基包含以下：＝O、＝S、＝NNR^*₂、＝NNHC(O)R^*、＝NNHC(O)OR^*、＝NNHS(O)₂R^*、＝NR^*、＝NOR^*、-O(C(R^*₂))_2-3O-或-S(C(R^*₂))_2-3S-，其中每个独立出现的R^*选自氢、C_1-6脂肪族，其可以如下文所定义被取代或为具有0-4个独立地选自氮、氧或硫的杂原子的未被取代的5至6元饱和、部分不饱和或芳基环。与“任选地取代的”基团的邻位可取代碳键合的合适的二价取代基包括：–O(CR^*₂)_2–3O–，其中每个独立出现的R^*选自氢、可如下文定义取代的C_1–6脂肪族，或具有0-4个独立地选自氮、氧或硫的杂原子的未取代的5至6元饱和、部分不饱和或芳基环。Suitable divalent substituents include the following: =O, =S, =NNR^*₂ , =NNHC(O)R^* , =NNHC(O)OR^* , =NNHS(O)_2R^* , =NR^* , =NOR^* , -O(C(R^*₂ ))_2-3O- or -S(C(R^*₂ ))_2-3S- , wherein each independent occurrence of R^* is selected from hydrogen,_C1-6 aliphatic which may be substituted as defined below or is an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bonded to an ortho-substitutable carbon of an "optionally substituted" group include: -O(CR^*₂ )_2-3 O-, wherein each independent occurrence of R^* is selected from hydrogen, a C_1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

R^*的脂肪族基团上的合适的取代基包括卤素、-R^·、-(卤代R^·)、-OH、-OR^·、-O(卤代R^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^·₂或-NO₂，其中每个R^·未被取代的或在前缀有“卤代”的情况下仅被一个或多个卤素取代，并且独立地为C_1-4脂肪族、-CH₂Ph、-O(CH₂)_0-1Ph、或具有0-4个独立地选自氮、氧或硫的杂原子的未被取代的5至6元饱和、部分不饱和或芳基环。Suitable substituents on the aliphatic group of R^* include halogen, -R^· , -(haloR^· ), -OH, -OR^· , -O(haloR^· ), -CN, -C(O)OH, -C(O)OR^· , -NH₂ , -NHR^· , -NR^·₂ or -NO₂ , wherein each R^· is unsubstituted or, when prefixed with "halo", is substituted only by one or more halogens, and is independently C_1-4 aliphatic, -CH₂ Ph, -O(CH₂ )_0-1 Ph, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

在一些实施例中，可取代氮上的合适取代基包含或其中每个独立地为氢，可以如下文所定义被取代的C_1-6脂肪族，未被取代的-OPh，或具有0-4个独立地选自氮、氧或硫的杂原子的未被取代的5至6元饱和、部分不饱和或芳基环；或除上述定义以外，两个独立出现的与其中间原子一起形成具有0-4个独立地选自氮、氧或硫的杂原子的未被取代的3至12元饱和、部分不饱和或芳基单环或双环。In some embodiments, suitable substituents on the substitutable nitrogen include or Each of these independently hydrogen, optionally substituted C_1-6 aliphatic as defined below, unsubstituted -OPh, or an unsubstituted 5- to 6-membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; or, in addition to the above definitions, two independent occurrences of Together with its central atoms, it forms an unsubstituted 3- to 12-membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

的脂肪族基团上的合适的取代基独立地为卤素、-R^·、-(卤代R^·)、-OH、-OR^·、-O(卤代R^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^·₂或-NO₂，其中每个R^·为取代的或在前缀有“卤代”的情况下仅经一个或多个卤素取代，并且独立地为C_1-4脂肪族、-CH₂Ph、-O(CH₂)_0-1Ph、或具有0-4个独立地选自氮、氧或硫的杂原子的未被取代的5至6元饱和、部分饱和或芳基环。 Suitable substituents on the aliphatic group are independently halogen, -R^· , -(haloR^· ), -OH, -OR^· , -O(haloR^· ), -CN, -C(O)OH, -C(O)OR^· , -NH₂ , -NHR^· , -NR^·₂ or -NO₂ , wherein each R^· is substituted or, when prefixed with "halo", is substituted only with one or more halogens, and is independently C_1-4 aliphatic, -CH₂ Ph, -O(CH₂ )_0-1 Ph, or an unsubstituted 5- to 6-membered saturated, partially saturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

除非另有说明，本文描述的结构还意在包含该结构的所有异构体(例如，对映体、非对映体和几何(或构象))形式；例如，每个不对称中心的R和S构型、Z和E双键异构体以及Z和E构象异构体。因此，本发明化合物的单一立体化学异构体以及对映异构、非对映异构和几何异构(或构象异构)混合物都在本发明的范围内。除非另有说明，否则本发明化合物的所有互变异构形式都在本发明的范围内。另外，除非另外说明，否则本文中所描绘的结构还意味着包含不同之处仅在于存在一个或多个同位素增浓原子的化合物。例如，具有本结构(包含由氘或氚置换氢或由¹³C或¹⁴C增浓的碳置换碳)的化合物在本发明的范围内。这类化合物适用作例如分析工具，用作生物分析中的探针，或用作根据本发明的治疗剂。Unless otherwise stated, the structures depicted herein are also intended to include all isomeric (e.g., enantiomers, diastereomers, and geometric (or conformational)) forms of the structure; for example, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers of the compounds of the invention as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise stated, the structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure (including replacement of hydrogen by deuterium or tritium or replacement of carbon by carbon enriched with¹³ C or¹⁴ C) are within the scope of the invention. Such compounds are useful, for example, as analytical tools, as probes in biological analysis, or as therapeutic agents according to the invention.

易患：“易患”疾病、病症或病状的受试者处于该疾病、病症或病状发展的风险下。在一些实施例中，易患疾病、病症或病状的个体没有显示出所述疾病、病症或病状的任何症状。在一些实施例中，易患疾病、病症或病状的个体尚未诊断患有所述疾病、病症和/或病状。在一些实施例中，易患疾病、病症或病状的个体是已暴露于与疾病、病症或病状的发展相关的条件的个体。在一些实施例中，疾病、病症和/或病状发展的风险是基于群体的风险(例如患有所述疾病、病症或病状的个体的家族成员)。Susceptible to: A subject who is "susceptible" to a disease, disorder, or condition is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with the development of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and/or condition is based on the risk of a population (e.g., family members of an individual who suffers from the disease, disorder, or condition).

全身性：如本文中所使用，短语“全身性施用”、“全身性地施用”、“外周施用”以及“外周地施用”具有其在本领域中所理解的含义，是指施用化合物或组合物，使其进入接受者的系统。Systemic: As used herein, the phrases "systemic administration," "administering systemically," "peripheral administration," and "peripherally administered" have their art-understood meanings and refer to administering a compound or composition so that it enters the recipient's system.

互变异构形式：如本文中所使用，短语“互变异构形式”用于描述有机化合物的能够容易地相互转化的不同异构形式。互变异构体的特征可以在于氢原子或质子的形式迁移，伴随着单键和相邻双键的转换。在一些实施例中，互变异构体可以由质子互变异构(即，质子的重新定位)产生。在一些实施例中，互变异构体可以由价互变异构(即，成键电子的快速重组)产生。所有这类互变异构形式均旨在包含在本公开的范围内。在一些实施例中，化合物的互变异构形式彼此以动态平衡存在，因此尝试制备单独物质时会形成混合物。在一些实施例中，化合物的互变异构形式是可分开的和可分离的化合物。在本公开的一些实施例中，可以提供如下化学组合物：其是或包含化合物的单一互变异构形式的纯制剂。在一些实施例中，化学组合物可以按化合物的两种或更多种互变异构形式的混合物形式提供。在某些实施例中，此类混合物含有等量的不同互变异构形式；在某些实施例中，此类混合物含有不同量的至少两种不同互变异构形式的化合物。在本公开的一些实施例中，化学组合物可以含有化合物的所有互变异构形式。在本公开的一些实施例中，化学组合物可以含有化合物的不到全部的互变异构形式。在本公开的一些实施例中，化学组合物可以含有化合物的一种或多种互变异构形式，其量因为相互转化而随时间推移改变。在本公开的一些实施例中，互变异构是酮-烯醇互变异构。化学领域的技术人员将想到，酮-烯醇互变异构体可以使用化学领域中已知的任何合适的试剂“捕获”(即，化学改性以使其保持“烯醇”形式)以提供随后可以使用本领域中已知的一种或多种合适的技术分离的烯醇衍生物。除非另有指示，否则本公开涵盖相关化合物的所有互变异构形式，无论是纯形式还是彼此混合。Tautomeric forms: As used herein, the phrase "tautomeric forms" is used to describe different isomeric forms of organic compounds that can be easily interconverted. Tautomers can be characterized by the migration of the form of hydrogen atoms or protons, accompanied by the conversion of single bonds and adjacent double bonds. In some embodiments, tautomers can be produced by proton tautomerism (i.e., the relocation of protons). In some embodiments, tautomers can be produced by valence tautomerism (i.e., the rapid reorganization of bonding electrons). All such tautomeric forms are intended to be included within the scope of the present disclosure. In some embodiments, the tautomeric forms of the compound exist in dynamic equilibrium with each other, so a mixture is formed when attempting to prepare a separate substance. In some embodiments, the tautomeric forms of the compound are separable and separable compounds. In some embodiments of the present disclosure, a chemical composition may be provided as follows: it is or contains a pure preparation of a single tautomeric form of a compound. In some embodiments, a chemical composition may be provided in the form of a mixture of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain embodiments, such mixtures contain different amounts of compounds of at least two different tautomeric forms. In some embodiments of the present disclosure, chemical composition can contain all tautomeric forms of compound.In some embodiments of the present disclosure, chemical composition can contain less than all tautomeric forms of compound.In some embodiments of the present disclosure, chemical composition can contain one or more tautomeric forms of compound, and its amount changes over time because of mutual conversion.In some embodiments of the present disclosure, tautomerism is keto-enol tautomerism.It will be appreciated by those skilled in the art of chemistry that keto-enol tautomer can be "captured" (that is, chemically modified to keep "enol" form) using any suitable reagent known in the chemical field to provide the enol derivatives that can be separated using one or more suitable techniques known in the art subsequently.Unless otherwise indicated, the present disclosure encompasses all tautomeric forms of related compounds, whether pure form or mixed with each other.

治疗剂：如本文中所使用，短语“治疗剂”是指当施用于受试者时具有治疗作用和/或引发所期望的生物学和/或药理学作用的药剂。在一些实施例中，治疗剂是任何可用于缓解、改善、减轻、抑制、预防疾病、病症和/或病状的一个或多个症状或特征、延迟其发作、降低其严重程度，和/或降低其发病率的物质。Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and/or induces a desired biological and/or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.

治疗有效量：如本文中所使用，术语“治疗有效量”意指物质(例如，治疗剂、组合物和/或调配物)的在作为治疗方案的一部分施用时能引发所期望的生物反应的量。在一些实施例中，物质的治疗有效量是当向患有或易患疾病、病症和/或病状的受试者施用时足以治疗、诊断、抑制、缓解、预防所述疾病、病症和/或病状和/或延迟其发作的量。如本领域普通技术人员将理解的，物质的有效量可以根据诸如期望的生物学终点、待递送的物质、靶细胞或组织等的因素而变化。例如，调配物中用于治疗疾病、病症和/或病状的化合物的有效量是减轻、改善、缓解、抑制、预防、延迟该疾病、病症和/或病状的一种或多种症状或特征的发作、降低其严重性和/或降低其发生率的量。在一些实施例中，以单次剂量施用治疗有效量；在一些实施例中，需要多个单位剂量来递送治疗有效量。精确剂量将根据各种因素变化，如受试者相关性变量(例如，年龄、免疫系统健康等)、疾病和所进行的治疗。Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that can elicit a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, inhibit, alleviate, prevent, and/or delay the onset of a disease, disorder, and/or condition when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition. As will be appreciated by one of ordinary skill in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound for treating a disease, disorder, and/or condition in a formulation is an amount that alleviates, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. The exact dosage will vary according to factors such as subject-related variables (eg, age, health of immune system, etc.), the disease, and the treatment being administered.

“组织”和/或“器官”：如本文中所使用，除非另有规定，否则术语“组织”和/或“器官”是指呈聚合形式的存活细胞材料，例如小部分器官，以及分散细胞，例如从肌肉、心肌、肝脏或肾脏分散、分离和/或生长的细胞，包含骨髓细胞和子代细胞、血源干细胞和子代以及各种其它血液元件。在一些实施例中，组织和/或器官是指肾、心脏、肝、胃、脾、胰腺、肺、大脑、眼睛、肠道、膀胱、皮肤或皮肤组织、血管、静脉、动脉、心脏瓣膜、精子和卵母细胞。如本文所使用的术语“器官”涵盖实体器官，例如肾、心脏、肝、肺，以及器官的功能部分，例如皮肤段、动脉段、静脉段、可移植肝叶、肾叶、肺叶等。"Tissue" and/or "organ": As used herein, unless otherwise specified, the terms "tissue" and/or "organ" refer to viable cell material in aggregated form, such as a small portion of an organ, as well as dispersed cells, such as cells dispersed, separated and/or grown from muscle, myocardium, liver or kidney, including bone marrow cells and progeny cells, hematogenous stem cells and progeny, and various other blood elements. In some embodiments, tissue and/or organ refers to kidney, heart, liver, stomach, spleen, pancreas, lung, brain, eye, intestine, bladder, skin or skin tissue, blood vessels, veins, arteries, heart valves, sperm and oocytes. As used herein, the term "organ" encompasses solid organs, such as kidney, heart, liver, lung, and functional parts of organs, such as skin segments, arterial segments, venous segments, transplantable liver lobes, kidney lobes, lung lobes, etc.

治疗(treatment)：如本文中所使用，术语“治疗(treatment)”(还有“治疗(treat/treating)”是指施用一种疗法，其部分或完全缓解、改善、减轻、抑制特定疾病、病症和/或病状的一种或多种症状、特征和/或成因，延迟其发作，降低其严重程度和/或降低其发病率。在一些实施例中，治疗可针对未出现相关疾病、病症和/或病状的病征的受试者和/或仅出现疾病、病症和/或病状的早期病征的受试者。或者或另外，这种治疗可以是针对出现相关疾病、病症和/或病状的一种或多种确定病征的受试者。在一些实施例中，治疗可针对已诊断为患有相关疾病、病症和/或病状的受试者。在一些实施例中，治疗可针对已知具有一种或多种易感因素的受试者，所述易感因素在统计学上与相关疾病、病症和/或病状的发展风险增加相关。因此，在一些实施例中，治疗可以是预防性的；在一些实施例中，治疗可以是治疗性的。Treatment: As used herein, the term "treatment" (also "treat" and "treating") refers to the administration of a therapy that partially or completely alleviates, ameliorates, relieves, suppresses, delays the onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, treatment may be directed to subjects who do not develop signs of the relevant disease, disorder, and/or condition and/or subjects who only develop early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be directed to subjects who develop one or more established signs of the relevant disease, disorder, and/or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and/or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

某些实施例的详细描述Detailed Description of Certain Embodiments

本公开描述了所述组合物、制剂、纳米粒子和/或纳米材料的组分中的一种或多种组分的选择和组合会影响脂质纳米粒子的功能活性，诸如期望的向性、稳定性和药物递送功效。此外，本发明提供用于将治疗剂和/或预防剂递送到靶细胞和/或组织的组合物、制剂、纳米粒子和/或纳米材料。例如，本公开描述适用于组合物、制剂、纳米粒子和/或纳米材料的脂质化合物。在一些实施例中，组合物、制剂和/或纳米材料包含携带货物到指定靶细胞、组织和/或器官的LNP。The present disclosure describes that the selection and combination of one or more components in the components of the compositions, preparations, nanoparticles and/or nanomaterials will affect the functional activity of lipid nanoparticles, such as the desired tropism, stability and drug delivery efficacy. In addition, the present invention provides compositions, preparations, nanoparticles and/or nanomaterials for delivering therapeutic agents and/or preventive agents to target cells and/or tissues. For example, the present disclosure describes lipid compounds suitable for compositions, preparations, nanoparticles and/or nanomaterials. In certain embodiments, compositions, preparations and/or nanomaterials include LNPs that carry goods to specified target cells, tissues and/or organs.

I.脂质纳米粒子(LNP)I. Lipid Nanoparticles (LNP)

本发明提供包含脂质纳米粒子的组合物、制剂和/或纳米材料。在一些实施例中，脂质纳米粒子包括一种或多种组分。在一些实施例中，脂质纳米粒子包括一种或多种组分，如化合物、可电离脂质、甾醇、缀合物-连接子脂质和磷脂。此外，本公开描述如本文所述的组分中的一种或多种的选择和组合会影响脂质纳米粒子的特征，如直径、pKa、稳定性和可电离性。The present invention provides compositions, preparations and/or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticles include one or more components. In some embodiments, the lipid nanoparticles include one or more components, such as compounds, ionizable lipids, sterols, conjugate-linker lipids and phospholipids. In addition, the present disclosure describes that one or more of the selections and combinations of the components as described herein will affect the characteristics of the lipid nanoparticles, such as diameter, pKa, stability and ionizability.

此外，本公开描述如本文所述的组分中的一种或多种的选择和组合会影响脂质纳米粒子的功能活性，如向性、稳定性和药物递送功效。例如，本公开描述组分的组合可以更好地适合siRNA的递送。作为另一实例，本公开描述组分的组合可以更好地适合mRNA的递送。作为另一实例，本公开描述组分的组合可以更好地适合DNA的递送。In addition, the present disclosure describes one or more of the selection and combination of components as described herein that can affect the functional activity of lipid nanoparticles, such as tropism, stability and drug delivery efficacy. For example, the present disclosure describes the combination of components that can be better suited for the delivery of siRNA. As another example, the present disclosure describes the combination of components that can be better suited for the delivery of mRNA. As another example, the present disclosure describes the combination of components that can be better suited for the delivery of DNA.

在一些实施例中，脂质纳米粒子包括一种或多种如本文所述的化合物。在一些实施例中，脂质纳米粒子包含一种或多种如本文所述的可电离脂质。在一些实施例中，脂质纳米粒子包含一种或多种如本文所述的甾醇。在一些实施例中，脂质纳米粒子包含一种或多种如本文所述的缀合物-连接子脂质。在一些实施例中，脂质纳米粒子包含一种或多种如本文所述的磷脂。In some embodiments, the lipid nanoparticles include one or more compounds as described herein. In some embodiments, the lipid nanoparticles comprise one or more ionizable lipids as described herein. In some embodiments, the lipid nanoparticles comprise one or more sterols as described herein. In some embodiments, the lipid nanoparticles comprise one or more conjugate-linker lipids as described herein. In some embodiments, the lipid nanoparticles comprise one or more phospholipids as described herein.

A.化合物A. Compounds

此外，本公开描述包括一种或多种如本文所述的化合物的组合物、制剂、纳米粒子和/或纳米材料。Additionally, the present disclosure describes compositions, formulations, nanoparticles, and/or nanomaterials comprising one or more compounds as described herein.

在一些实施例中，本公开提供一种式I化合物：In some embodiments, the present disclosure provides a compound of Formula I:

或其N-氧化物、或其药学上可接受的盐，其中：or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein:

L¹、L^1'和L^1”中的每一者独立地为不存在的、-C(O)-或-OC(O)-；Each of L¹ , L^1′ and L^1″ is independently absent, —C(O)— or —OC(O)—;

L²、L^2'和L^2”中的每一者独立地为不存在的、任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-；Each of L² , L^2′ and L^2″ is independently absent, an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_m′ -;

每个Cy^A独立地为选自亚苯基和3至12元饱和或部分不饱和的亚碳环基的任选地被取代的环；each Cy^A is independently an optionally substituted ring selected from phenylene and a 3- to 12-membered saturated or partially unsaturated carbocyclylene;

m和m'中的每一者独立地为0、1或2；Each of m and m' is independently 0, 1 or 2;

L³、L^3'和L^3”中的每一者独立地为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-；each of L³ , L^3′, and L^3″ is independently absent, —C(O)—, —C(O)O—, —OC(O)—, —O—, or —OC(O)O—;

R¹、R^1'和R^1”中的每一者独立地为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each of R¹ , R^1′ and R^1″ is independently -(CH₂ )_p -Cy^B , or an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;

每个Cy^B独立地为选自以下的任选地被取代的环：3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧或硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each Cy^B is independently an optionally substituted ring selected from: a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

每个p独立地为0、1、2或3；Each p is independently 0, 1, 2 or 3;

每个L^3a独立地为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each L^3a is independently absent or optionally substituted divalent saturated or unsaturated linear or branched C_1-10 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;

每个R^a独立地为氢或选自以下的任选地被取代的基团：C_6-20脂肪族、3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧和硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each^{Ra is} independently hydrogen or an optionally substituted group selected from the group consisting of a_C6-20 aliphatic, a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

X¹为不存在的、-O-、-S-或-NR-；^X1 is absent, -O-, -S- or -NR-;

X²为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换；^X2 is absent or optionally substituted divalent saturated or unsaturated linear or branched_C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or^-CyC- ;

Cy^C为选自以下的任选地被取代的环：3至7元饱和或部分不饱和的亚碳环、亚苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的亚杂环或具有1-3个独立地选自氮、氧和硫的杂原子的5至6元亚杂芳基；Cy^C is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5- to 6-membered heteroarylene group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur;

X³为氢或选自以下的任选地被取代的环：3至7元饱和或部分不饱和的碳环基、苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的杂环基以及具有1-3个独立地选自氮、氧和硫的杂原子的5至6元杂芳基；并且^X3 is hydrogen or an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a phenyl, a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and

每个R独立地为氢或任选地被取代的C_1-6脂肪族基团。Each R is independently hydrogen or an optionally substituted C_1-6 aliphatic group.

在一些实施例中，本公开提供了一种式I-A化合物：In some embodiments, the present disclosure provides a compound of formula I-A:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、L^3'、L^3”、R¹、R^1'、R^1”、X¹、X²和X³中的每一者单独地或组合地如上所述并在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L³ , L^3′ , L^3″ , R¹ , R^1′ , R^1″ , X¹ , X² , and X³ , individually or in combination, is as described above and in the classes and subclasses herein.

在一些实施例中，本公开提供了一种式I-A-a化合物：In some embodiments, the present disclosure provides a compound of formula I-A-a:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、L^3'、L^3”、R¹、R^1'、R^1”、X²和X³中的每一者单独地或组合地如上所述并在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L 3 , L^3′ , L^3″ , R¹ , R^1′ , R^1″ , X² and X³ , individually or in combination, is as described above and in classes and subclasses herein.

在一些实施例中，本公开提供了一种式I-A-b化合物：In some embodiments, the present disclosure provides a compound of formula I-A-b:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、L^3'、L^3”、R、R¹、R^1'、R^1”、X²和X³中的每一者单独地或组合地如上所述并在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L 3 , L^3′ , L^3″ , R, R¹ , R^1′ , R^1″ , X² and X³ , individually or in combination, is as described above and in the classes and subclasses herein.

在一些实施例中，本公开提供了一种式I-A-c化合物：In some embodiments, the present disclosure provides a compound of formula I-A-c:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、L^3'、L^3”、R¹、R^1'、R^1”、X²和X³中的每一者单独地或组合地如上所述并在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L 3 , L^3′ , L^3″ , R¹ , R^1′ , R^1″ , X² and X³ , individually or in combination, is as described above and in classes and subclasses herein.

在一些实施例中，本公开提供一种式I-B化合物：In some embodiments, the present disclosure provides a compound of formula I-B:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、R¹、R^1'、R^1”、X¹、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , R 1 , R^1′ , R^1″ , X¹ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供了一种式I-B-a化合物：In some embodiments, the present disclosure provides a compound of formula I-B-a:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、R¹、R^1'、R^1”、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , R¹ , R^1′ , R^1″ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供一种式I-B-b化合物：In some embodiments, the present disclosure provides a compound of formula I-B-b:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、R¹、R^1'、R^1”、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , R¹ , R^1′ , R^1″ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供一种式I-B-c化合物：In some embodiments, the present disclosure provides a compound of formula I-B-c:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、R¹、R^1'、R^1”、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , R¹ , R^1′ , R^1″ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供一种式I-C化合物：In some embodiments, the present disclosure provides a compound of formula I-C:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、R¹、R^1'、R^1”、X¹、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L³ , R¹ , R^1′ , R^1″ , X¹ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供一种式I-D化合物：In some embodiments, the present disclosure provides a compound of formula I-D:

或其N-氧化物、或其药学上可接受的盐，其中L²、L^2'、L^2”、L³、R¹、R^1'、R^1”、X¹、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L^2′ , L^2″ , L³ , R¹ , R^1′ , R^1″ , X¹ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在一些实施例中，本公开提供一种式I-E化合物：In some embodiments, the present disclosure provides a compound of Formula I-E:

或其N-氧化物、或其药学上可接受的盐，其中L²、L³、R¹、R^1'、R^1”、X¹、X²和X³中的每一者单独地和组合地如上所述且在本文的类别和子类别中。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein each of L² , L³ , R¹ , R^1′ , R^1″ , X¹ , X² , and X³ is as described above and in classes and subclasses herein, individually and in combination.

在本文所述的任何式的一些实施例中，L¹为不存在的、-C(O)-或-OC(O)-。在一些实施例中，L¹为不存在的。在一些实施例中，L¹为-C(O)-或-OC(O)-。在一些实施例中，L¹为-C(O)-。在一些实施例中，L¹为-OC(O)-。应当理解，当L¹为-OC(O)-时，L¹的羰基连接至式I中描绘的氧并且L¹的氧连接至L²，使得形成碳酸酯部分(例如，-O-C(O)-O-L²-)。In some embodiments of any formula described herein,^L1 is absent, -C(O)-, or -OC(O)-. In some embodiments,^L1 is absent. In some embodiments,^L1 is -C(O)- or -OC(O)-. In some embodiments,^L1 is -C(O)-. In some embodiments,^L1 is -OC(O)-. It is understood that when^L1 is -OC(O)-, the carbonyl of^L1 is connected to the oxygen depicted in Formula I and the oxygen of^L1 is connected to^L2 , so that a carbonate moiety is formed (e.g., -OC(O)^-OL2- ).

在本文所述的任何式的一些实施例中，L^1'为不存在的、-C(O)-或-OC(O)-。在一些实施例中，L^1'为不存在的。在一些实施例中，L^1'为-C(O)-或-OC(O)-。在一些实施例中，L^1'为-C(O)-。在一些实施例中，L^1'为-OC(O)-。应当理解，当L^1'为-OC(O)-时，L^1'的羰基连接至式I中描绘的氧并且L^1'的氧连接至L^2'，使得形成碳酸酯部分(例如，-O-C(O)-O-L^2'-)。In some embodiments of any formula described herein, L^1' is absent, -C(O)-, or -OC(O)-. In some embodiments, L^1' is absent. In some embodiments, L^1' is -C(O)- or -OC(O)-. In some embodiments, L 1^{' is -C(O)-. In some embodiments, L 1'} is -OC(O)-. It should be understood that when L^1' is -OC(O)-, the carbonyl of L^1' is connected to the oxygen depicted in Formula I and the oxygen of L^1' is connected to L^2' , so that a carbonate moiety is formed (e.g., -OC(O)-OL^2'- ).

在本文所述的任何式的一些实施例中，L^1”为不存在的、-C(O)-或-OC(O)-。在一些实施例中，L^1”为不存在的。在一些实施例中，L^1”为-C(O)-或-OC(O)-。在一些实施例中，L^1”为-C(O)-。在一些实施例中，L^1”为-OC(O)-。应当理解，当L^1”为-OC(O)-时，L^1”的羰基连接至式I中描绘的氧并且L^1”的氧连接至L^2”，使得形成碳酸酯部分(例如，-O-C(O)-O-L^2”-)。In some embodiments of any of the formulae described herein, L^1″ is absent, -C(O)-, or -OC(O)-. In some embodiments, L^1″ is absent. In some embodiments, L^1″ is -C(O)- or -OC(O)-. In some embodiments, L 1″ is -C(O)-. In some embodiments, L^1″ is -OC(O)-. It should be understood that when L¹ ″ is -OC(O)-, the carbonyl of L^1″ is connected to the oxygen depicted in Formula I and the oxygen of L^1″ is connected to L^2″ , such that a carbonate moiety is formed (e.g., -OC(O)-OL^2″ -).

在一些实施例中，L¹、L^1'和L^1”是相同的。在一些实施例中，L¹和L^1'是相同的，但不同于L^1”。在一些实施例中，L^1'和L^1”是相同的，但不同于L¹。在一些实施例中，L¹和L^1”是相同的，但不同于L^1'。在一些实施例中，L¹、L^1'和L^1”是彼此不同的。In some embodiments, L¹ , L^1′ , and L^1″ are the same. In some embodiments, L¹ and L^1′ are the same, but different from L^1″ . In some embodiments, L^1′ and L^1″ are the same, but different from L¹ . In some embodiments, L¹ and L^1″ are the same, but different from L^1′ . In some embodiments, L¹ , L^1′ , and L^1″ are different from each other.

在本文所述的任何式的一些实施例中，L²为不存在的，任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L²为不存在的。在一些实施例中，L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。在一些实施例中，L²为-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。在一些实施例中，L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-6烃链。在一些实施例中，L²为任选地被取代的二价饱和的直链或支链C_1-12烃链。在一些实施例中，L²为任选地被取代的二价饱和的直链或支链C_1-8烃链。在一些实施例中，L²为任选地被取代的二价饱和的直链或支链C_1-6烃链。在一些实施例中，L²为-CH₂-、-(CH₂)₂-、-(CH₂)₃-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-或-(CH₂)₇-。在一些实施例中，L²为-CH₂-。在一些实施例中，L²为-(CH₂)₂-。在一些实施例中，L²为-(CH₂)₃-。在一些实施例中，L²为-(CH₂)₄-。在一些实施例中，L²为-(CH₂)₅-。在一些实施例中，L²为-(CH₂)₆-。在一些实施例中，L²为-(CH₂)₇-。In some embodiments of any formula described herein, L² is absent, an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_{m '} -. In some embodiments, L² is absent. In some embodiments, L² is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain. In some embodiments, L² is -(CH₂ )_m -Cy^A -(CH₂ )_{m '} -. In some embodiments, L² is an optionally substituted divalent saturated or unsaturated straight or branched C_1-8 hydrocarbon chain. In some embodiments, L² is an optionally substituted divalent saturated or unsaturated straight or branched C_1-6 hydrocarbon chain. In some embodiments, L² is an optionally substituted divalent saturated straight or branched C_1-12 hydrocarbon chain. In some embodiments,^L2 is an optionally substituted divalent saturated straight or branched_C1-8 hydrocarbon chain. In some embodiments,^L2 is an optionally substituted divalent saturated straight or branched_C1-6 hydrocarbon chain. In some embodiments,^L2 is_-CH2- , -(_CH2 )_2- , -(_CH2 )_3- , -(_CH2 )_4- , -(_CH2 )_5- , -(_CH2 )_6- , or -(_CH2 )_7- . In some embodiments, L2 is_-CH2- . In some embodiments,^L2 is -(_CH2 )_2- . In some embodiments,^L2 is -(_CH2 )_3- . In some embodiments,^L2 is -(_CH2 )_4- . In some embodiments,^L2 is -(_CH2 )_5- . In some embodiments,^L2 is -(_CH2⁾_6- . In some embodiments, L² is -(CH₂ )₇ -.

在本文所述的任何式的一些实施例中，L^2'为不存在的，任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L^2'为不存在的。在一些实施例中，L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。在一些实施例中，L^2'为-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。在一些实施例中，L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-6烃链。在一些实施例中，L^2'为任选地被取代的二价饱和的直链或支链C_1-12烃链。在一些实施例中，L^2'为任选地被取代的二价饱和的直链或支链C_1-8烃链。在一些实施例中，L^2'为任选地被取代的二价饱和的直链或支链C_1-6烃链。在一些实施例中，L^2'为-CH₂-、-(CH₂)₂-、-(CH₂)₃-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-或-(CH₂)₇-。在一些实施例中，L^2'为-CH₂-。在一些实施例中，L^2'为-(CH₂)₂-。在一些实施例中，L^2'为-(CH₂)₃-。在一些实施例中，L^2'为-(CH₂)₄-。在一些实施例中，L^2'为-(CH₂)₅-。在一些实施例中，L^2'为-(CH₂)₆-。在一些实施例中，L^2'为-(CH₂)₇-。In some embodiments of any formula described herein, L^2' is absent, an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_m'- . In some embodiments, L^2' is absent. In some embodiments, L^2' is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain. In some embodiments, L^2' is -(CH₂ )_m -Cy^A -(CH₂ )_m'- . In some embodiments, L^2' is an optionally substituted divalent saturated or unsaturated straight or branched C_1-8 hydrocarbon chain. In some embodiments, L 2' is an optionally substituted divalent saturated or unsaturated straight or branched C_1-6 hydrocarbon chain. In some embodiments, L^2'is an optionally substituted divalent saturated straight or branched C_1-12 hydrocarbon chain. In some embodiments, L^2' is an optionally substituted divalent saturated straight or branched C_1-8 hydrocarbon chain. In some embodiments, L^2' is an optionally substituted divalent saturated straight or branched C_1-6 hydrocarbon chain. In some embodiments, L^2' is -CH₂ -, -(CH₂ )₂ -, -(CH₂ )₃ -, -(CH₂ )₄ -, -(CH₂ )₅ -, -(CH₂ )₆ -, or -(CH₂ )₇ -. In some embodiments, L^2' is -CH₂ -. In some embodiments, L^2' is -(CH₂ )₂ -. In some embodiments, L^2' is -(CH₂ )₃ -. In some embodiments, L^2' is -(CH₂ )₄ -. In some embodiments, L^2' is -(CH₂ )₅ -. In some embodiments, L^2' is -(CH₂ )₆ -. In some embodiments, L^2' is -(CH₂ )₇ -.

在本文所述的任何式的一些实施例中，L^2”为不存在的，任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L^2”为不存在的。在一些实施例中，L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。在一些实施例中，L^2”为-(CH₂)_m-Cy^A-(CH₂)_m'-。在一些实施例中，L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。在一些实施例中，L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-6烃链。在一些实施例中，L^2”为任选地被取代的二价饱和的直链或支链C_1-12烃链。在一些实施例中，L^2”为任选地被取代的二价饱和的直链或支链C_1-8烃链。在一些实施例中，L^2”为任选地被取代的二价饱和的直链或支链C_1-6烃链。在一些实施例中，L^2”为-CH₂-、-(CH₂)₂-、-(CH₂)₃-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-或-(CH₂)₇-。在一些实施例中，L^2”为-CH₂-。在一些实施例中，L^2”为-(CH₂)₂-。在一些实施例中，L^2”为-(CH₂)₃-。在一些实施例中，L^2”为-(CH₂)₄-。在一些实施例中，L^2”为-(CH₂)₅-。在一些实施例中，L^2”为-(CH₂)₆-。在一些实施例中，L^2”为-(CH₂)₇-。In some embodiments of any formula described herein, L^2" is absent, an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_m'- . In some embodiments, L^2" is absent. In some embodiments, L^2" is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain. In some embodiments, L^2" is -(CH₂ )_m -Cy^A -(CH₂ )_m'- . In some embodiments, L^2" is an optionally substituted divalent saturated or unsaturated straight or branched C_1-8 hydrocarbon chain. In some embodiments, L^2" is an optionally substituted divalent saturated or unsaturated straight or branched C_1-6 hydrocarbon chain. In some embodiments, L^2" is an optionally substituted divalent saturated linear or branched C_1-12 hydrocarbon chain. In some embodiments, L 2^" is an optionally substituted divalent saturated linear or branched C_1-8 hydrocarbon chain. In some embodiments, L 2" is an optionally substituted divalent saturated linear or branched C_1-6 hydrocarbon chain. In some embodiments, L^2" is -CH₂ -, -(CH₂ )₂ -, -(CH 2 )₃ -, -(CH₂ )₄ -, -(CH₂ )₅ -, -(CH₂ )₆ -, or -(CH₂ )₇ -. In some embodiments, L^2" is -CH₂ -. In some embodiments, L^2" is -(CH₂ )₂ -. In some embodiments, L^2" is -(_{CH 2)3} -. In some embodiments, L^2" is -(CH₂ )₄ -. In some embodiments, L^2" is -(CH₂ )₅ -. In some embodiments, L^2" is -(CH₂ )₆ -. In some embodiments, L^2" is -(CH₂ )₇ -.

在一些实施例中，L²、L^2'和L^2”是相同的。在一些实施例中，L²和L^2'是相同的，但不同于L^2”。在一些实施例中，L^2'和L^2”是相同的，但不同于L²。在一些实施例中，L²和L^2”是相同的，但不同于L^2'。在一些实施例中，L²、L^2'和L^2”是彼此不同的。In some embodiments, L² , L^2' , and L^2" are the same. In some embodiments, L² and L^2' are the same, but different from L^2" . In some embodiments, L^2' and L^2" are the same, but different from L^2. In some embodiments, L² and L^2" are the same, but different from L^2' . In some embodiments, L² , L^2' , and L^2" are different from each other.

在本文所述的任何式的一些实施例中，每个Cy^A独立地为选自亚苯基和3至12元饱和或部分不饱和的亚碳环基的任选地被取代的环。在一些实施例中，Cy^A为亚苯基。在一些实施例中，Cy^A为3至12元饱和或部分不饱和的亚碳环基。In some embodiments of any formula described herein, each Cy^{A is} independently an optionally substituted ring selected from phenylene and 3 to 12 membered saturated or partially unsaturated carbocyclylene. In some embodiments, Cy^A is phenylene. In some embodiments, Cy^A is 3 to 12 membered saturated or partially unsaturated carbocyclylene.

在本文所述的任何式的一些实施例中，每个m独立地为0、1或2。在一些实施例中，m为0。在一些实施例中，m为1。在一些实施例中，m为2。In some embodiments of any of the formulae described herein, each m is independently 0, 1, or 2. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2.

在本文所述的任何式的一些实施例中，每个m'独立地为0、1或2。在一些实施例中，m'为0。在一些实施例中，m'为1。在一些实施例中，m'为2。In some embodiments of any of the formulae described herein, each m' is independently 0, 1, or 2. In some embodiments, m' is 0. In some embodiments, m' is 1. In some embodiments, m' is 2.

在一些实施例中，m和m'是相同的。在一些实施例中，m和m'是不同的。In some embodiments, m and m' are the same. In some embodiments, m and m' are different.

在本文所述的任何式的一些实施例中，L³为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L³为不存在的。在一些实施例中，L³为-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L³为-C(O)-、-C(O)O-、-OC(O)-或-OC(O)O-。在一些实施例中，L³为-C(O)O-或-OC(O)-。在一些实施例中，L³为-C(O)-。在一些实施例中，L³为-C(O)O-。在一些实施例中，L³为-OC(O)-。在一些实施例中，L³为-O-。在一些实施例中，L³为-OC(O)O-。In some embodiments of any formula described herein, L³ is absent, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L^{3 is absent. In some embodiments, L 3} is -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L³ is -C(O)-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L³ is -C(O)O- or -OC(O)-. In some embodiments, L³ is -C(O)-. In some embodiments, L³ is -C(O)O-.^{In some embodiments, L 3 is -OC(O)-. In some embodiments, L 3 is -O-. In some embodiments, L 3is-OC(} O)O-.

在本文所述的任何式的一些实施例中，L^3'为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L^3'为不存在的。在一些实施例中，L^3'为-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L^3'为-C(O)-、-C(O)O-、-OC(O)-或-OC(O)O-。在一些实施例中，L^3'为-C(O)O-或-OC(O)-。在一些实施例中，L^3'为-C(O)-。在一些实施例中，L^3'为-C(O)O-。在一些实施例中，L^3'为-OC(O)-。在一些实施例中，L^3'为-O-。在一些实施例中，L^3'为-OC(O)O-。In some embodiments of any formula described herein, L^3' is absent, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L 3' is absent. In some embodiments, L^3'is -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L^3' is -C(O)-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L^3' is -C(O)O- or -OC(O)-. In some embodiments, L^3' is -C(O)-. In some embodiments, L^3' is -C(O)O-. In some embodiments, L^3' is -OC(O)-. In some embodiments, L^3' is -O-. In some embodiments, L^3' is -OC(O)O-.

在本文所述的任何式的一些实施例中，L^3”为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L^3”为不存在的。在一些实施例中，L^3”为-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-。在一些实施例中，L^3”为-C(O)-、-C(O)O-、-OC(O)-或-OC(O)O-。在一些实施例中，L^3”为-C(O)O-或-OC(O)-。在一些实施例中，L^3”为-C(O)-。在一些实施例中，L^3”为-C(O)O-。在一些实施例中，L^3”为-OC(O)-。在一些实施例中，L^3”为-O-。在一些实施例中，L^3”为-OC(O)O-。In some embodiments of any of the formulae described herein, L^3" is absent, -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L^3" is absent. In some embodiments, L^3" is -C(O)-, -C(O)O-, -OC(O)-, -O-, or -OC(O)O-. In some embodiments, L^3" is -C(O)-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L^3" is -C(O)O- or -OC(O)-. In some embodiments, L^3" is -C(O)-. In some embodiments, L^3" is -C(O)O-. In some embodiments, L^3" is -OC(O)-. In some embodiments, L^3" is -O-. In some embodiments, L^3" is -OC(O)O-.

在一些实施例中，L³、L^3'和L^3”是相同的。在一些实施例中，L³和L^3'是相同的，但不同于L^3”。在一些实施例中，L^3'和L^3”是相同的，但不同于L³。在一些实施例中，L³和L^3”是相同的，但不同于L^3'。在一些实施例中，L³、L^3'和L^3”是彼此不同的。In some embodiments, L³ , L^3' and L^3" are the same. In some embodiments, L³ and L^3' are the same but different from L^3" . In some embodiments, L^3' and L^3" are the same but different from L^3. In some embodiments, L³ and L^3" are the same but different from L^3' . In some embodiments, L³ , L^3' and L^3" are different from each other.

在本文所述的任何式的一些实施例中，R¹为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换。在一些实施例中，R¹为-(CH₂)_p-Cy^B。在一些实施例中，R¹为在一些实施例中，R¹为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。在一些实施例中，R¹为任选地被取代的饱和或不饱和的直链或支链C_6-20烃链。在一些实施例中，R¹为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。在一些实施例中，R¹为任选地被取代的饱和或不饱和的直链或支链C_8-9烃链。在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为在一些实施例中，R¹为In some embodiments of any of the formulas described herein, R¹ is -(CH₂ )_p -Cy^B , or an optionally substituted saturated or unsaturated straight or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-. In some embodiments, R¹ is -(CH₂ )_p -Cy^B . In some embodiments, R¹ is In some embodiments, R¹ is an optionally substituted saturated or unsaturated straight or branched C_1-20 hydrocarbon chain. In some embodiments, R¹ is an optionally substituted saturated or unsaturated straight or branched C_6-20 hydrocarbon chain. In some embodiments, R^{1 is an optionally substituted saturated or unsaturated straight or branched C 6-12 hydrocarbon chain. In some embodiments, R 1}_{is an optionally substituted saturated or unsaturated straight or branched C 8-9}^hydrocarbon_chain . In some embodiments, R¹ is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is In some embodiments,^R1 is

在本文所述的任何式的一些实施例中，R^1'为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换。在一些实施例中，R^1'为-(CH₂)_p-Cy^B。在一些实施例中，R^1'为在一些实施例中，R^1'为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。在一些实施例中，R^1'为任选地被取代的饱和或不饱和的直链或支链C_6-20烃链。在一些实施例中，R^1'为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。在一些实施例中，R^1'为任选地被取代的饱和或不饱和的直链或支链C_8-9烃链。在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为在一些实施例中，R^1'为In some embodiments of any of the formulae described herein, R^1′ is —(CH₂ )_p —Cy^B , or an optionally substituted saturated or unsaturated straight or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-. In some embodiments, R^1' is -(CH₂ )_p -Cy^B . In some embodiments, R^1' is In some embodiments, R^1' is an optionally substituted saturated or unsaturated straight or branched C_1-20 hydrocarbon chain. In some embodiments, R^1' is an optionally substituted saturated or unsaturated straight or branched C_6-20 hydrocarbon chain. In some embodiments, R 1' is an optionally substituted saturated or unsaturated straight or branched C_6-12 hydrocarbon chain. In some embodiments, R¹ ' is an optionally substituted saturated or unsaturated straight or branched C_8-9 hydrocarbon chain. In some embodiments, R^1'is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is In some embodiments, R^1' is

在本文所述的任何式的一些实施例中，R^1”为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换。在一些实施例中，R^1”为-(CH₂)_p-Cy^B。在一些实施例中，R^1”为在一些实施例中，R^1”为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。在一些实施例中，R^1”为任选地被取代的饱和或不饱和的直链或支链C_6-20烃链。在一些实施例中，R^1”为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。在一些实施例中，R^1”为任选地被取代的饱和或不饱和的直链或支链C_8-9烃链。在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为在一些实施例中，R^1”为In some embodiments of any of the formulae described herein, R^1″ is -(CH₂ )_p -Cy^B , or an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-. In some embodiments, R^1" is -(CH₂ )_p -Cy^B . In some embodiments, R^1" is In some embodiments, R^1" is an optionally substituted saturated or unsaturated straight or branched C_1-20 hydrocarbon chain. In some embodiments, R 1^" is an optionally substituted saturated or unsaturated straight or branched C_6-20 hydrocarbon chain. In some embodiments, R 1" is an optionally substituted saturated or unsaturated straight or branched C_6-12 hydrocarbon chain. In some embodiments, R¹ " is an optionally substituted saturated or unsaturated straight or branched C_8-9 hydrocarbon chain. In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is In some embodiments, R^1" is

在一些实施例中，R¹、R^1'和R^1”是相同的。在一些实施例中，R¹和R^1'是相同的，但不同于R^1”。在一些实施例中，R^1'和R^1”是相同的，但不同于R¹。在一些实施例中，R¹和R^1”是相同的，但不同于R^1'。在一些实施例中，R¹、R^1'和R^1”是彼此不同的。In some embodiments, R¹ , R^1′ , and R^1″ are the same. In some embodiments, R¹ and R^1′ are the same, but different from R^1″ . In some embodiments, R^1′ and R^1″ are the same, but different from R¹ . In some embodiments, R¹ and R^1″ are the same, but different from R^1′ . In some embodiments, R¹ , R^1′ , and R^1″ are different from each other.

在本文所述的任何式的一些实施例中，每个Cy^B独立地为选自以下的任选地被取代的环：3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧和硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基。在一些实施例中，Cy^B为1-金刚烷基。In some embodiments of any formula described herein, each Cy^B is independently an optionally substituted ring selected from: a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic radical having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, 1-adamantyl, 2-adamantyl, sterol, and phenyl. In some embodiments, Cy^B is 1-adamantyl.

在本文所述的任何式的一些实施例中，每个p独立地为0、1、2或3。在一些实施例中，p为0。在一些实施例中，p为1、2或3。在一些实施例中，p为0或1。在一些实施例中，p为1。在一些实施例中，p为2。在一些实施例中，p为3。In some embodiments of any of the formulae described herein, each p is independently 0, 1, 2, or 3. In some embodiments, p is 0. In some embodiments, p is 1, 2, or 3. In some embodiments, p is 0 or 1. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3.

在本文所述的任何式的一些实施例中，每个L^3a独立地为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换。在一些实施例中，L^3a为不存在的。在一些实施例中，L^3a为任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换。在一些实施例中，L^3a为任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链。In some embodiments of any formula described herein, each L^3a is independently absent or optionally substituted divalent saturated or unsaturated straight or branched C_1-10 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-. In some embodiments, L^{3a is absent. In some embodiments, L 3a} is optionally substituted divalent saturated or unsaturated straight or branched C_1-10 hydrocarbon chain, wherein 1-3 methylene units^{are optionally and independently replaced by -O- or -NR-. In some embodiments, L 3ais} optionally substituted divalent saturated or unsaturated straight or branched C_1-10 hydrocarbon chain.

在本文所述的任何式的一些实施例中，每个R^a独立地为氢或选自以下的任选地被取代的基团：C_6-20脂肪族、3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧和硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基。在一些实施例中，R^a为任选地被取代的C_6-20脂肪族。在一些实施例中，R^a为任选地被取代的C_6-12脂肪族。在一些实施例中，R^a为任选地被取代的C_6-20烯基。在一些实施例中，R^a为任选地被取代的C_6-12烯基。在一些实施例中，R^a为In some embodiments of any formula described herein, each Ra^is independently hydrogen or an optionally substituted group selected from the following:_C6-20 aliphatic, 3 to 12-membered saturated or partially unsaturated carbocyclyl, 7 to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl. In some embodiments, Ra^is an optionally substituted_C6-20 aliphatic. In some embodiments, Ra^is an optionally substituted_C6-12 aliphatic. In some embodiments, Ra^is an optionally substituted_C6-20 alkenyl. In some embodiments, Ra^is an optionally substituted_C6-12 alkenyl. In some embodiments, Ra^is

在一些实施例中，每个为In some embodiments, each for

在本文所述的任何式的一些实施例中，X¹为不存在的、-O-、-S-或-NR-。在一些实施例中，X¹为不存在的。在一些实施例中，X¹为-O-、-S-或-NR-。在一些实施例中，X¹为-O-。在一些实施例中，X¹为-NR-。在一些实施例中，X¹为-S-。In some embodiments of any formula described herein,^Xi is absent, -O-, -S-, or -NR-. In some embodiments,^Xi is absent. In some embodiments,^Xi is -O-, -S-, or -NR-. In some embodiments,^Xi is -O-. In some embodiments,^Xi is -NR-. In some embodiments,^Xi is -S-.

在本文所述的任何式的一些实施例中，X²为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换。在一些实施例中，X²为不存在的。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-NR-置换。在一些实施例中，X²为任选地被取代的二价饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-NR-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1个亚甲基单元被-NR-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_4-8烃链，其中1个亚甲基单元被-NR-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-Cy^C-置换。在一些实施例中，X²为任选地被取代的二价饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-Cy^C-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1个亚甲基单元被-Cy^C-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_4-8烃链，其中1个亚甲基单元被-Cy^C-置换。在一些实施例中，X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。在一些实施例中，X²为任选地被取代的二价饱和的直链或支链C_1-12烃链。在一些实施例中，X²为任选地被取代的二价饱和的直链或支链C_1-6烃链。在一些实施例中，X²为任选地被取代的二价饱和的直链或支链C_2-3烃链。In some embodiments of any formula described herein,^X is absent or optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or -Cy^C -. In some embodiments,^X is absent. In some embodiments,^X is optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or -Cy^C -. In some embodiments,^X is optionally substituted divalent saturated or unsaturated straight or branched C 1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -NR-. In some embodiments,^X is optionally substituted divalent saturated straight or branched C_1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -NR-_. In some embodiments,^X is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1 methylene unit is replaced by -NR-. In some embodiments,^X is an optionally substituted divalent saturated or unsaturated straight or branched C_4-8 hydrocarbon chain, wherein 1 methylene unit is replaced by -NR-. In some embodiments, X is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein^1-3 methylene units are optionally and independently replaced by -Cy^C -. In some embodiments, X^is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -Cy^C -. In some embodiments, X^is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1 methylene unit is replaced by -Cy^C -. In some embodiments,^X is an optionally substituted divalent saturated or unsaturated straight or branched C_4-8 hydrocarbon chain, wherein one methylene unit is replaced by -Cy^C -. In some embodiments,^X is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain. In some embodiments,^X is an optionally substituted divalent saturated straight or branched C 1-12 hydrocarbon chain. In some embodiments,^X is an optionally substituted divalent saturated straight or branched C_1-6 hydrocarbon chain. In some embodiments, X^is_{an optionally substituted divalent saturated straight or branched C 2-3hydrocarbon} chain.

在本文所述的任何式的一些实施例中，Cy^C为选自以下的任选地被取代的环：3至7元饱和或部分不饱和的亚碳环、亚苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的亚杂环或具有1-3个独立地选自氮、氧和硫的杂原子的5至6元亚杂芳基。在一些实施例中，Cy^C为具有1-3个独立地选自氮、氧和硫的杂原子的任选地被取代的3至7元饱和或部分不饱和的亚杂环基。在一些实施例中，Cy^C为具有1-3个独立地选自氮、氧和硫的杂原子的任选地被取代的5至6元饱和或部分不饱和的亚杂环基。在一些实施例中，Cy^C为具有1-2个独立地选自氮、氧和硫的杂原子的任选地被取代的5至6元饱和的亚杂环基。在一些实施例中，Cy^C为具有1个氮的5至6元饱和的杂环烯。在一些实施例中，Cy^C为任选地被取代的哌啶环。在一些实施例中，Cy^C为哌啶环。In some embodiments of any formula described herein, Cy^C is an optionally substituted ring selected from the following: a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy^C is an optionally substituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy^C is an optionally substituted 5- to 6-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy^C is an optionally substituted 5- to 6-membered saturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy^C is a 5- to 6-membered saturated heterocyclic ring having 1 nitrogen. In some embodiments, Cy^C is an optionally substituted piperidine ring. In some embodiments, Cy^C is a piperidine ring.

在本文所述的任何式的一些实施例中，X³为氢或选自以下的任选地被取代的环：3至7元饱和或部分不饱和的碳环基、苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的杂环基以及具有1-3个独立地选自氮、氧和硫的杂原子的5至6元杂芳基。在一些实施例中，X³为氢。在一些实施例中，X³为具有1-3个独立地选自氮、氧和硫的杂原子的任选地被取代的3至7元饱和或部分不饱和的杂环基。在一些实施例中，X³为具有1-3个独立地选自氮、氧和硫的杂原子的任选地被取代的5至6元饱和或部分不饱和的杂环基。在一些实施例中，X³为具有1-2个独立地选自氮、氧和硫的杂原子的任选地被取代的5至6元杂环基。在一些实施例中，X³为具有1-2个氮原子的任选地被取代的5元饱和或部分不饱和的杂环基。在一些实施例中，X³为吡咯烷基。在一些实施例中，X³为具有1-2个氮原子的任选地被取代的6元饱和或部分不饱和的杂环基。在一些实施例中，X³为任选地被取代的哌啶基(例如，被C_1-6烷基取代的哌啶基)。In some embodiments of any formula described herein, X is hydrogen or an optionally substituted ring selected from the following:³ to 7 membered saturated or partially unsaturated carbocyclyl, phenyl, 3 to 7 membered saturated or partially unsaturated heterocyclyl with 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5 to 6 membered heteroaryl with 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments,^X is hydrogen. In some embodiments,^X is an optionally substituted 3 to 7 membered saturated or partially unsaturated heterocyclyl with 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments,^X is an optionally substituted 5 to 6 membered saturated or partially unsaturated heterocyclyl with 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments,^X is an optionally substituted 5 to 6 membered heterocyclyl with 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments,^X is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic radical with 1-2 nitrogen atoms. In some embodiments,^X is a pyrrolidinyl radical. In some embodiments,^X is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic radical with 1-2 nitrogen atoms. In some embodiments,^X is an optionally substituted piperidinyl radical (e.g., a piperidinyl radical substituted with C_1-6 alkyl).

在一些实施例中，-X²-X³为在一些实施例中，-X²-X³为在一些实施例中，-X²-X³为在一些实施例中，-X²-X³为在一些实施例中，-X²-X³为在一些实施例中，-X²-X³为In some embodiments, -X² -X³ is In some embodiments, -X² -X³ is In some embodiments, -X² -X³ is In some embodiments, -X² -X³ is In some embodiments, -X² -X³ is In some embodiments, -X² -X³ is

在本文所述的任何式的一些实施例中，每个R独立地为氢或任选地被取代的C_1-6脂肪族基团。在一些实施例中，R为氢。在一些实施例中，R为任选地被取代的C_1-6脂肪族。在一些实施例中，R为任选地被取代的C_1-3脂肪族。在一些实施例中，R是-CH₃或-CH₂CH₃。In some embodiments of any formula described herein, each R is independently hydrogen or an optionally substituted C_1-6 aliphatic group. In some embodiments, R is hydrogen. In some embodiments, R is an optionally substituted C_{1-6 aliphatic group. In some embodiments, R is an optionally substituted C 1-3aliphatic} group. In some embodiments, R is -CH₃ or -CH₂ CH₃ .

应当理解，如本文中所使用，“[化合物/式]或其N-氧化物或其药学上可接受的盐”是指i)各化合物或式或ii)此类化合物或式的N-氧化物的药学上可接受的盐。It should be understood that as used herein, "[compound/formula] or its N-oxide or a pharmaceutically acceptable salt thereof" refers to i) the respective compound or formula or ii) a pharmaceutically acceptable salt of such compound or formula's N-oxide.

在一些实施例中，本公开提供选自表1的化合物。In some embodiments, the present disclosure provides a compound selected from Table 1.

表1.Table 1.

或其药学上可接受的盐。or a pharmaceutically acceptable salt thereof.

应当理解，除非式I、I-A、I-A-a、I-A-b、I-A-c、I-B、I-B-a、I-B-b、I-B-c、I-C、I-D和I-E中的任一者的前述定义另有规定或禁止，如上所定义并在本文类别和子类别中描述的变量L¹、L^1'、L^1”、L²、L^2'、L^2”、L³、L^3'、L^3”、R¹、R^1'、R^1”、Cy^A、Cy^B、Cy^C、m、m'、p、L^3a、R^a、R、X¹、X²和X³的实施例单独地和组合地适用于式I、I-A、I-A-a、I-A-b、I-A-c、I-B、I-B-a、I-B-b、I-B-c、I-C、I-D和I-E中任一者的化合物。It should be understood that unless otherwise specified or prohibited by the foregoing definition of any of Formula I, IA, IAa, IAb, IAc, IB, IBa, IBb, IBc, IC, ID, and IE, the embodiments for the variables^L1 , L1^' , L1^" ,^L2 , L2',^L2" ,^L3 , L3', L3^" ,^R1 , R1^' ,^R1", CyA,^CyB ,^CyC , m, m', p,^L3a ,^Ra , R,^Xi ,^X2 , and^X3 , individually and in combination, apply to compounds of any of Formula I, IA, IAa, IAb, IAc,^IB , IBa, IBb, IBc, IC, ID, and IE, as defined above and described in classes and subclasses herein.

在一些实施例中，所提供的化合物是式I、I-A、I-A-a、I-A-b、I-A-c、I-B、I-B-a、I-B-b、I-B-c、I-C、I-D和I-E中任一者的化合物或其药学上可接受的盐。In some embodiments, provided compounds are compounds of any of Formula I, I-A, I-A-a, I-A-b, I-A-c, I-B, I-B-a, I-B-b, I-B-c, I-C, I-D, and I-E, or pharmaceutically acceptable salts thereof.

在一些实施例中，所提供的化合物以盐形式(例如药学上可接受的盐形式)提供和/或利用所提供的化合物。除非另外指明，否则对本文所提供的化合物的提及应理解为包含对其盐的提及。In some embodiments, provided compounds are provided and/or utilized in salt form (e.g., pharmaceutically acceptable salt form). Unless otherwise indicated, reference to a compound provided herein should be understood to include reference to a salt thereof.

应当理解，在整个本公开中，除非另有说明，提及式I的化合物还旨在包含式I-A、I-A-a、I-A-b、I-A-c、I-B、I-B-a、I-B-b、I-B-c、I-C、I-D和I-E中的任一者的化合物以及本文公开的此类式的化合物种类。It should be understood that throughout this disclosure, unless otherwise indicated, reference to compounds of Formula I is also intended to include compounds of any of Formulas I-A, I-A-a, I-A-b, I-A-c, I-B, I-B-a, I-B-b, I-B-c, I-C, I-D, and I-E, as well as species of compounds of such formulae disclosed herein.

在一些实施例中，本公开涵盖如下认识：例如与参考化合物或其它已知化合物相比，所提供的化合物显示某些所需特征。例如，在一些实施例中，所提供的化合物在本文所述的一项或多项实验中表现出更有效地递送至各种细胞类型，和/或具有一种或多种使得其比起其它已知化合物更适合于递送诸如治疗剂或预防剂等货物的其它特征。以不受任何理论约束为前提，本公开涵盖以下认识：与缺乏相同四价核心特征的相应化合物相比，所提供的包括四价核心(例如，来源于2,2-双(羟甲基)丙烷-1,3-二醇的核心)特征的化合物显示出某些更理想的特性(例如，在本文所述的一项或多项实验中更有效递送至各种细胞类型)。In some embodiments, the present disclosure encompasses the recognition that provided compounds display certain desired characteristics, for example, compared to reference compounds or other known compounds. For example, in some embodiments, provided compounds exhibit more effective delivery to various cell types in one or more experiments described herein, and/or have one or more other characteristics that make them more suitable for delivery of goods such as therapeutic agents or prophylactic agents than other known compounds. Without being bound by any theory, the present disclosure encompasses the recognition that provided compounds including tetravalent core (e.g., a core derived from 2,2-bis(hydroxymethyl)propane-1,3-diol) features display certain more desirable properties (e.g., more effective delivery to various cell types in one or more experiments described herein) compared to corresponding compounds lacking the same tetravalent core features.

B.制备所提供的化合物B. Preparation of provided compounds

所提供的化合物通常可以通过随后的方案和实例中描述的方法来制备。The provided compounds can generally be prepared by the methods described in the Schemes and Examples which follow.

C.可电离脂质C. Ionizable lipids

此外，本公开描述包括一种或多种如本文所述的可电离脂质的组合物、制剂、纳米粒子和/或纳米材料。Additionally, the present disclosure describes compositions, formulations, nanoparticles, and/or nanomaterials comprising one or more ionizable lipids as described herein.

此外，出人意料地发现，可电离脂质的不同比率会影响一种或多种功能活性，如本文所述的组合物、制剂、纳米粒子和/或纳米材料的所需向性、稳定性和药物递送功效。例如，本公开展示了一个令人惊讶的发现，即可电离脂质的量不同于本领域中描述的那些量(例如，参见美国专利第8,058,069B2号，或参见例如美国专利第9,364,435号，两者的内容特此通过全文引用的方式并入本文中)对于本文所述的组合物、制剂、纳米粒子和/或纳米材料的一种或多种功能活性很重要，和/或会影响所述一种或多种功能活性。例如，在一些实施例中，发现具有以脂质纳米粒子组分的总摩尔数计约50mol％或更少的可电离脂质的组合物、制剂、纳米粒子和/或纳米材料对脂质纳米粒子的功能活性(诸如本文所述的所需向性、稳定性和药物递送功效)有用和/或至关重要。In addition, it is unexpectedly found that different ratios of ionizable lipids can affect one or more functional activities, such as the desired tropism, stability and drug delivery efficacy of compositions, formulations, nanoparticles and/or nanomaterials described herein. For example, the present disclosure shows a surprising discovery that the amount of ionizable lipids is different from those described in the art (e.g., see U.S. Patent No. 8,058,069B2, or see, for example, U.S. Patent No. 9,364,435, the contents of both of which are hereby incorporated herein by reference in their entirety) for compositions, formulations, nanoparticles and/or nanomaterials described herein One or more functional activities are important, and/or can affect the one or more functional activities. For example, in some embodiments, it is found that compositions, formulations, nanoparticles and/or nanomaterials with about 50 mol% or less ionizable lipids based on the total molar number of lipid nanoparticle components are useful and/or essential for the functional activities of lipid nanoparticles (such as the desired tropism, stability and drug delivery efficacy described herein).

在一些实施例中，可电离脂质可包含头部基团上的含胺基团。在一些实施例中，可电离脂质为或包含本文所述的化合物。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约30摩尔％至约70摩尔％存在于脂质纳米粒子(LNP)制剂中。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约33摩尔％至约60摩尔％存在。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约34摩尔％至约55摩尔％存在。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约33摩尔％至约51摩尔％存在。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约34.7摩尔％存在。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约50摩尔％存在。In some embodiments, ionizable lipids may include amine-containing groups on the head group. In some embodiments, ionizable lipids are or include compounds as described herein. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present in lipid nanoparticle (LNP) preparations with about 30 mol % to about 70 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 33 mol % to about 60 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 34 mol % to about 55 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 33 mol % to about 51 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 34.7 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 50 mol %.

此外，在一些实施例中，脂质纳米粒子组合物包括可电离脂质。在一些实施例中，脂质纳米粒子制剂包含可电离脂质；磷脂；缀合物-连接子脂质；以及胆固醇。在一些实施例中，可电离脂质为或包含根据本文所述的化合物的结构。在一些实施例中，以脂质纳米粒子组分的总摩尔数计，可电离脂质以约30摩尔％至约70摩尔％存在于LNP制剂中。In addition, in some embodiments, the lipid nanoparticle composition includes an ionizable lipid. In some embodiments, the lipid nanoparticle formulation comprises an ionizable lipid; a phospholipid; a conjugate-linker lipid; and cholesterol. In some embodiments, the ionizable lipid is or comprises a structure according to a compound described herein. In some embodiments, the ionizable lipid is present in the LNP formulation at about 30 mol % to about 70 mol % based on the total moles of the lipid nanoparticle components.

D.甾醇D. Sterol

此外，本公开描述包括一种或多种如本文所述的甾醇的组合物、制剂、纳米粒子和/或纳米材料。Additionally, the present disclosure describes compositions, formulations, nanoparticles, and/or nanomaterials comprising one or more sterols as described herein.

在一些实施例中，甾醇为胆甾醇或其变体或衍生物。在一些实施例中，胆甾醇经过改性。在一些实施例中，胆甾醇为氧化胆甾醇。在一些实施例中，胆甾醇为酯化胆甾醇。未经改性的胆甾醇可以通过酶作用形成侧链或环氧化的变体。在一些实施例中，胆甾醇可在β环结构上或烃尾结构上氧化。在一些实施例中，甾醇为植物甾醇。被视为适用于所公开的脂质纳米粒子的示例性甾醇包含但不限于25-羟基胆甾醇(25-OH)、20α-羟基胆甾醇(20α-OH)、27-羟基胆甾醇、6-酮基-5α-羟基胆甾醇、7-酮基胆甾醇、7β-羟基胆甾醇、7α-羟基胆甾醇基、7β-25-二羟基胆甾醇、β-谷甾醇、豆甾醇、菜籽甾醇、菜油甾醇或其组合。在一些实施例中，侧链氧化胆甾醇相对于其它胆甾醇变体可以增强货物递送。在一些实施例中，胆甾醇为未经改性的胆甾醇。In some embodiments, sterol is cholesterol or its variant or derivative. In some embodiments, cholesterol is modified. In some embodiments, cholesterol is oxidized cholesterol. In some embodiments, cholesterol is esterified cholesterol. Unmodified cholesterol can form side chain or epoxidized variants by enzyme action. In some embodiments, cholesterol can be oxidized on the β ring structure or on the hydrocarbon tail structure. In some embodiments, sterol is phytosterol. Exemplary sterols considered to be suitable for the disclosed lipid nanoparticles include but are not limited to 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesteryl, 7β-25-dihydroxycholesterol, β-sitosterol, stigmasterol, rapeseed sterol, campesterol or its combination. In some embodiments, side chain oxidized cholesterol can enhance cargo delivery relative to other cholesterol variants. In some embodiments, the cholesterol is unmodified cholesterol.

在一些实施例中，LNP组合物包括约20mol％至约50mol％甾醇。在一些实施例中，LNP组合物包括约38mol％甾醇。在一些实施例中，LNP组合物包括约38.5mol％甾醇。在一些实施例中，LNP组合物包括约33.8mol％胆甾醇。In some embodiments, the LNP composition includes about 20mol% to about 50mol% sterol. In some embodiments, the LNP composition includes about 38mol% sterol. In some embodiments, the LNP composition includes about 38.5mol% sterol. In some embodiments, the LNP composition includes about 33.8mol% cholesterol.

E.缀合物-连接子脂质E. Conjugate-Linker Lipid

此外，本公开描述包括一种或多种如本文所述的缀合物-连接子脂质的组合物、制剂、纳米粒子和/或纳米材料。Additionally, the present disclosure describes compositions, formulations, nanoparticles, and/or nanomaterials comprising one or more conjugate-linker lipids as described herein.

在一些实施例中，缀合物-连接子脂质为或包括聚乙二醇(PEG)-脂质或PEG修饰的脂质。在一些实施例中，PEG或PEG修饰的脂质可替代地被称作聚乙二醇化脂质或PEG-脂质。包含聚乙二醇化脂质可以用于增强体外脂质纳米粒子的体外胶体稳定性和体内循环时间。在一些实施例中，因为PEG部分在血液循环中逐步释放，所以聚乙二醇化是可逆的。示范性PEG脂质包含但不限于与长度为C6-C20的饱和或不饱和烷基链缀合的PEG。PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺(PEG-CER)、PEG修饰的二烷基胺、PEG修饰的二酰基甘油(PEG-DAG)、PEG修饰的二烷基甘油及其混合物。例如，在一些实施例中，PEG脂质可以是PEG-c-DOMG、PEG-DMG、PEG-DLPE、PEG-DMPE、PEG-DPPE、PEG-DSG或PEG-DSPE脂质。In certain embodiments, conjugate-linker lipid is or includes polyethylene glycol (PEG)-lipid or PEG-modified lipid.In certain embodiments, PEG or PEG-modified lipid is alternatively referred to as PEGylated lipid or PEG-lipid.Comprise PEGylated lipid can be used to enhance the in vitro colloidal stability and in vivo circulation time of lipid nanoparticle in vitro.In certain embodiments, because PEG part is progressively released in blood circulation, PEGization is reversible.Exemplary PEG lipid includes but is not limited to the PEG conjugated with saturated or unsaturated alkyl chain of length C6-C20.PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol and its mixture.For example, in certain embodiments, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or PEG-DSPE lipid.

在一些实施例中，缀合物-连接子脂质包括聚乙二醇脂质。在一些实施例中，缀合物-连接子脂质包括二肉豆蔻基甘油(DMG)、1,2-二棕榈酰基-rac-甘油、甲氧基聚乙二醇(DPG-PEG)或1,2-二硬脂酰基-rac-甘油-3-甲基聚氧乙烯(DSG-PEG)。在一些实施例中，缀合物-连接子脂质的平均分子量为约500Da至约5000Da。在一些实施例中，缀合物-连接子脂质的平均分子量为约2000Da。在一些实施例中，LNP组合物包含约0mol％至约5mol％的缀合物-连接子脂质。在一些实施例中，LNP组合物包括约1.5mol％缀合物-连接子脂质。在一些实施例中，LNP组合物包括约3mol％缀合物-连接子脂质。In some embodiments, conjugate-linker lipids include polyethylene glycol lipids. In some embodiments, conjugate-linker lipids include dimyristyl glycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxy polyethylene glycol (DPG-PEG) or 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, the average molecular weight of conjugate-linker lipids is about 500Da to about 5000Da. In some embodiments, the average molecular weight of conjugate-linker lipids is about 2000Da. In some embodiments, LNP compositions include about 0mol% to about 5mol% of conjugate-linker lipids. In some embodiments, LNP compositions include about 1.5mol% conjugate-linker lipids. In some embodiments, LNP compositions include about 3mol% conjugate-linker lipids.

F.磷脂F. Phospholipids

此外，本公开描述包括一种或多种如本文所述的磷脂的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，本公开描述包括一种或多种(多)不饱和脂质的组合物、制剂、纳米粒子和/或纳米材料。In addition, the present disclosure describes compositions, formulations, nanoparticles and/or nanomaterials comprising one or more phospholipids as described herein. In some embodiments, the present disclosure describes compositions, formulations, nanoparticles and/or nanomaterials comprising one or more (poly)unsaturated lipids.

在一些实施例中，一种或多种磷脂可组装成一种或多种脂质双层。在一些实施例中，一种或多种磷脂可包含磷脂部分。在一些实施例中，一种或多种磷脂可包含一种或多种脂肪酸部分。在一些实施例中，一种或多种磷脂可包含磷脂部分和一种或多种脂肪酸部分。在一些实施例中，磷脂部分包含但不限于磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油、磷脂酰丝氨酸、磷脂酸、2-溶血磷脂酰胆碱和鞘磷脂。在一些实施例中，脂肪酸部分包含但不限于月桂酸、肉豆蔻酸、肉豆蔻油酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚麻酸、α亚麻酸、芥酸、植烷酸、花生酸、花生四烯酸、二十碳五烯酸、山萮酸、二十二碳五烯酸和二十二碳六烯酸。还涵盖非天然物质，包含具有包括分支、氧化、环化和炔烃的改性和取代的天然物质。例如，磷脂可以用一个或多个炔烃(例如,一个或多个双键被三键置换的烯基)官能化或与其交联。在适当反应条件下，炔基可在暴露于叠氮化物时进行铜催化的环加成。此类反应可适用于官能化纳米粒子组合物的脂质双层以促进膜渗透或细胞识别，或适用于将纳米粒子组合物与有用组分，如靶向或成像部分(例如,染料)缀合。In some embodiments, one or more phospholipids can be assembled into one or more lipid bilayers. In some embodiments, one or more phospholipids can include a phospholipid moiety. In some embodiments, one or more phospholipids can include one or more fatty acid moieties. In some embodiments, one or more phospholipids can include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, the phospholipid moiety includes but is not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. In some embodiments, the fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural substances are also contemplated, including natural substances with modifications and substitutions including branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkyne group can undergo a copper-catalyzed cycloaddition when exposed to an azide. Such reactions can be useful for functionalizing the lipid bilayer of a nanoparticle composition to facilitate membrane penetration or cell recognition, or for conjugating a nanoparticle composition with a useful component, such as a targeting or imaging moiety (e.g., a dye).

示例性磷脂包含但不限于1,2-二硬脂酰基-sn甘油-3-磷酸胆碱(DSPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二亚油酰基-sn-甘油-3-磷酸胆碱(DLPC)、1,2-二肉豆蔻酰基-sn-甘油磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-双十一烷酰基-sn-甘油磷酸胆碱(DUPC)、l-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二-O-十八碳烯基-sn-甘油-3-磷酸胆碱(18:0二醚PC)、1-油酰基-2-胆甾醇半琥珀酰基l-sn-甘油-3-磷酸胆碱(OChemsPC)、1-十六烷基sn甘油-3-磷酸胆碱(C16 Lyso PC)、1,2-二亚麻酰基-sn-甘油-3-磷酸胆碱、1,2-二花生四烯酰基-sn-甘油-3-磷酸胆碱、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸胆碱、1,2-二植烷酰基-sn-甘油-3-磷酸乙醇胺(ME 16.0PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚油酰基-sn-甘油-3-磷酸乙醇胺、1,2-二亚麻酰基-sn-甘油-3-磷酸乙醇胺、1,2-二花生四烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-双二十二碳六烯酰基-sn-甘油-3-磷酸乙醇胺、1,2-二油酰基-sn-甘油-3-磷酰-rac-(1-甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基-磷脂酰-乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)、1-硬脂酰基-2油酰基磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)或它们的组合。在一些实施例中，磷脂为DSPC。在一些实施例中，磷脂为DMPC。Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestyrene hemisuccinyl l-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oil Phosphatidylcholine (POPE), phosphatidylcholine (DSPE), phosphatidylethanolamine (DPPE), phosphatidylethanolamine (DMPE), phosphatidylcholine (SOPE), phosphatidylethanolamine (SOPC), phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC.

在一些实施例中，磷脂包括1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-(琥珀酰基)(琥珀酰基PE)、1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、胆甾醇、1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺(DSPE)、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-(琥珀酰基)(琥珀酰基-DPPE)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱(DMPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)或它们的组合。In some embodiments, the phospholipid includes 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

G.直径G. Diameter

此外，本公开描述了平均流体动力学直径为约30至约220nm的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的平均流体动力学直径为约30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm、150nm、155nm、160nm、165nm、170nm、175nm、180nm、185nm、190nm、195nm、200nm、205nm、210nm、215nm、220nm或具有由前述各值中的任何两个限定的端点的任何范围。例如，在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的平均流体动力学直径在50nm到200nm之间。In addition, the present disclosure describes compositions, formulations, nanoparticles and/or nanomaterials having an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the average hydrodynamic diameter of the compositions, formulations, nanoparticles and/or nanomaterials described herein is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, the compositions, formulations, nanoparticles, and/or nanomaterials described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

在一些实施例中，本文所述的脂质纳米粒子的平均流体动力学直径为约30至约220nm。在一些实施例中，本文所述的脂质纳米粒子的平均流体动力学直径为约30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm、150nm、155nm、160nm、165nm、170nm、175nm、180nm、185nm、190nm、195nm、200nm、205nm、210nm、215nm、220nm或具有由前述各值中的任何两个值限定的端点的任何范围。例如，在一些实施例中，本文所述的脂质纳米粒子的平均流体动力学直径在50nm与200nm之间。In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein is about 30 to about 220 nm. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein is between 50 nm and 200 nm.

H.多分散性H. Polydispersity

此外，本公开描述多分散指数(PDI)为约0.01至约0.3的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的PDI为约0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.15、0.2、0.25、0.3或具有由前述各值中的任何两个限定的端点的任何范围。例如，在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的PDI为约0.05至约0.2、约0.06至约0.1或约0.07至约0.09。In addition, the present disclosure describes a composition, formulation, nanoparticle and/or nanomaterial having a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, the PDI of the composition, formulation, nanoparticle and/or nanomaterial described herein is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3 or any range with endpoints defined by any two of the aforementioned values. For example, in some embodiments, the PDI of the composition, formulation, nanoparticle and/or nanomaterial described herein is about 0.05 to about 0.2, about 0.06 to about 0.1 or about 0.07 to about 0.09.

在一些实施例中，本文所述的脂质纳米粒子的PDI为约0.01至约0.3。在一些实施例中，本文所述的脂质纳米粒子的PDI为约0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.15、0.2、0.25、0.3或具有由前述各值中的任何两个限定的端点的任何范围。例如，在一些实施例中，本文所述的脂质纳米粒子的PDI为约0.05至约0.2、约0.06至约0.1、或约0.07至约0.09。In some embodiments, the PDI of the lipid nanoparticles described herein is about 0.01 to about 0.3. In some embodiments, the PDI of the lipid nanoparticles described herein is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, the PDI of the lipid nanoparticles described herein is about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

I.包裹效率I. Packaging efficiency

此外，本公开描述了组合物、制剂、纳米粒子和/或纳米材料，其中所提供的组合物、制剂、纳米粒子和/或纳米材料的包裹效率为约80％至约100％。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的包裹效率为约80％、85％、90％、91％、92％、93％、94％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％、100％或具有由前述各值中的任何两个值定义的端点的任何范围。例如，在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的包裹效率为约90％至约100％、约95％至约100％、约95％至约98％或约95.5％至约97.5％。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的包裹效率为至少约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。In addition, the present disclosure describes compositions, preparations, nanoparticles and/or nanomaterials, wherein the encapsulation efficiency of the compositions, preparations, nanoparticles and/or nanomaterials provided is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles and/or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% or any range with endpoints defined by any two values of the aforementioned values. For example, in some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles and/or nanomaterials described herein is about 90% to about 100%, about 95% to about 100%, about 95% to about 98% or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the compositions, formulations, nanoparticles and/or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

在一些实施例中，本文所述的脂质纳米粒子的包裹效率为约80％至约100％。在一些实施例中，本文所述的脂质纳米粒子的包裹效率为约80％、85％、90％、91％、92％、93％、94％、95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％、99.5％、100％或具有由前述各值中的任何两个值所限定的端点的任何范围。例如，在一些实施例中，本文所述的脂质纳米粒子的包裹效率为约90％至约100％、约95％至约100％、约95％至约98％或约95.5％至约97.5％。在一些实施例中，本文所述的脂质纳米粒子的包裹效率为至少约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％。In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100% or any range of endpoints defined by any two values of the aforementioned values. For example, in some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 90% to about 100%, about 95% to about 100%, about 95% to about 98% or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

J.pKaJ.pKa

此外，本公开描述pKa为约5至约9的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的pKa为约5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5或具有由前述各值中的任何两个所限定的端点的任何范围。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料的pKa为约6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0或具有由前述各值中的任何两个所限定的端点的任何范围。Additionally, the present disclosure describes compositions, formulations, nanoparticles, and/or nanomaterials having a pKa of about 5 to about 9. In some embodiments, the pKa of the compositions, formulations, nanoparticles, and/or nanomaterials described herein is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints bounded by any two of the foregoing values. In some embodiments, the pKa of the compositions, formulations, nanoparticles, and/or nanomaterials described herein is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints bounded by any two of the foregoing values.

在一些实施例中，本文所述的脂质纳米粒子的pKa为约5至约9。在一些实施例中，本文所述的脂质纳米粒子的pKa为约5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5或具有由前述各值中的任何两个所限定的端点的任何范围。在一些实施例中，本文所述的脂质纳米粒子的pKa为约6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0或具有由前述各值中的任何两个所限定的端点的任何范围。In some embodiments, the pKa of the lipid nanoparticles described herein is about 5 to about 9. In some embodiments, the pKa of the lipid nanoparticles described herein is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the foregoing values. In some embodiments, the pKa of the lipid nanoparticles described herein is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or any range having endpoints defined by any two of the foregoing values.

II.示范性LNP制剂II. Exemplary LNP Formulations

本发明提供包括脂质纳米粒子的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，脂质纳米粒子制剂包括约30摩尔％至约70摩尔％的可电离脂质、约5摩尔％至约25摩尔％的磷脂、约25摩尔％至约45摩尔％的胆甾醇和约0摩尔％至约5摩尔％的缀合物-连接子脂质。The present invention provides compositions, preparations, nanoparticles and/or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle preparation comprises about 30 mol % to about 70 mol % of ionizable lipids, about 5 mol % to about 25 mol % of phospholipids, about 25 mol % to about 45 mol % of cholesterol and about 0 mol % to about 5 mol % of conjugate-linker lipids.

在一些实施例中，脂质纳米粒子制剂包括约45摩尔％的可电离脂质、约9摩尔％的磷脂、约44摩尔％的胆甾醇和约2摩尔％的缀合物-连接子脂质。在一些实施例中，脂质纳米粒子制剂包括约50摩尔％的可电离脂质、约9摩尔％的磷脂、约38摩尔％的胆甾醇和约3摩尔％的缀合物-连接子脂质。In some embodiments, the lipid nanoparticle formulation comprises about 45 mol % ionizable lipid, about 9 mol % phospholipid, about 44 mol % cholesterol and about 2 mol % conjugate-linker lipid. In some embodiments, the lipid nanoparticle formulation comprises about 50 mol % ionizable lipid, about 9 mol % phospholipid, about 38 mol % cholesterol and about 3 mol % conjugate-linker lipid.

在一些实施例中，脂质纳米粒子制剂包括基于以下四种成分的总摩尔数计约40摩尔％至约60摩尔％的任何所提供的化合物的可电离脂质、约5摩尔％至约15摩尔％的1-2-二硬脂酰基-sn-甘油-3-磷酸胆碱、约1摩尔％至约5摩尔％的C14PEG2000和约30摩尔％至约47摩尔％的胆甾醇。In some embodiments, the lipid nanoparticle formulation includes about 40 mol % to about 60 mol % of an ionizable lipid of any provided compound, about 5 mol % to about 15 mol % of 1-2-distearoyl-sn-glycero-3-phosphocholine, about 1 mol % to about 5 mol % of C14PEG2000, and about 30 mol % to about 47 mol % of cholesterol, based on the total moles of the following four components.

在一些实施例中，脂质纳米粒子(LNP)制剂包括质量比为约2:1至50:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):mRNA。在一些实施例中，LNP制剂包括质量比为约2:1、约3:1、约4:1、约5:1、约6:1、约7:1、约8:1、约9:1、约10:1、约11:1、约12:1、约13:1、约14:1、约15:1、约16:1、约17:1、约18:1、约19:1、约20:1、约21:1、约22:1、约23:1、约24:1、约25:1、约26:1、约27:1、约28:1、约29:1、约30:1、约31:1、约32:1、约33:1、约34:1、约35:1、约36:1、约37:1、约38:1、约39:1、约40:1、约41:1、约42:1、约43:1、约44:1、约45:1、约46:1、约47:1、约48:1、约49:1、约50:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):mRNA。在一些实施例中，脂质纳米粒子(LNP)制剂包括质量比为约11.7:1及19:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):mRNA。In some embodiments, the lipid nanoparticle (LNP) formulation comprises (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA in a mass ratio of about 2: 1 to 50: 1. In some embodiments, the LNP formulation comprises a mass ratio of about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21: 1, about 22: 1, about 23: 1, about 24: 1, about 25: 1, about 26: 1, about 27: 1 , about 28: 1, about 29: 1, about 30: 1, about 31: 1, about 32: 1, about 33: 1, about 34: 1, about 35: 1, about 36: 1, about 37: 1, about 38: 1, about 39: 1, about 40: 1, about 41: 1, about 42: 1, about 43: 1, about 44: 1, about 45: 1, about 46: 1, about 47: 1, about 48: 1, about 49: 1, about 50: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA. In some embodiments, the lipid nanoparticle (LNP) formulation comprises a mass ratio of about 11.7: 1 and 19: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA.

在一些实施例中，脂质纳米粒子制剂包括质量比为约2:1至50:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):siRNA。在一些实施例中，LNP制剂包括质量比为约2:1、约3:1、约4:1、约5:1、约6:1、约7:1、约8:1、约9:1、约10:1、约11:1、约12:1、约13:1、约14:1、约15:1、约16:1、约17:1、约18:1、约19:1、约20:1、约21:1、约22:1、约23:1、约24:1、约25:1、约26:1、约27:1、约28:1、约29:1、约30:1、约31:1、约32:1、约33:1、约34:1、约35:1、约36:1、约37:1、约38:1、约39:1、约40:1、约41:1、约42:1、约43:1、约44:1、约45:1、约46:1、约47:1、约48:1、约49:1、约50:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):mRNA。在一些实施例中，脂质纳米粒子(LNP)制剂包括质量比为约11.7:1及19:1的(可电离脂质、胆甾醇、脂质-PEG和磷脂):mRNA。In some embodiments, the lipid nanoparticle formulation comprises a mass ratio of about 2: 1 to 50: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): siRNA. In some embodiments, the LNP formulation comprises a mass ratio of about 2: 1, about 3: 1, about 4: 1, about 5: 1, about 6: 1, about 7: 1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 21: 1, about 22: 1, about 23: 1, about 24: 1, about 25: 1, about 26: 1, about 27: 1 , about 28: 1, about 29: 1, about 30: 1, about 31: 1, about 32: 1, about 33: 1, about 34: 1, about 35: 1, about 36: 1, about 37: 1, about 38: 1, about 39: 1, about 40: 1, about 41: 1, about 42: 1, about 43: 1, about 44: 1, about 45: 1, about 46: 1, about 47: 1, about 48: 1, about 49: 1, about 50: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA. In some embodiments, the lipid nanoparticle (LNP) formulation comprises a mass ratio of about 11.7: 1 and 19: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA.

在一些实施例中，LNP制剂包含一种或多种核酸，诸如RNA。在一些实施例中，可以选择LNP制剂中的一种或多种核酸(例如，RNA)、脂质及其量以提供特定的N/P比。N/P比可以选自约1至约30。N/P比可以选自约2至约12。在一些实施例中，N/P比为约0.1至约50。在一些实施例中，N/P比为约2至约8。在一些实施例中，N/P比为约2至约15、约2至约10、约2至约8、约2至约6、约3至约15、约3至约10、约3至约8、约3至约6、约4至约15、约4至约10、约4至约8、约4至约6或约5至约7。在一些实施例中，N/P比为约2、约2.5、约3、约3.5、约4、约4.5、约5、约5.5、约6、约6.5、约7、约7.5、约8、约9或约10。在一些实施例中，N/P比为约4至约6。在一些实施例中，N/P比为约4、约4.5、约5、约5.5或约6。In some embodiments, the LNP formulation comprises one or more nucleic acids, such as RNA. In some embodiments, one or more nucleic acids (e.g., RNA), lipids and their amounts in the LNP formulation can be selected to provide a specific N/P ratio. The N/P ratio can be selected from about 1 to about 30. The N/P ratio can be selected from about 2 to about 12. In some embodiments, the N/P ratio is about 0.1 to about 50. In some embodiments, the N/P ratio is about 2 to about 8. In some embodiments, the N/P ratio is about 2 to about 15, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 15, about 3 to about 10, about 3 to about 8, about 3 to about 6, about 4 to about 15, about 4 to about 10, about 4 to about 8, about 4 to about 6 or about 5 to about 7. In some embodiments, the N/P ratio is about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 9 or about 10. In some embodiments, the N/P ratio is about 4 to about 6. In some embodiments, the N/P ratio is about 4, about 4.5, about 5, about 5.5, or about 6.

如本文中所使用，“N/P比”是一种脂质(或多种脂质)中的可电离(例如，在生理pH范围内)氮原子与一个核酸分子实体(或多个核酸分子实体)中的磷酸基团的摩尔比，例如，在包含脂质组分和RNA的纳米粒子组合物中。可电离氮原子可以包含例如可以在约pH 1、约pH 2、约pH 3、约pH 4、约pH 4.5、约pH 5、约pH 5.5、约pH6、约pH 6.5、约pH 7、约pH 7.5或约pH 8或更高下被质子化的氮原子。生理pH范围可以包含例如不同细胞区室(诸如器官、组织和细胞)和体液(诸如血液、CSF、胃液、乳汁、胆汁、唾液、眼泪和尿液)的pH范围。在某些具体实施例中，生理pH范围是指哺乳动物的血液的pH范围，例如，约7.35至约7.45。类似地，对于具有一个或多个可电离氮原子的磷酸盐电荷中和剂，N/P比可以是指该磷酸盐电荷中和剂中的可电离氮原子与核酸中的磷酸基团的摩尔比。在一些实施例中，可电离氮原子是指在5至14之间的pH范围内可电离的那些氮原子。As used herein, "N/P ratio" is the molar ratio of ionizable (e.g., within the physiological pH range) nitrogen atoms in a lipid (or multiple lipids) to phosphate groups in a nucleic acid molecule entity (or multiple nucleic acid molecule entities), for example, in a nanoparticle composition comprising a lipid component and RNA. Ionizable nitrogen atoms can include, for example, nitrogen atoms that can be protonated at about pH 1, about pH 2, about pH 3, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, or about pH 8 or higher. Physiological pH ranges can include, for example, pH ranges of different cell compartments (such as organs, tissues, and cells) and body fluids (such as blood, CSF, gastric juice, milk, bile, saliva, tears, and urine). In certain specific embodiments, physiological pH ranges refer to the pH range of mammalian blood, for example, from about 7.35 to about 7.45. Similarly, for a phosphate charge neutralizer having one or more ionizable nitrogen atoms, the N/P ratio can refer to the molar ratio of ionizable nitrogen atoms in the phosphate charge neutralizer to phosphate groups in the nucleic acid. In some embodiments, ionizable nitrogen atoms refer to those nitrogen atoms that are ionizable within a pH range of 5 to 14.

III.药物组合物III. Pharmaceutical Compositions

本发明提供包括药物组合物的组合物、制剂、纳米粒子和/或纳米材料。此外，在一些实施例中，药物组合物包括本文所述的脂质纳米粒子和脂质纳米粒子制剂。例如，在一些实施例中，本文所述的脂质纳米粒子和脂质纳米粒子制剂可整体或部分调配为药物组合物。The present invention provides compositions, preparations, nanoparticles and/or nanomaterials including pharmaceutical compositions. In addition, in some embodiments, the pharmaceutical compositions include lipid nanoparticles and lipid nanoparticle preparations described herein. For example, in some embodiments, the lipid nanoparticles and lipid nanoparticle preparations described herein can be formulated as pharmaceutical compositions in whole or in part.

在一些实施例中，药物组合物可包含本文所述的一种或多种纳米粒子组合物。例如，药物组合物可包括一种或多种纳米粒子组合物(包含一种或多种不同的治疗剂和/或预防剂，包含但不限于一种或多种不同类型的核酸)或编码不同药剂。在一些实施例中，药物组合物包括一种或多种药学上可接受的赋形剂或助剂，包含但不限于药学上可接受的载剂。In some embodiments, the pharmaceutical composition may include one or more nanoparticle compositions described herein. For example, the pharmaceutical composition may include one or more nanoparticle compositions (including one or more different therapeutic and/or preventive agents, including but not limited to one or more different types of nucleic acids) or encode different agents. In some embodiments, the pharmaceutical composition includes one or more pharmaceutically acceptable excipients or adjuvants, including but not limited to pharmaceutically acceptable carriers.

可以向受试者施用药物组合物。在一些实施例中，如本文所述施用药物组合物。在一些体内方法中，以如本文所述的治疗有效量向受试者施用本文所公开的纳米粒子组合物。The pharmaceutical composition can be administered to a subject. In some embodiments, the pharmaceutical composition is administered as described herein. In some in vivo methods, the nanoparticle composition disclosed herein is administered to a subject in a therapeutically effective amount as described herein.

在一些实施例中，考虑到接受者的治疗表环境、年龄和一般健康状况，普通技术人员将能够使用本文所述的药物组合物设计适当剂量水平和给药方案以治疗各种患者的各种病状。例如，在一些实施例中，所选择的剂量取决于所需的治疗效果、施用途径和所需治疗的持续时间。在一些实施例中，通常以每剂每公斤体重约0.001mg至约5mg核酸的剂量水平向哺乳动物施用。更具体地说，在一些实施例中，所公开的纳米粒子内核酸的优选剂量为约0.1mg/kg至约1.0mg/kg。对于所公开的纳米粒子，通常将每公斤体重约0.2mg至约100mg四种组分(可电离脂质、胆甾醇、缀合物-连接缀合物和磷脂)的剂量水平施加至哺乳动物。更具体地说，在一些实施例中，所公开的纳米粒子的优选剂量为每公斤体重约0.5mg/kg至约5mg/kg四种组分。In some embodiments, taking into account the recipient's treatment environment, age and general health, ordinary technicians will be able to use the pharmaceutical compositions described herein to design appropriate dosage levels and dosage regimens to treat various conditions of various patients. For example, in some embodiments, the selected dosage depends on the desired therapeutic effect, route of administration and the duration of the desired treatment. In some embodiments, it is usually administered to mammals at a dosage level of about 0.001 mg to about 5 mg of nucleic acid per kg of body weight per dose. More specifically, in some embodiments, the preferred dosage of nucleic acid in the disclosed nanoparticles is about 0.1 mg/kg to about 1.0 mg/kg. For the disclosed nanoparticles, dosage levels of about 0.2 mg to about 100 mg of four components (ionizable lipids, cholesterol, conjugate-connected conjugates and phospholipids) per kg of body weight are usually applied to mammals. More specifically, in some embodiments, the preferred dosage of the disclosed nanoparticles is about 0.5 mg/kg to about 5 mg/kg of four components per kg of body weight.

在一些实施例中，局部施用本文所述的药物组合物，例如通过直接注射到待治疗部位中施用。通常，注射引起组合物的局部浓度增加，其大于可以通过全身性施用实现的局部浓度。在一些实施例中，本文所述的药物组合物可与本文所述的基质组合，以通过减少多肽被动扩散出待治疗部位来帮助产生局部浓度增加的多肽组合物。In some embodiments, the pharmaceutical compositions described herein are administered topically, for example, by direct injection into the site to be treated. Typically, injection causes an increase in the local concentration of the composition that is greater than the local concentration that can be achieved by systemic administration. In some embodiments, the pharmaceutical compositions described herein can be combined with a matrix as described herein to help produce a polypeptide composition with an increased local concentration by reducing passive diffusion of the polypeptide out of the site to be treated.

A.用于肠胃外施用的制剂A. Formulations for Parenteral Administration

在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料(包含含有脂质纳米粒子的组合物、制剂、纳米粒子和/或纳米材料)以水溶液形式通过肠胃外注射施用。在一些实施例中，制剂还可呈悬浮液或乳液的形式。一般来说，提供药物组合物，其包含有效量的脂质纳米粒子，并且任选地包含药学上可接受的稀释剂、防腐剂、增溶剂、乳化剂、佐剂和/或载剂。此类组合物任选地包含以下的一者或多者：稀释剂、无菌水、各种缓冲液含量(例如，Tris-HCl、乙酸盐、磷酸盐)、pH和离子强度的缓冲盐水；以及添加剂，诸如洗涤剂和增溶剂(例如，TWEEN 20(聚山梨酯-20)、TWEEN 80(聚山梨酯-80))、抗氧化剂(例如，抗坏血酸、焦亚硫酸钠)和防腐剂(例如，硫柳汞、苯甲醇)以及填充物质(例如，乳糖、甘露醇)。非水溶剂或媒剂的实例为丙二醇；聚乙二醇；植物油，如橄榄油和玉米油；明胶；以及可注射有机酯，如油酸乙酯。调配物可以冻干并在即将使用之前再溶解/再悬浮。可以通过例如通过细菌截留过滤器过滤、通过将灭菌剂并入到组合物中、通过照射组合物或通过加热组合物对调配物灭菌。In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein (compositions, formulations, nanoparticles and/or nanomaterials containing lipid nanoparticles) are administered by parenteral injection in the form of aqueous solutions. In some embodiments, the formulations may also be in the form of suspensions or emulsions. In general, a pharmaceutical composition is provided, which comprises an effective amount of lipid nanoparticles, and optionally comprises a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and/or carrier. Such compositions optionally include one or more of the following: diluent, sterile water, buffered saline of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium pyrosulfite) and preservatives (e.g., thimerosal, benzyl alcohol) and filler materials (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol; polyethylene glycol; vegetable oils, such as olive oil and corn oil; gelatin; and injectable organic esters, such as ethyl oleate. The formulation can be lyophilized and redissolved/resuspended immediately prior to use. The formulation can be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

B.控制递送聚合基质B. Controlled Delivery Polymeric Matrices

在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料还可以控制释放调配物形式施用。在一些实施例中，可以制造控制释放聚合装置，用于在植入聚合装置(如棒、圆柱、膜、盘)或注射(如微米粒子)之后长期全身性地释放。在一些实施例中，基质可呈微米粒子(如微球)的形式。在一些实施例中，药剂分散于固体聚合基质或微胶囊内。在一些实施例中，核是与所描述的组合物、制剂、纳米粒子和/或纳米材料中的任一者的聚合物壳不同的材料。在一些实施例中，肽分散或悬浮于所描述的组合物、制剂、纳米粒子和/或纳米材料中的任一种的核中，核本质上可以是液体或固体。除非本文中特别地定义，否则微米粒子、微球和微胶囊可互换地使用。在一些实施例中，聚合物可以浇铸成范围为几纳米到四厘米的薄板或薄膜、通过研磨或其它标准技术产生的粉末或甚至凝胶(如水凝胶)。In some embodiments, the compositions, preparations, nanoparticles and/or nanomaterials disclosed herein can also be administered in the form of controlled release formulations. In some embodiments, controlled release polymeric devices can be manufactured for long-term systemic release after implantation of polymeric devices (such as rods, cylinders, membranes, disks) or injections (such as microparticles). In some embodiments, the matrix can be in the form of microparticles (such as microspheres). In some embodiments, the agent is dispersed in a solid polymeric matrix or microcapsule. In some embodiments, the core is a material different from the polymer shell of any one of the described compositions, preparations, nanoparticles and/or nanomaterials. In some embodiments, the peptide is dispersed or suspended in the core of any one of the described compositions, preparations, nanoparticles and/or nanomaterials, and the core can be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres and microcapsules can be used interchangeably. In some embodiments, the polymer can be cast into a thin plate or film ranging from a few nanometers to four centimeters, a powder or even a gel (such as a hydrogel) produced by grinding or other standard techniques.

在一些实施例中，不可生物降解的基质用于递送所描述的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，可生物降解的基质用于递送所描述的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，可生物降解的基质为优选的。在一些实施例中，可生物降解的基质包括天然或合成聚合物。在一些实施例中，由于能更好地表征降解和释放曲线，所以合成聚合物是优选的。在一些实施例中，基于期望释放的时间段来选择聚合物。在一些实施例中，线性释放可以是最有用的，但在其它实施例中，脉冲释放或“批量释放(bulkrelease)”可以提供更有效的结果。在一些实施例中，聚合物可呈水凝胶(通常吸收高达约90重量％的水)的形式，且可任选地与多价离子或聚合物交联。In some embodiments, non-biodegradable substrates are used to deliver the described compositions, preparations, nanoparticles and/or nanomaterials. In some embodiments, biodegradable substrates are used to deliver the described compositions, preparations, nanoparticles and/or nanomaterials. In some embodiments, biodegradable substrates are preferred. In some embodiments, biodegradable substrates include natural or synthetic polymers. In some embodiments, synthetic polymers are preferred because they can better characterize degradation and release curves. In some embodiments, polymers are selected based on the time period of the desired release. In some embodiments, linear release can be the most useful, but in other embodiments, pulse release or "bulk release" can provide more effective results. In some embodiments, polymers can be in the form of hydrogels (usually absorbing up to about 90% by weight of water), and can be optionally cross-linked with multivalent ions or polymers.

基质可以通过溶剂蒸发、喷雾干燥、溶剂萃取以及本领域的普通技术人员已知的其它方法形成。可生物蚀解的微球可以使用为制备用于药物递送的微球而开发的方法中的任何方法来制备，例如，如Mathiowitz和Langer,J.Controlled Release,5:13-22(1987)；Mathiowitz等人，Reactive Polymers,6:275-283(1987)；和Mathiowitz等人，J.Appl.Polymer Sci.,35:755-774(1988)中所述，其公开内容通过引用整体并入本文。The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those of ordinary skill in the art. Bioerodible microspheres can be prepared using any of the methods developed for preparing microspheres for drug delivery, for example, as described in Mathiowitz and Langer, J. Controlled Release, 5: 13-22 (1987); Mathiowitz et al., Reactive Polymers, 6: 275-283 (1987); and Mathiowitz et al., J. Appl. Polymer Sci., 35: 755-774 (1988), the disclosures of which are incorporated herein by reference in their entirety.

在一些实施例中，所述组合物、制剂、纳米粒子和/或纳米材料可以调配成用于局部释放以治疗植入或注射区域，这将通常递送比用于治疗整个身体或全身递送的剂量小得多的剂量。这些可以植入或皮下注射到肌肉、脂肪中或被吞咽。In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials can be formulated for local release to treat an implanted or injected area, which will typically deliver a much smaller dose than that used to treat the entire body or systemic delivery. These can be implanted or injected subcutaneously into muscle, fat or swallowed.

C.货物C. Goods

此外，本发明提供包括如本文所述的货物的组合物、制剂、纳米粒子和/或纳米材料。在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包含用于递送到受试者的治疗剂或预防剂。在一些实施例中，治疗剂或预防剂被脂质纳米粒子包裹。在一些实施例中，脂质纳米粒子负载有一种或多种核酸。In addition, the present invention provides compositions, preparations, nanoparticles and/or nanomaterials comprising goods as described herein. In some embodiments, compositions, preparations, nanoparticles and/or nanomaterials comprise therapeutic agents or prophylactic agents for delivery to a subject. In some embodiments, the therapeutic agent or prophylactic agent is wrapped by lipid nanoparticles. In some embodiments, the lipid nanoparticles are loaded with one or more nucleic acids.

D.治疗剂和/或预防剂D. Therapeutic and/or preventive agents

通过LNP组合物递送的货物可以是生物活性剂。在一些实施例中，货物为或包含一种或多种生物活性剂，如mRNA、向导RNA(gRNA)、核酸、RNA导向的DNA结合剂、表达载体、模板核酸、抗体(例如，单克隆抗体、嵌合抗体、人源化抗体、纳米抗体及其片段等)、胆甾醇、激素、肽、蛋白质、化学治疗剂和其它类型的抗肿瘤剂、低分子量药物、维生素、辅因子、核苷、核苷酸、寡核苷酸、酶性核酸、反义核酸、三链体形成寡核苷酸、反义DNA或RNA组合物、嵌合DNA:RNA组合物、同种异型酶、适体、核糖酶、陷阱(decoy)及其类似物、质粒和其它类型的体、及小核酸分子、RNAi剂、短干扰核酸酸(siNA)、短干扰RNA(siRNA)、双链RNA(dsRNA)、微小RNA(miRNA)、短发夹RNA(shRNA)和“自我复制RNA”(编码复制酶活性并能够引导其自身在体内复制或扩增)分子、肽核酸(PNA)、锁核酸核糖核苷酸(LNA)、吗啉代核苷酸、苏糖核酸(TNA)、乙二醇核酸(GNA)、sisiRNA(小型内部分段干扰RNA)以及iRNA(不对称干扰RNA)。以上生物活性剂列表仅为示范性的，且并不旨在是限制性的。此类化合物可以经纯化或部分纯化，并且可以是天然存在或合成的，并且可以经化学改性。The cargo delivered by the LNP composition can be a bioactive agent. In some embodiments, the cargo is or comprises one or more bioactive agents, such as mRNA, guide RNA (gRNA), nucleic acid, RNA-guided DNA binding agent, expression vector, template nucleic acid, antibody (e.g., monoclonal antibody, chimeric antibody, humanized antibody, nanobody and fragments thereof, etc.), cholesterol, hormone, peptide, protein, chemotherapeutic agent and other types of anti-tumor agent, low molecular weight drug, vitamin, cofactor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA: RNA composition, allozyme, aptamer, ribozyme, trap (dec oy) and its analogs, plasmids and other types of bodies, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and "self-replicating RNA" (encoding replicase activity and capable of guiding its own replication or amplification in vivo) molecules, peptide nucleic acids (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acids (TNA), glycol nucleic acids (GNA), sisiRNA (small internal segmented interfering RNA) and iRNA (asymmetric interfering RNA). The above list of bioactive agents is exemplary only and is not intended to be limiting. Such compounds can be purified or partially purified, and can be naturally occurring or synthesized, and can be chemically modified.

通过LNP组合物递送的货物可以是RNA，例如编码所关注蛋白质的mRNA分子。例如，在一些实施例中，本文描述用于表达如绿色荧光蛋白(GFP)、RNA引导的DNA结合剂或Cas核酸酶等蛋白质的mRNA。提供LNP组合物，其包含Cas核酸酶mRNA，例如允许在2类Cas核酸酶(如Cas9或Cpf1蛋白)的细胞中表达的2类Cas核酸酶mRNA。此外，货物可以含有一种或多种编码向导RNA的向导RNA或核酸。模板核酸(例如用于修复或重组)还可以包含在组合物中，或模板核酸可以用于本文所述的方法中。在一些实施例中，货物包含编码任选的化脓性链球菌Cas9和化脓性链球菌gRNA的mRNA。在一些实施例中，货物包含编码任选的脑膜炎奈瑟菌Cas9和nme gRNA的mRNA。The goods delivered by the LNP composition can be RNA, such as mRNA molecules encoding proteins of interest. For example, in some embodiments, mRNA for expressing proteins such as green fluorescent protein (GFP), RNA-guided DNA binders or Cas nucleases is described herein. LNP compositions are provided, which include Cas nuclease mRNA, such as 2 types of Cas nuclease mRNAs allowed to be expressed in cells of 2 types of Cas nucleases (such as Cas9 or Cpf1 proteins). In addition, goods can contain one or more guide RNAs or nucleic acids encoding guide RNAs. Template nucleic acids (such as for repair or reorganization) can also be included in the composition, or template nucleic acids can be used in the methods described herein. In some embodiments, goods include mRNA encoding optional Streptococcus pyogenes Cas9 and Streptococcus pyogenes gRNA. In some embodiments, goods include mRNA encoding optional Neisseria meningitidis Cas9 and nme gRNA.

“mRNA”是指多核苷酸并且包括可以翻译成多肽的开放阅读框架(即，可以用作通过核糖体和氨基酰化tRNA进行翻译的底物)。mRNA可以包含磷酸-糖主链，其包含核糖残基或其类似物，例如，2'-甲氧基核糖残基。在一些实施例中，mRNA磷酸-糖主链的糖基本上由核糖残基、2'-甲氧基核糖残基或其组合组成。一般来说，mRNA不含有大量的胸苷残基(例如，0个残基或少于30个、20个、10个、5个、4个、3个或2个胸苷残基；或小于10％、9％、8％、7％、6％、5％、4％、4％、3％、2％、1％、0.5％、0.2％或0.1％的胸苷含量)。mRNA可以在其一些或全部尿苷位置含有修饰的尿苷。"mRNA" refers to a polynucleotide and includes an open reading frame that can be translated into a polypeptide (i.e., can be used as a substrate for translation by ribosomes and aminoacylated tRNA). mRNA can contain a phosphate-sugar backbone comprising a ribose residue or an analog thereof, for example, a 2'-methoxyribose residue. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of a ribose residue, a 2'-methoxyribose residue, or a combination thereof. In general, mRNA does not contain a large amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3 or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1% thymidine content). mRNA can contain modified uridines at some or all of its uridine positions.

E.CRISPR/Cas货物E. CRISPR/Cas cargo

在一些实施例中，所公开的组合物、制剂、纳米粒子和/或纳米材料包括编码RNA引导的DNA结合剂(如Cas核酸酶)的mRNA。在特定实施例中，所公开的组合物、制剂、纳米粒子和/或纳米材料包括编码2类Cas核酸酶(如化脓性链球菌Cas9)的mRNA。In some embodiments, the disclosed compositions, formulations, nanoparticles and/or nanomaterials include mRNA encoding RNA-guided DNA binders (such as Cas nucleases). In specific embodiments, the disclosed compositions, formulations, nanoparticles and/or nanomaterials include mRNA encoding Class 2 Cas nucleases (such as Streptococcus pyogenes Cas9).

如本文所使用，“RNA引导的DNA结合剂”意指具有RNA和DNA结合活性的多肽或多肽复合物，或此类复合物的DNA结合亚单元，其中DNA结合活性具有序列特异性并取决于RNA的序列。示范性RNA引导的DNA结合剂包含Cas切割酶/切口酶及其失活形式(“dCas DNA结合剂”)。如本文所使用，“Cas核酸酶”涵盖Cas切割酶、Cas切口酶和dCas DNA结合剂。Cas切割酶/切口酶和dCas DNA结合剂包含III型CRISPR系统的Csm或Cmr复合物、其CaslO、Csm1或Cmr2亚单位、I型CRISPR系统的级联复合物、其Cas3亚单位和2类Cas核酸酶。如本文所使用，“2类Cas核酸酶”是具有RNA引导的DNA结合活性的单链多肽。2类Cas核酸酶包含：2类Cas切割酶/切口酶(例如，H840A、D10A或N863 A变体)，所述切割酶/切口酶进一步具有RNA引导的DNA切割酶或切口酶活性；以及2类dCas DNA结合剂，其中切割酶/切口酶活性失活。2类Cas核酸酶包含例如Cas9、Cpf1、C2c1、C2c2、C2c3、HF Cas9(例如，N497A、R661A、Q695A、Q926A变体)、HypaCas9(例如，N692A、M694A、Q695A、H698A变体)、eSPCas9(1.0)(例如，K810A、K1003A、R1060A变体)和eSPCas9(1.1)(例如，K848A、K1003A、R1060A变体)蛋白及其修饰。Cpf1蛋白(Zetsche等人，Cell,163:1-13(2015))与Cas9同源，并含有RuvC样核酸酶域。Zetsche的Cpf1序列通过全文引用的方式并入本文中。参见例如，Zetsche，表S1和S3。参见例如，Makarova等人，Nat Rev Microbiol,13(11):722-36(2015)；Shmakov等人，MolecularCell,60:385-397(2015)，其内容整体并入本文。As used herein, "RNA-guided DNA binders" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders include Cas cleavage enzymes/nickases and inactivated forms thereof ("dCas DNA binders"). As used herein, "Cas nucleases" encompass Cas cleavage enzymes, Cas nickases, and dCas DNA binders. Cas cleavage enzymes/nickases and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, Cas10, Csm1, or Cmr2 subunits thereof, Cascade complexes of type I CRISPR systems, Cas3 subunits thereof, and class 2 Cas nucleases. As used herein, "class 2 Cas nucleases" are single-chain polypeptides with RNA-guided DNA binding activity. Class 2 Cas nucleases include: Class 2 Cas cleavage enzymes/nickases (e.g., H840A, D10A or N863 A variants), the cleavage enzymes/nickases further having RNA-guided DNA cleavage enzyme or nickase activity; and Class 2 dCas DNA binders, wherein the cleavage enzyme/nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants) and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated herein by reference in its entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60: 385-397 (2015), the contents of which are incorporated herein in their entirety.

如本文所使用，“核糖核蛋白”(RNP)或“RNP复合物”是指向导RNA以及RNA引导的DNA结合剂，如Cas核酸酶、例如Cas切割酶、Cas切口酶或dCas DNA结合剂(例如，Cas9)。在一些实施例中，向导RNA将RNA引导的DNA结合剂(如Cas9)引导到靶序列，并且向导RNA与靶序列杂交并且所述结合剂与靶序列结合；在试剂为切割酶或切口酶的情况下，结合后可以进行切割或切口。As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas nuclease, for example, a Cas cleavage enzyme, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (e.g., Cas9) to the target sequence, and the guide RNA hybridizes to the target sequence and the binder binds to the target sequence; in the case where the agent is a cleavage enzyme or a nickase, cleavage or nicking can be performed after binding.

在一些实施例中，LNP组合物的货物包含至少一种包括引导序列的向导RNA，所述引导序列将RNA引导的DNA结合剂导向至靶DNA，所述RNA引导的DNA结合剂可以是核酸酶(例如Cas核酸酶，如Cas9)。gRNA可以将Cas核酸酶或2类Cas核酸酶引导至靶核酸分子上的靶序列。在一些实施例中，gRNA与2类Cas核酸酶结合并且通过2类Cas核酸酶提供切割的特异性。在一些实施例中，gRNA和Cas核酸酶可以形成核糖核蛋白(RNP)，例如，CRISPR/Cas复合物，诸如CRISPR/Cas9复合物。在一些实施例中，CRISPR/Cas复合物可以是II型CRISPR/Cas9复合物。在一些实施例中，CRISPR/Cas复合物可以是V型CRISPR/Cas复合物，如Cpf1/向导RNA复合物。Cas核酸酶和同源gRNA可以配对。与每个2类Cas核酸酶配对的gRNA支架结构随特定的CRISPR/Cas系统变化。In some embodiments, the cargo of the LNP composition comprises at least one guide RNA including a guide sequence, which guides the RNA-guided DNA binder to the target DNA, and the RNA-guided DNA binder can be a nuclease (e.g., Cas nuclease, such as Cas9). The gRNA can guide the Cas nuclease or 2 types of Cas nucleases to the target sequence on the target nucleic acid molecule. In some embodiments, the gRNA binds to 2 types of Cas nucleases and provides the specificity of cutting by 2 types of Cas nucleases. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), for example, a CRISPR/Cas complex, such as a CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex can be a type II CRISPR/Cas9 complex. In some embodiments, the CRISPR/Cas complex can be a type V CRISPR/Cas complex, such as a Cpf1/guide RNA complex. The Cas nuclease and the homologous gRNA can be paired. The gRNA scaffold structure paired with each type 2 Cas nuclease varies with the specific CRISPR/Cas system.

“向导RNA”、“gRNA”和简单的“向导”在本文中可互换使用以指代crRNA(也被称为CRISPR RNA)，或crRNA和trRNA的组合(也被称为tracrRNA)。向导RNA可以包含如本文所述的经修饰的RNA。crRNA和trRNA可以作为单个RNA分子(单向导RNA、sgRNA)或在两个单独的RNA分子(双向导RNA、dgRNA)中缔合。“引导RNA”或“gRNA”是指每种类型。trRNA可以是天然存在的序列，或与天然存在的序列相比具有修饰或变异的trRNA序列。"Guide RNA", "gRNA" and simply "guide" are used interchangeably herein to refer to crRNA (also known as CRISPR RNA), or a combination of crRNA and trRNA (also known as tracrRNA). The guide RNA may comprise a modified RNA as described herein. The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally occurring sequence, or a trRNA sequence that has a modification or variation compared to a naturally occurring sequence.

如本文所使用，“向导序列”是指在向导RNA内与靶序列互补并且用作将向导RNA导向到靶序列以通过RNA引导的DNA结合剂进行结合或修饰(例如，切割)的序列。“向导序列”也可以被称为“靶向序列”或“间隔序列”。向导序列的长度可以是20个碱基对，例如，在酿脓链球菌(即Spy Cas9)和相关的Cas9同源物/直系同源物的情况下。较短或较长的序列还可以用作向导，例如，长度为15个、16个、17个、18个、19个、21个、22个、23个、24个或25个核苷酸。在一些实施例中，靶序列例如是位于基因中或染色体上，并且与向导序列互补。在一些实施例中，向导序列与其对应靶序列之间的互补性或一致性程度可为约或至少75％、80％、85％、90％、95％、96％、97％、98％、99％或100％。在一些实施例中，向导序列和靶区在至少15、16、17、18、19或20个连续核苷酸的区域内可以是100％互补或一致的。在其它实施例中，向导序列和靶区可以含有至少一个错配。例如，向导序列和靶序列可以含有1个、2个、3个或4个错配，其中靶序列的总长度为至少17个、18个、19个、20个或更多个碱基对。在一些实施例中，向导序列和靶区可以含有1至4个错配，其中向导序列包括至少17个、18个、19个、20个或更多个核苷酸。在一些实施例中，向导序列和靶区可以含有1个、2个、3个或4个错配，其中向导序列包括20个核苷酸。As used herein, "guide sequence" refers to a sequence that is complementary to a target sequence within a guide RNA and is used to guide the guide RNA to the target sequence for binding or modification (e.g., cutting) by an RNA-guided DNA binder. "Guide sequence" may also be referred to as a "targeting sequence" or "spacer sequence". The length of the guide sequence may be 20 base pairs, for example, in the case of Streptococcus pyogenes (i.e. Spy Cas9) and related Cas9 homologs/orthologs. Shorter or longer sequences may also be used as guides, for example, with a length of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the target sequence is, for example, located in a gene or on a chromosome and is complementary to the guide sequence. In some embodiments, the degree of complementarity or consistency between the guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region can be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides. In other embodiments, the guide sequence and the target region can contain at least one mismatch. For example, the guide sequence and the target sequence can contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region can contain 1 to 4 mismatches, wherein the guide sequence includes at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region can contain 1, 2, 3, or 4 mismatches, wherein the guide sequence includes 20 nucleotides.

RNA引导的DNA结合蛋白(如Cas蛋白)的靶序列包含基因组DNA的正链和负链两者(即，给定的序列和所述序列的反向补体)，因为Cas蛋白的核酸底物是双链核酸。因此，在向导序列被称为“与靶序列互补”的情况下，应当理解，向导序列可以将向导RNA引导为与靶序列的反向补体结合。因此，在一些实施例中，在向导序列结合靶序列的反向补体的情况下，除了向导序列中的U取代T之外，向导序列与靶序列的某些核苷酸(例如，不包含PAM的靶序列)一致。The target sequence of RNA-guided DNA binding proteins (such as Cas proteins) contains both the positive and negative strands of genomic DNA (i.e., a given sequence and the reverse complement of the sequence), because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Therefore, in the case where the guide sequence is referred to as "complementary to the target sequence", it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Therefore, in some embodiments, in the case where the guide sequence binds to the reverse complement of the target sequence, in addition to the substitution of U for T in the guide sequence, the guide sequence is consistent with certain nucleotides of the target sequence (e.g., a target sequence that does not contain PAM).

靶向序列的长度可以取决于CRISPR/Cas系统和所使用的组分。例如，来自不同细菌物种的不同的2类Cas核酸酶具有不同的最佳靶向序列长度。因此，靶向序列的长度可以包括5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、35个、40个、45个、50个或超过50个核苷酸。在一些实施例中，靶向序列长度比天然存在的核苷酸序列的向导序列长或短0、1、2、3、4或5个核苷酸。The length of the targeting sequence can depend on the CRISPR/Cas system and the components used. For example, different 2 types of Cas nucleases from different bacterial species have different optimal targeting sequence lengths. Therefore, the length of the targeting sequence can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more than 50 nucleotides. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4 or 5 nucleotides longer or shorter than the guide sequence of the naturally occurring nucleotide sequence.

在某些实施例中，Cas核酸酶和gRNA支架将来源于同一CRISPR/Cas系统。在一些实施例中，靶向序列可以包含18-24个核苷酸或由其组成。在一些实施例中，靶向序列可以包含19-21个核苷酸或由其组成。在一些实施例中，靶向序列可以包括20个核苷酸或由20个核苷酸组成。In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR/Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

在一些实施例中，sgRNA是能够通过Cas9蛋白介导RNA引导的DNA切割的“Cas9sgRNA”。在一些实施例中，sgRNA是能够通过Cpf1蛋白介导RNA引导的DNA切割的“Cpf1sgRNA”。在一些实施例中，gRNA包括足以与Cas9蛋白形成活性复合物并介导RNA引导的DNA切割的crRNA和tracr RNA。在一些实施例中，gRNA包括足以与Cpf1蛋白形成活性复合物并介导RNA引导的DNA切割的crRNA。参见Zetsche2015。In some embodiments, the sgRNA is a "Cas9sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In some embodiments, the gRNA includes a crRNA and a tracr RNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA includes a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

本发明的某些实施例还提供了编码本文所述的gRNA的核酸，例如，表达盒。本文所使用的“向导RNA核酸”是指向导RNA(例如，sgRNA或dgRNA)和向导RNA表达盒，其是编码一个或多个向导RNA的核酸。Certain embodiments of the present invention also provide nucleic acids encoding gRNAs described herein, e.g., expression cassettes. As used herein, "guide RNA nucleic acid" refers to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes, which are nucleic acids encoding one or more guide RNAs.

本公开的某些实施例也提供了使用本文所述的LNP组合物、制剂、纳米粒子和/或纳米材料递送腺嘌呤碱基编辑器(“ABE”)。ABE及其使用方法描述于例如美国专利第10,113,163号和美国专利公开案第2021/0130805号中，其中每一者的内容以全文引用的方式并入本文中。Certain embodiments of the present disclosure also provide for the delivery of adenine base editors ("ABEs") using LNP compositions, formulations, nanoparticles and/or nanomaterials described herein. ABEs and methods of using them are described, for example, in U.S. Pat. No. 10,113,163 and U.S. Patent Publication No. 2021/0130805, the contents of each of which are incorporated herein by reference in their entirety.

本公开的某些实施例也提供了使用本文所述的LNP组合物、制剂、纳米粒子和/或纳米材料递送胞嘧啶碱基编辑器(“CBE”)。ABE及其使用方法描述于例如美国专利第10,167,457号和第9,840,699号中，其中每一者的内容以全文引用的方式并入本文中。Certain embodiments of the present disclosure also provide for the delivery of cytosine base editors ("CBEs") using LNP compositions, formulations, nanoparticles and/or nanomaterials described herein. ABEs and methods of using them are described, for example, in U.S. Pat. Nos. 10,167,457 and 9,840,699, the contents of each of which are incorporated herein by reference in their entirety.

术语“碱基编辑器(BE)”或“核碱基编辑器(NBE)”是指包括能够对核酸序列(例如DNA或RNA)内的碱基(例如A、T、C、G或U)进行修饰的多肽的试剂。在一些实施例中，碱基编辑器能够使核酸内的碱基脱氨基。在一些实施例中，碱基编辑器能够使DNA分子内的碱基脱氨基。在一些实施例中，碱基编辑器能够使DNA内的腺嘌呤(A)脱氨基。在一些实施例中，脱氨酶为胞嘧啶脱氨酶或胞苷脱氨酶。在一些实施例中，碱基编辑器为融合蛋白，其包括与腺苷脱氨酶融合的核酸可编程DNA结合蛋白(napDNAbp)。在一些实施例中，碱基编辑器为与腺苷脱氨酶融合的Cas9蛋白。在一些实施例中，碱基编辑器为与腺苷脱氨酶融合的Cas9切口酶(nCas9)。在一些实施例中，碱基编辑器为与腺苷脱氨酶融合的无核酸酶活性的Cas9(dCas9)。在一些实施例中，碱基编辑器与碱基切除修复抑制剂(例如UGI结构域或dISN结构域)融合。在一些实施例中，融合蛋白包括与脱氨酶融合的Cas9切口酶和碱基切除修复抑制剂，如UGI或dISN结构域。术语“核酸可编程的DNA结合蛋白”或“napDNAbp”是指与核酸(例如DNA或RNA)(如将napDNAbp引导到特定核酸序列的向导核酸)缔合的蛋白质。例如，Cas9蛋白可与向导RNA缔合，所述向导RNA将Cas9蛋白引导到与向导RNA互补的特定DNA序列。在一些实施例中，napDNAbp为2类微生物CRISPR-Cas效应子。在一些实施例中，napDNAbp为Cas9域，例如核酸酶活性Cas9、Cas9切口酶(nCas9)或无核酸酶活性的Cas9(dCas9)。核酸可编程的DNA结合蛋白的实例包含但不限于Cas9(例如，dCas9和nCas9)、CasX、CasY、Cpf1、C2c1、C2c2、C2C3和阿高蛋白(Argonaute)。然而，应了解，核酸可编程的DNA结合蛋白还包含结合RNA的核酸可编程蛋白。例如，napDNAbp可以与将napDNAbp引导到RNA的核酸缔合。其它核酸可编程的DNA结合蛋白也在本公开的范围内，但其可能未在本公开中具体列出。The term "base editor (BE)" or "nucleobase editor (NBE)" refers to an agent comprising a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, a base editor is capable of deaminating a base within a nucleic acid. In some embodiments, a base editor is capable of deaminating a base within a DNA molecule. In some embodiments, a base editor is capable of deaminating adenine (A) within DNA. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, a base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. In some embodiments, a base editor is a Cas9 protein fused to an adenosine deaminase. In some embodiments, a base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, a base editor is a nuclease-free Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, a base editor is fused to a base excision repair inhibitor (e.g., a UGI domain or a dISN domain). In some embodiments, the fusion protein includes a Cas9 nickase fused to a deaminase and a base excision repair inhibitor, such as a UGI or dISN domain. The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein associated with a nucleic acid (e.g., DNA or RNA) (e.g., a guide nucleic acid that guides napDNAbp to a specific nucleic acid sequence). For example, the Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, napDNAbp is a Class 2 microbial CRISPR-Cas effector. In some embodiments, napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a Cas9 without nuclease activity (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. However, it should be understood that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, napDNAbp can be associated with a nucleic acid that guides napDNAbp to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure.

F.经修饰的RNAF. Modified RNA

在某些实施例中，所公开的组合物、制剂、纳米粒子和/或纳米材料包括经修饰的核酸，包含经修饰的RNA。In certain embodiments, the disclosed compositions, formulations, nanoparticles, and/or nanomaterials include modified nucleic acids, including modified RNA.

经修饰的核苷或核苷酸可以存在于RNA，例如gRNA或mRNA中。包括一个或多个经修饰的核苷或核苷酸的gRNA或mRNA例如被称为“经修饰的”RNA，用以描述一个或多个非天然和/或天然存在的用于替代或除了规范的A、G、C和U残基之外使用的组分或构型的存在。在一些实施例中，经修饰的RNA是用非规范核苷或核苷酸合成的，这里称为“经修饰的”。Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides is, for example, referred to as "modified" RNA to describe the presence of one or more non-natural and/or naturally occurring components or configurations used to replace or in addition to canonical A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with non-canonical nucleosides or nucleotides, referred to herein as "modified".

经修饰的核苷和核苷酸可以包含以下中的一者或多者：(i)磷酸二酯主链键合中非连接磷酸氧中的一个或两个非连接磷酸氧和/或连接磷酸氧中的一个或多个连接磷酸氧的改变(例如，置换)(示例性主链修饰)；(ii)核糖的成分(例如，核糖上的2'羟基)的改变(例如，置换)(示例性糖修饰)；(iii)用“去磷酸”连接子大规模置换磷酸部分(示例性主链修饰)；(iv)天然存在的核碱基的修饰或置换，包含用非规范核碱基(示例性碱基修饰)；(v)核糖-磷酸主链的置换或修饰(示例性主链修饰)；(vi)寡核苷酸的3'末端或5'末端的修饰，例如，末端磷酸基团的去除、修饰或置换或者部分、帽或连接子的缀合(此类3'或5'帽修饰可以包括糖和/或主链修饰)；以及(vii)糖的修饰或置换(示例性的糖修饰)。某些实施例包括对mRNA、gRNA或核酸的5'端修饰。某些实施例包括对mRNA、gRNA或核酸的3'端修饰。经修饰的RNA可以含有5'端和3'端修饰。经修饰的RNA可以在非末端位置处含有一个或多个经修饰的残基。在某些实施例中，gRNA包含至少一个经修饰的残基。在某些实施例中，mRNA包含至少一个经修饰的残基。Modified nucleosides and nucleotides can comprise one or more of the following: (i) an alteration (e.g., substitution) of one or both of the non-linking phosphate oxygens and/or one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration (e.g., substitution) of the composition of the ribose sugar (e.g., the 2' hydroxyl group on the ribose sugar) (exemplary sugar modifications); (iii) large-scale replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modification); (iv) modification or replacement of naturally occurring nucleobases, including with non-canonical nucleobases (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification or replacement of the terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications can include sugar and/or backbone modifications); and (vii) modification or replacement of the sugar (exemplary sugar modifications). Certain embodiments include 5' end modifications to mRNA, gRNA or nucleic acid. Certain embodiments include 3' end modifications to mRNA, gRNA or nucleic acid. The modified RNA may contain 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA comprises at least one modified residue. In certain embodiments, the mRNA comprises at least one modified residue.

未经修饰的核酸可能易于被例如胞内核酸酶或在血清中发现的核酸酶降解。例如，核酸酶可以水解核酸磷酸二酯键。因此，在一方面，本文所述的gRNA(例如mRNA、gRNA)可以含有一种或多种经修饰的核苷或核苷酸，例如用以引入对胞内或基于血清的核酸酶的稳定性。在一些实施例中，本文所述的经修饰的gRNA分子在被体内和离体引入细胞群体时可以表现出降低的先天免疫应答。术语“先天免疫应答”包含对外源性核酸的细胞应答，所述外源性核酸包含单链核酸，所述细胞应答涉及诱导细胞因子表达和释放，特别是干扰素和细胞死亡。Unmodified nucleic acids may be susceptible to degradation by, for example, intracellular nucleases or nucleases found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Therefore, on the one hand, gRNA (e.g., mRNA, gRNA) described herein may contain one or more modified nucleosides or nucleotides, for example to introduce stability to intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population in vivo and in vitro. The term "innate immune response" includes a cellular response to an exogenous nucleic acid, the exogenous nucleic acid comprising a single-stranded nucleic acid, and the cellular response involves inducing cytokine expression and release, particularly interferon and cell death.

因此，在一些实施例中，所公开所公开的组合物、制剂、纳米粒子和/或纳米材料中的RNA或核酸包括至少一种修饰，其赋予核酸增加或增强的稳定性，包含例如改善的核酸酶体内消化抗性。如本文所使用的术语“修饰”和“经修饰的”当与本文所提供的核酸相关时，此类术语包含至少一种改变，其优选增强稳定性并使RNA或核酸比野生型或天然存在的RNA或核酸型式更稳定(例如，对核酸酶消化具有抗性)。如本文所使用的术语“稳定的”和“稳定性”当与本发明的核酸相关并且特别是关于RNA时，此类术语是指例如通过通常能够降解此类RNA的核酸酶(即，核酸内切酶或核酸外切酶)使降解抗性增加或增强。稳定性增加可以包含例如对由内源性酶(例如核酸内切酶或核酸外切酶)或靶细胞或组织内的条件引起的水解或其它破坏的敏感性降低，从而增加或增强此类RNA在靶细胞、组织、受试者和/或细胞质中的停留。本文提供的稳定RNA分子相对于其天然存在的未修饰对应物(例如mRNA的野生型型式)表现出更长的半衰期。术语“修饰”和“经修饰的”当与本文所公开的LNP组合物的mRNA相关时，此类术语还涵盖改善或增强mRNA核酸翻译的改变，包含例如：包含在蛋白质翻译起始中起作用的序列(例如Kozac共有序列)。(Kozak,M.,Nucleic Acids Res 15(20):8125-48(1987)，其内容特此通过全文引用的方式并入本文中)。Therefore, in some embodiments, the RNA or nucleic acid in the disclosed compositions, formulations, nanoparticles and/or nanomaterials include at least one modification that imparts increased or enhanced stability to the nucleic acid, including, for example, improved resistance to in vivo digestion by nucleases. The terms "modification" and "modified" as used herein, when associated with the nucleic acids provided herein, include at least one change that preferably enhances stability and makes the RNA or nucleic acid more stable (for example, resistant to nuclease digestion) than wild-type or naturally occurring RNA or nucleic acid patterns. The terms "stable" and "stability" as used herein, when associated with the nucleic acids of the present invention and particularly with respect to RNA, refer to, for example, an increase or enhancement of degradation resistance by nucleases (i.e., endonucleases or exonucleases) that are generally capable of degrading such RNA. Increased stability may include, for example, reduced sensitivity to hydrolysis or other destruction caused by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions in target cells or tissues, thereby increasing or enhancing the stay of such RNA in target cells, tissues, subjects and/or cytoplasm. The stable RNA molecules provided herein exhibit a longer half-life relative to their naturally occurring unmodified counterparts (e.g., wild-type forms of mRNA). The terms "modification" and "modified" when related to the mRNA of the LNP compositions disclosed herein, such terms also encompass changes that improve or enhance mRNA nucleic acid translation, including, for example, sequences that play a role in protein translation initiation (e.g., Kozak consensus sequences). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987), the contents of which are hereby incorporated herein by reference in their entirety).

在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料的RNA或核酸已经历化学或生物改性以使得其更稳定。对RNA的示范性修饰包含碱基耗尽(例如，通过缺失或通过一个核苷酸取代另一个)或碱基修饰，例如碱基的化学修饰。如本文所使用的短语“化学修饰”包含引入与天然存在的RNA中可见的化学物质的化学物质的修饰，例如共价修饰，如引入经修饰的核苷酸(例如，核苷酸类似物，或包含并非天然存在于此类RNA分子中的侧基)。In some embodiments, the RNA or nucleic acid of the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein has undergone chemical or biological modification to make it more stable. Exemplary modifications to RNA include base depletion (e.g., by deletion or by substitution of one nucleotide for another) or base modification, such as chemical modification of bases. As used herein, the phrase "chemical modification" includes the introduction of chemical substances visible in naturally occurring RNA, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or comprising side groups that are not naturally present in such RNA molecules).

在主链修饰的一些实施例中，可以通过用不同的取代基置换一个或多个氧来修饰经修饰的残基的磷酸基团。另外，经修饰的残基(例如经修饰的核酸中存在的经修饰的残基)可以包含用如本文所述的经修饰的磷酸基团大规模置换未经修饰的磷酸部分。在一些实施例中，磷酸主链的主链修饰可以包含导致不带电荷的连接子或具有不对称电荷分布的带电荷连接子的改变。经修饰的磷酸基团的实例包含硫代磷酸酯、硒代磷酸酯、硼烷磷酸(borano phosphate)、硼烷磷酸酯(borano phosphate ester)、膦酸氢酯、氨基磷酸酯、烷基或芳基膦酸酯和磷酸三酯。未经修饰的磷酸基团中的磷原子是非手性的。然而，用上述原子或基团中的一个原子或基团置换非桥氧中的一个非桥氧可以使磷原子具有手性。立构磷原子可以具有“R”构型(此处为Rp)或“S”构型(此处为Sp)。还可以通过用氮(桥接的氨基磷酸酯)、硫(桥接的硫代磷酸酯)和碳(桥接的亚甲基膦酸酯)置换桥氧(即，连接磷酸酯与核苷的氧)来修饰主链。置换可以发生在任一连接氧处或两个连接氧处。在某些主链修饰中，磷酸基团可以被不含磷的接头置换。在一些实施例中，带电荷的磷酸基团可以被中性部分置换。可以置换磷酸基团的部分的实例可以包含但不限于，例如膦酸甲酯、羟氨基、硅氧烷、碳酸酯、羧甲基、氨基甲酸酯、酰胺、硫醚、环氧乙烷连接子、磺酸盐、磺酰胺、硫代甲缩醛化物、甲缩醛化物、肟、亚甲基亚氨基、亚甲基甲亚氨基、亚甲基亚肼基(methylenehydrazo)、亚甲基二甲亚肼基(methylenedimethylhydrazo)和亚甲基氧甲亚氨基。In some embodiments of backbone modification, the phosphate group of modified residue can be modified by replacing one or more oxygen with different substituents. In addition, modified residues (such as modified residues present in modified nucleic acids) can include large-scale replacement of unmodified phosphate moieties with modified phosphate groups as described herein. In certain embodiments, the backbone modification of the phosphate backbone can include changes that cause uncharged linkers or charged linkers with asymmetric charge distribution. Examples of modified phosphate groups include thiophosphates, selenophosphates, boranophosphates, boranophosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom in the unmodified phosphate group is achiral. However, replacing a non-bridge oxygen in the non-bridge oxygen with an atom or group in the above-mentioned atoms or groups can make the phosphorus atom have chirality. Stereo phosphorus atoms can have "R" configuration (here Rp) or "S" configuration (here Sp). The main chain can also be modified by replacing the bridge oxygen (i.e., the oxygen connecting phosphate and nucleoside) with nitrogen (phosphoramidate of bridged connection), sulfur (phosphorothioate of bridged connection) and carbon (methylene phosphonate of bridged connection). Replacement can occur at any connection oxygen place or two connection oxygen places. In some main chain modifications, the phosphate group can be replaced by a phosphorus-free joint. In certain embodiments, the charged phosphate group can be replaced by a neutral part. The example of the part that can replace the phosphate group can include but is not limited to, such as methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformylation, methylation, oxime, methylene imino, methylene methyl imino, methylene hydrazo (methylenehydrazo), methylene dimethyl hydrazo (methylenedimethylhydrazo) and methylene oxygen methyl imino.

G.mRNAG.mRNA

在一些实施例中，所公开的组合物、制剂、纳米粒子和/或纳米材料包括mRNA，所述mRNA包括编码RNA引导的DNA结合剂的开放阅读框架(ORF)，如本文所述的Cas核酸酶或2类Cas核酸酶。在一些实施例中，提供、使用或施用包括编码RNA引导的DNA结合剂(如Cas核酸酶或2类Cas核酸酶)的ORF的mRNA。mRNA可以包括5'帽、5'非翻译区(UTR)、3'UTR和聚腺嘌呤尾中的一个或多个。mRNA可以包括经修饰的开放阅读框架，例如用以编码核定位序列或使用替代密码子编码蛋白质。In some embodiments, the disclosed compositions, formulations, nanoparticles and/or nanomaterials include mRNA, the mRNA including an open reading frame (ORF) encoding an RNA-guided DNA binder, such as a Cas nuclease or a Class 2 Cas nuclease as described herein. In some embodiments, an mRNA including an ORF encoding an RNA-guided DNA binder (such as a Cas nuclease or a Class 2 Cas nuclease) is provided, used or administered. The mRNA may include one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may include a modified open reading frame, for example, to encode a nuclear localization sequence or to encode a protein using alternative codons.

所公开的组合物、制剂、纳米粒子和/或纳米材料中的mRNA可以编码例如所分泌的激素、酶、受体、多肽、肽或正常所分泌的其它所关注蛋白质。在本发明的一个实施方面中，mRNA可以任选地具有化学或生物修饰，其例如改善此类mRNA的稳定性和/或半衰期，或改善或以其它方式促进蛋白质产生。The mRNA in the disclosed compositions, formulations, nanoparticles and/or nanomaterials can encode, for example, secreted hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest that are normally secreted. In one embodiment of the invention, the mRNA can optionally have a chemical or biological modification that, for example, improves the stability and/or half-life of such mRNA, or improves or otherwise promotes protein production.

此外，适合的修饰包含密码子的一个或多个核苷酸的改变，使得所述密码子编码相同氨基酸，但比在mRNA的野生型型式中发现的密码子更稳定。例如，已经证明RNA的稳定性与更多的胞苷(C)和/或尿苷(U)残基之间存在反比关系，并且已经发现不含C和U残基的RNA对大多数RNA酶都是稳定的(Heidenreich等人，J Biol Chem 269,2131-8(1994)，其公开内容通过引用整体并入本文中)。在一些实施例中，mRNA序列中的C和/或U残基的数量减少。在另一个实施例中，通过用编码特定氨基酸的一个密码子取代编码相同或相关氨基酸的另一个密码子来减少C和/或U残基的数量。对本发明的mRNA核酸的预期修饰还包括并入假尿苷。将假尿苷并入本发明的mRNA核酸中可以增强稳定性和翻译能力，以及降低体内免疫原性。参见例如Kariko,K.等人，Molecular Therapy 16(11):1833-1840(2008)，其内容特此以全文引用的方式并入本文中。可以通过本领域的普通技术人员容易了解的方法对本发明的mRNA进行取代和修饰。In addition, suitable modification comprises the change of one or more nucleotides of codon, so that the codon encodes the same amino acid, but is more stable than the codon found in the wild-type form of mRNA. For example, it has been shown that there is an inverse relationship between the stability of RNA and more cytidine (C) and/or uridine (U) residues, and it has been found that RNA without C and U residues is stable to most RNases (Heidenreich et al., J Biol Chem 269, 2131-8 (1994), the disclosure of which is incorporated herein by reference in its entirety). In certain embodiments, the number of C and/or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and/or U residues is reduced by replacing another codon encoding the same or related amino acids with a codon encoding a specific amino acid. The expected modification of the mRNA nucleic acid of the present invention also includes the incorporation of pseudouridine. Pseudouridine is incorporated into the mRNA nucleic acid of the present invention to enhance stability and translation ability, and reduce immunogenicity in vivo. See, for example, Kariko, K. et al., Molecular Therapy 16(11):1833-1840 (2008), the contents of which are hereby incorporated by reference in their entirety. The mRNA of the present invention can be substituted and modified by methods readily understood by those of ordinary skill in the art.

与非翻译区相比，对于减少序列中C和U残基数量的限制在mRNA编码区内可能更多(即，可能无法消除所有存在于消息中的C和U残基，同时仍保留消息编码所需氨基酸序列的能力)。然而，遗传密码的简并提供了如下机会：使序列中存在的C和/或U残基的数量减少，同时保持相同的编码能力(即，取决于哪些氨基酸由密码子编，可能有几种不同的RNA序列修饰可能性)。The constraints on reducing the number of C and U residues in the sequence may be greater within the coding region of the mRNA than in the untranslated regions (i.e., it may not be possible to eliminate all C and U residues present in the message while still retaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code provides the opportunity to reduce the number of C and/or U residues present in the sequence while maintaining the same coding capacity (i.e., depending on which amino acids are encoded by the codons, there may be several different possibilities for RNA sequence modifications).

术语修饰还包含，例如，将非核苷酸键或经修饰的核苷酸并入本发明的mRNA序列中(例如，对编码功能性分泌蛋白或酶的mRNA分子的3'端和5'端中的一个或两个进行修饰)。此类修饰包含向mRNA序列添加碱基(例如，包含poly A尾或更长的poly A尾)、改变3'UTR或5'UTR、使mRNA与试剂(例如、蛋白质或互补核酸分子)复合，以及包含改变mRNA分子结构的元件(例如，其形成二级结构)。The term modification also includes, for example, incorporating non-nucleotide bonds or modified nucleotides into the mRNA sequence of the invention (e.g., modifying one or both of the 3' and 5' ends of the mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include adding bases to the mRNA sequence (e.g., including a poly A tail or a longer poly A tail), changing the 3'UTR or 5'UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and including elements that change the structure of the mRNA molecule (e.g., it forms a secondary structure).

poly A尾被认为可以稳定自然信使。因此，在一个实施例中，长poly A尾可以添加到mRNA分子中，因此使mRNA更稳定。可使用多种本领域公认的技术添加Poly A尾。例如，可以使用poly A聚合酶将长poly A尾添加到合成或体外转录的mRNA中(Yokoe等人，NatureBiotechnology.1996；14:1252-1256，其内容据此通过引用整体并入本文)。转录载体还可编码长poly A尾。此外，可以通过直接从PCR产物转录来添加poly A尾。在一个实施例中，poly A尾的长度为至少约90个、200个、300个、400个、至少500个核苷酸。在一个实施例中，调节poly A尾的长度以控制本发明的经修饰mRNA分子的稳定性，并且因此控制蛋白质的转录。例如，由于poly A尾的长度可影响mRNA分子的半衰期，因此可调节poly A尾的长度以改变mRNA对核酸酶的抗性水平，且由此控制细胞中蛋白质表达的时程。在一个实施例中，稳定mRNA分子对体内降解(例如，通过核酸酶降解)具有足够抗性，使得其可以在无转移媒剂的情况下递送至靶细胞。The poly A tail is considered to stabilize natural messengers. Therefore, in one embodiment, a long poly A tail can be added to an mRNA molecule, thereby making the mRNA more stable. Poly A tails can be added using a variety of techniques recognized in the art. For example, a long poly A tail can be added to a synthetic or in vitro transcribed mRNA using poly A polymerase (Yokoe et al., Nature Biotechnology. 1996; 14: 1252-1256, the contents of which are hereby incorporated by reference as a whole). The transcription vector can also encode a long poly A tail. In addition, the poly A tail can be added by directly transcribing from a PCR product. In one embodiment, the length of the poly A tail is at least about 90, 200, 300, 400, at least 500 nucleotides. In one embodiment, the length of the poly A tail is adjusted to control the stability of the modified mRNA molecule of the present invention, and thus control the transcription of the protein. For example, because the length of the poly A tail can affect the half-life of the mRNA molecule, the length of the poly A tail can be adjusted to change the resistance level of mRNA to nucleases, and thus control the time course of protein expression in the cell. In one embodiment, the stabilized mRNA molecule is sufficiently resistant to degradation in vivo (eg, by nucleases) such that it can be delivered to target cells in the absence of a transfer vehicle.

在一些实施例中，可以通过并入并非天然存在于野生型mRNA中的3'和/或5'非翻译(UTR)序列来修饰mRNA。在一个实施例中，天然侧接mRNA并编码第二个不相关蛋白的3'和/或5'侧接序列可以并入到编码治疗性或功能性蛋白的mRNA分子的核苷酸序列中以便对其进行修饰。例如来自稳定的mRNA分子(例如球蛋白、肌动蛋白、GAPDH、微管蛋白、组蛋白或柠檬酸循环酶)的3'或5'序列可以并入到有义mRNA核酸分子的3'和/或5'区中，以增加有义mRNA分子的稳定性。参见例如US2003/0083272，其内容特此以全文引用的方式并入本文中。mRNA修饰的更详细描述可以见US2017/0210698A1的第57至68页，所述内容以全文引用的方式并入本文中。In certain embodiments, mRNA can be modified by incorporating 3' and/or 5' untranslated (UTR) sequences that are not naturally present in wild-type mRNA. In one embodiment, natural flanking mRNA and encoding the 3' and/or 5' flanking sequences of the second unrelated protein can be incorporated into the nucleotide sequence of the mRNA molecule encoding therapeutic or functional protein to modify it. For example, 3' or 5' sequences from stable mRNA molecules (such as globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) can be incorporated into 3' and/or 5' regions of the mRNA nucleic acid molecules to increase the stability of the mRNA molecules. See, for example, US2003/0083272, the contents of which are hereby incorporated herein by reference in their entirety. A more detailed description of mRNA modifications can be found in pages 57 to 68 of US2017/0210698A1, which are incorporated herein by reference in their entirety.

H.模板核酸H. Template nucleic acid

本文所公开的组合物、制剂、纳米粒子和/或纳米材料和方法可以包含模板核酸。模板可以用于改变或插入RNA引导的DNA结合蛋白(如Cas核酸酶，例如2类Cas核酸酶)的靶位点处或附近的核酸序列。在一些实施例中，所述方法包括将模板引入细胞。在一些实施例中，可以提供单个模板。在一些实施例中，可以提供两个或更多个模板，使得可以在两个或更多个靶位点处进行编辑。例如，可以提供不同的模板来编辑细胞中的单个基因或细胞中的两个不同基因。The compositions, formulations, nanoparticles and/or nanomaterials and methods disclosed herein may include template nucleic acids. The template can be used to change or insert a nucleic acid sequence at or near the target site of an RNA-guided DNA binding protein (such as a Cas nuclease, for example a Class 2 Cas nuclease). In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In some embodiments, two or more templates may be provided so that editing can be performed at two or more target sites. For example, different templates may be provided to edit a single gene in a cell or two different genes in a cell.

在一些实施例中，模板可以用于同源重组。在一些实施例中，同源重组可以使模板序列或模板序列的一部分合并到靶核酸分子中。在一些实施例中，模板可以用于同源性定向修复，其涉及核酸中的切割位点处的DNA链侵入。在一些实施例中，同源性定向修复可以导致在经编辑的靶核酸分子中包含模板序列。在一些实施例中，模板可以用于由非同源末端连接介导的基因编辑。在一些实施例中，模板序列与切割位点附近的核酸序列没有相似性。在一些实施例中，并入模板或模板序列的一部分。在一些实施例中，模板包含侧接反向末端重复(ITR)序列。In some embodiments, the template can be used for homologous recombination. In some embodiments, homologous recombination can cause the template sequence or a portion of the template sequence to be incorporated into the target nucleic acid molecule. In some embodiments, the template can be used for homology directed repair, which involves the invasion of the DNA chain at the cleavage site in the nucleic acid. In some embodiments, homology directed repair can result in the inclusion of the template sequence in the edited target nucleic acid molecule. In some embodiments, the template can be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, a portion of the template or template sequence is incorporated. In some embodiments, the template comprises a flanking reverse terminal repeat (ITR) sequence.

在一些实施例中，模板序列可以对应于靶细胞的内源性序列、包括所述内源性序列或由所述内源性序列组成。其还可以或可替代地对应于靶细胞的外源性序列、包括所述外源性序列或由所述外源性序列组成。如本文所使用的术语“内源性序列”是指细胞固有的序列。术语“外源性序列”是指不是细胞固有的序列，或其在细胞基因组中的固有位置处于不同位置的序列。在一些实施例中，内源性序列可以是细胞的基因组序列。In some embodiments, the template sequence may correspond to, include, or consist of an endogenous sequence of the target cell. It may also or alternatively correspond to, include, or consist of an exogenous sequence of the target cell. The term "endogenous sequence" as used herein refers to a sequence that is intrinsic to a cell. The term "exogenous sequence" refers to a sequence that is not intrinsic to a cell, or a sequence whose intrinsic position in the cell genome is at a different position. In some embodiments, the endogenous sequence may be a genomic sequence of a cell.

在一些实施例中，内源性序列可以是染色体或染色体外序列。在一些实施例中，内源性序列可以是细胞的质粒序列。In some embodiments, the endogenous sequence can be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence can be a plasmid sequence of the cell.

在一些实施例中，模板包含含有侧接反向末端重复(ITR)序列的ssDNA或dsDNA。在一些实施例中，模板作为载体、质粒、微环、纳米环或PCR产物提供。In some embodiments, the template comprises ssDNA or dsDNA containing flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, plasmid, minicircle, nanocircle, or PCR product.

在一些实施例中，核酸经纯化。在一些实施例中，使用沉淀方法(例如，LiCl沉淀、醇沉淀或等效方法，例如，如本文所述)纯化核酸。在一些实施例中，使用基于色谱的方法，如基于HPLC的方法或等效方法(例如，如本文所述)纯化核酸。在一些实施例中，使用沉淀方法(例如，LiCl沉淀)和基于HPLC的方法两者纯化核酸。在一些实施例中，通过切向流动过滤(tangential flow filtration；TFF)纯化核酸。In some embodiments, nucleic acid is purified. In some embodiments, nucleic acid is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or an equivalent method, e.g., as described herein). In some embodiments, nucleic acid is purified using a chromatography-based method, such as an HPLC-based method or an equivalent method (e.g., as described herein). In some embodiments, nucleic acid is purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, nucleic acid is purified by tangential flow filtration (TFF).

IV.制造LNP的方法IV. Methods of Making LNPs

制造脂质纳米粒子的方法为本领域中已知的。在一些实施例中，使用微流体制造所描述的组合物、制剂、纳米粒子和/或纳米材料。例如，Leung,A.K.K等人，J Phys Chem,116:18440-18450(2012)、Chen,D.等人，J Am Chem Soc,134:6947-6951(2012)和Belliveau,N.M.等人，Molecular Therapy-Nucleic Acids,1:e37(2012)中描述了使用微流体形成脂质纳米粒子的示例性方法，其公开内容据此通过引用整体并入。Methods for making lipid nanoparticles are known in the art. In some embodiments, microfluidics are used to make the described compositions, formulations, nanoparticles and/or nanomaterials. For example, Leung, A.K.K et al., J Phys Chem, 116: 18440-18450 (2012), Chen, D. et al., J Am Chem Soc, 134: 6947-6951 (2012) and Belliveau, N.M. et al., Molecular Therapy-Nucleic Acids, 1: e37 (2012) describe exemplary methods for forming lipid nanoparticles using microfluidics, the disclosures of which are hereby incorporated by reference as a whole.

简单来说，在第一缓冲溶液中制备货物，如本文所述的货物。在第二缓冲溶液中制备其它脂质纳米粒子组分(诸如可电离脂质、缀合物-连接子脂质、胆甾醇和磷脂)。在一些实施例中，注射器泵将两种溶液引入微流体装置中。两种溶液在微流体装置内接触，形成包裹货物的脂质纳米粒子。In brief, a cargo, such as the cargo described herein, is prepared in a first buffer solution. Other lipid nanoparticle components (such as ionizable lipids, conjugate-linker lipids, cholesterol, and phospholipids) are prepared in a second buffer solution. In some embodiments, a syringe pump introduces the two solutions into a microfluidic device. The two solutions are contacted within the microfluidic device to form lipid nanoparticles that encapsulate the cargo.

筛选所公开的脂质纳米粒子的方法描述于国际专利申请案第PCT/US2018/058171号中，所述申请案是以全文引用的方式并入本文中。在一些实施例中，筛选方法表征媒剂递送制剂，以识别具有所需向性并将功能性货物递送到特定细胞的细胞质的制剂。在一些实施例中，筛选方法使用具有当递送到细胞时可以检测到的功能性的报告基因(reporter)。例如，检测到细胞中的功能性报告基因表明LNP制剂将功能性货物递送至细胞。此外，在一些实施例中，化学成分标识符包含在每种不同的递送媒剂调配物中，以跟踪特定于每种不同递送媒剂调配物的化学组成。在一些实施例中，化学组成识别符是核酸条形码。在一些实施例中，使核酸条形码序列与用于调配其中负载化学组分的LNP制剂的所述化学组分配对，以便在测序核酸条形码时识别递送条形码的递送媒剂的化学组成。代表性条形码包含但不限于由Sago,2018PNAS,Sago,JACS2018描述的条形码，其公开内容特此以全文引用的方式并入。代表性报告基因包含但不限于siRNA、mRNA、核酸酶蛋白质、核酸酶mRNA、小分子、表观遗传修饰剂和表型修饰剂。DNA(基因组和DNA条形码)可以使用QuickExtract(Lucigen)进行分离，并使用Illumina MiniSeq进行测序，如Sago等人，PNAS2018；Sago等人，JACs 2018；Sago,Lokugamage等人，Nano Letters 2018，其公开内容据此通过引用整体并入)。The method for screening the disclosed lipid nanoparticles is described in International Patent Application No. PCT/US2018/058171, which is incorporated herein by reference in its entirety. In some embodiments, the screening method characterizes the medium delivery preparation to identify the preparation with the desired tropism and the functional cargo is delivered to the cytoplasm of a specific cell. In some embodiments, the screening method uses a reporter gene (reporter) with functionality that can be detected when delivered to the cell. For example, the functional reporter gene detected in the cell indicates that the LNP preparation delivers the functional cargo to the cell. In addition, in some embodiments, the chemical component identifier is included in each different delivery vehicle formulation to track the chemical composition specific to each different delivery vehicle formulation. In some embodiments, the chemical composition identifier is a nucleic acid barcode. In some embodiments, the nucleic acid barcode sequence is paired with the chemical component for preparing the LNP preparation of the chemical component loaded therein, so that the chemical composition of the delivery vehicle of the delivery barcode is identified when sequencing the nucleic acid barcode. Representative barcodes include, but are not limited to, those described by Sago, 2018 PNAS, Sago, JACS 2018, the disclosures of which are hereby incorporated by reference in their entirety. Representative reporter genes include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotypic modifiers. DNA (genomic and DNA barcodes) can be isolated using QuickExtract (Lucigen) and sequenced using Illumina MiniSeq, such as Sago et al., PNAS 2018; Sago et al., JACs 2018; Sago, Lokugamage et al., Nano Letters 2018, the disclosures of which are hereby incorporated by reference in their entirety).

V.使用方法V. How to use

此外，本公开描述使用本文所述的组合物、制剂、纳米粒子和/或纳米材料的方法。例如，在一些实施例中，本公开描述使用组合物、制剂、纳米粒子和/或纳米材料将货物传递到如本文所述的特定细胞、组织或器官的方法。作为另一实例，在一些实施例中，本公开描述使用如本文所述的组合物、制剂、纳米粒子和/或纳米材料治疗疾病或病症和/或延迟和/或遏制疾病或病症进展的方法。在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料适用于医学。In addition, the present disclosure describes methods of using the compositions, formulations, nanoparticles and/or nanomaterials described herein. For example, in some embodiments, the present disclosure describes methods of using compositions, formulations, nanoparticles and/or nanomaterials to deliver cargo to specific cells, tissues or organs as described herein. As another example, in some embodiments, the present disclosure describes methods of using compositions, formulations, nanoparticles and/or nanomaterials as described herein to treat diseases or conditions and/or delay and/or curb the progression of diseases or conditions. In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials described herein are suitable for medicine.

在一些实施例中，本文所述的组合物、制剂、纳米粒子和/或纳米材料将治疗剂或预防剂递送到有此需要的受试者的特定细胞或器官。在一些实施例中，所述组合物、制剂、纳米粒子和/或纳米材料在不存在靶向配体的情况下将治疗剂或预防剂递送到有此需要的受试者的特定细胞或器官。在一些实施例中，所述组合物、制剂、纳米粒子和/或纳米材料适用于治疗或预防有此需要的受试者的疾病。In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials described herein deliver therapeutic or prophylactic agents to specific cells or organs of a subject in need thereof. In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials deliver therapeutic or prophylactic agents to specific cells or organs of a subject in need thereof in the absence of a targeting ligand. In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials are suitable for treating or preventing a disease in a subject in need thereof.

A.将货物传递到细胞、组织或器官的方法A. Methods of delivering cargo to cells, tissues or organs

此外，在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料靶向特定类型或类别的细胞(例如，特定器官或其系统的细胞)、组织、细胞群体或器官。在一些实施例中，本公开提供向有此需要的受试者递送本文所述的一种或多种货物的方法。在一些实施例中，此类方法包括体内和/或体外递送。在一些实施例中，此类方法包括体内递送。在一些实施例中，此类方法包括体外递送。在一些实施例中，本公开提供本文描述向有此需要的受试者递送一种或多种治疗性和/或预防性核酸的方法。In addition, in some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein target specific types or categories of cells (e.g., cells of a specific organ or its system), tissues, cell colonies or organs. In some embodiments, the present disclosure provides methods for delivering one or more goods described herein to a subject in need thereof. In some embodiments, such methods include in vivo and/or in vitro delivery. In some embodiments, such methods include in vivo delivery. In some embodiments, such methods include in vitro delivery. In some embodiments, the present disclosure provides methods for delivering one or more therapeutic and/or prophylactic nucleic acids to a subject in need thereof as described herein.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包括可以特异性地递送到受试者的肝脏细胞的所关注的治疗剂和/或预防剂。示范性肝脏细胞包含但不限于肝细胞(hepatocyte)。In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials include therapeutic and/or prophylactic agents of interest that can be delivered specifically to liver cells of a subject. Exemplary liver cells include, but are not limited to, hepatocytes.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包含可以特异性地递送至受试者的肺细胞的所关注的治疗剂和/或预防剂。In some embodiments, the compositions, formulations, nanoparticles, and/or nanomaterials comprising therapeutic and/or prophylactic agents of interest can be specifically delivered to lung cells of a subject.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包括可以特异性递送到受试者的脾细胞的所关注的治疗剂和/或预防剂。示范性脾细胞包含但不限于脾单核细胞、脾T细胞、脾记忆B细胞或脾B细胞。In some embodiments, the composition, formulation, nanoparticle and/or nanomaterial includes a therapeutic and/or prophylactic agent of interest that can be specifically delivered to a subject's spleen cells. Exemplary spleen cells include, but are not limited to, spleen monocytes, spleen T cells, spleen memory B cells, or spleen B cells.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包括可以特异性地递送到受试者的骨髓细胞的所关注的治疗剂和/或预防剂。示范性骨髓细胞包含但不限于骨髓单核细胞、骨髓B细胞、骨髓记忆B细胞或骨髓T细胞。In some embodiments, the composition, formulation, nanoparticle and/or nanomaterial includes a therapeutic and/or prophylactic agent of interest that can be specifically delivered to the subject's bone marrow cells. Exemplary bone marrow cells include, but are not limited to, bone marrow mononuclear cells, bone marrow B cells, bone marrow memory B cells, or bone marrow T cells.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包括可以特异性递送到受试者的免疫细胞的所关注的治疗剂和/或预防剂。示范性免疫细胞包含但不限于CD8+、CD4+或CD8+CD4+细胞。In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials include therapeutic and/or prophylactic agents of interest that can be specifically delivered to immune cells of a subject. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包含可以特异性地递送至受试者的中枢神经系统的细胞的所关注的治疗剂和/或预防剂。In some embodiments, compositions, formulations, nanoparticles, and/or nanomaterials comprising therapeutic and/or prophylactic agents of interest can be specifically delivered to cells of the central nervous system of a subject.

在一些实施例中，组合物、制剂、纳米粒子和/或纳米材料包括可以特异性地递送到受试者的造血干细胞的所关注的治疗剂和/或预防剂。除非另有规定，否则应理解，术语“造血干细胞(HSC)”和“造血干细胞和祖细胞(HSPC)”在本公开中可互换地使用。In some embodiments, the composition, formulation, nanoparticle and/or nanomaterial includes a therapeutic agent and/or prophylactic agent of interest that can be specifically delivered to a subject's hematopoietic stem cell. Unless otherwise specified, it is understood that the terms "hematopoietic stem cell (HSC)" and "hematopoietic stem and progenitor cells (HSPC)" are used interchangeably in the present disclosure.

在一些实施例中，脂质纳米粒子可以调配成在不存在靶向配体的情况下递送到哺乳动物肝脏肝细胞、肝脏免疫细胞、脾T细胞或肺内皮细胞。特异性递送至特定类别或类型的细胞表明更高比例的脂质纳米粒子被递送至目标类型或类别的细胞。在一些实施例中，特异性递送可以导致比使用常规纳米粒子系统(例如，含有MC3的LNP)的递送大2倍、5倍、10倍、15倍或20倍。In certain embodiments, lipid nanoparticle can be formulated to be delivered to mammal liver hepatocyte, liver immune cell, spleen T cell or lung endothelial cell in the absence of targeting ligand.Specific delivery to the cell of particular classification or type shows that the lipid nanoparticle of higher ratio is delivered to the cell of target type or classification.In certain embodiments, specific delivery can cause than using conventional nanoparticle system (for example, the LNP containing MC3) delivery 2 times, 5 times, 10 times, 15 times or 20 times.

B.产生多肽的方法B. Methods of Producing Polypeptides

此外，在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料的使用方法被用于产生多肽的方法。此外，在一些实施例中，本文所述的脂质纳米粒子可以用于在有此需要的受试者的靶细胞中产生多肽。例如，在一些实施例中，本文所述的脂质纳米粒子可以用于在有此需要的受试者的靶细胞中产生多肽。在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料包括一种或多种待递送到细胞的核酸序列。In addition, in some embodiments, the methods of using the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein are used for methods of producing polypeptides. In addition, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells of a subject in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells of a subject in need thereof. In some embodiments, the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein include one or more nucleic acid sequences to be delivered to a cell.

在一些实施例中，一种或多种核酸表达于细胞中。在一些实施例中，核酸序列的表达涉及以下中的一者或多者：(1)从DNA序列产生RNA模板(例如，通过转录)；(2)处理RNA转录本(例如，通过剪接、编辑、5'帽形成和/或3'末端形成)；(3)将RNA翻译成多肽或蛋白质；且/或(4)翻译后修饰多肽或蛋白质。In some embodiments, one or more nucleic acids are expressed in a cell. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing the RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translationally modifying the polypeptide or protein.

C.基因调节方法C. Gene Regulation Methods

此外，在一些实施例中，本文所公开的组合物、制剂、纳米粒子和/或纳米材料的使用方法被用于基因调节。此外，在一些实施例中，本文所述的脂质纳米粒子可用于减少和/或增加有此需要的受试者的靶细胞中的基因表达。例如，在一些实施例中，本文所述的脂质纳米粒子可以在没有靶向配体的情况下将一种或多种核酸递送到受试者的靶细胞。在一些实施例中，核酸为抑制剂核酸。在一些实施例中，抑制性核酸为siRNA。在一些实施例中，核酸为本文所述的核酸。作为另一实例，在一些实施例中，本文所述的脂质纳米粒子可以在没有靶向配体的情况下将货物递送到受试者的靶细胞。在一些实施例中，货物是本文所述的任何货物。In addition, in some embodiments, the use methods of the compositions, formulations, nanoparticles and/or nanomaterials disclosed herein are used for gene regulation. In addition, in some embodiments, the lipid nanoparticles described herein can be used to reduce and/or increase gene expression in target cells of a subject in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can deliver one or more nucleic acids to a subject's target cells without a targeting ligand. In some embodiments, the nucleic acid is an inhibitor nucleic acid. In some embodiments, the inhibitory nucleic acid is an siRNA. In some embodiments, the nucleic acid is a nucleic acid described herein. As another example, in some embodiments, the lipid nanoparticles described herein can deliver goods to a subject's target cells without a targeting ligand. In some embodiments, the goods are any goods described herein.

此外，在一些实施例中，使用本文公开的组合物、制剂、纳米粒子和/或纳米材料来编辑有此需要的受试者的细胞中的基因的方法。Furthermore, in some embodiments, methods of using the compositions, formulations, nanoparticles, and/or nanomaterials disclosed herein to edit genes in cells of a subject in need thereof.

在一些实施例中，靶向用于基因调节的细胞是免疫细胞。免疫细胞可以是T细胞，如CD8+T细胞、CD4+T细胞或T调节细胞。用于基因编辑的其它示范性免疫细胞包含但不限于巨噬细胞、树突状细胞、B细胞或自然杀伤细胞。在一些实施例中，靶向用于在肝细胞中进行基因调节的细胞。In some embodiments, the cells targeted for gene regulation are immune cells. The immune cells can be T cells, such as CD8+T cells, CD4+T cells, or T regulatory cells. Other exemplary immune cells for gene editing include, but are not limited to, macrophages, dendritic cells, B cells, or natural killer cells. In some embodiments, cells targeted for gene regulation in hepatocytes.

可被靶向的示范性基因包含但不限于T细胞受体、B细胞受体、CTLA4、PD1、FOXO1、FOXO3、AKTs、CCR5、CXCR4、LAG3、TIM3、杀伤免疫球蛋白样受体、GITR、BTLA、LFA-4、T4、LFA-1、Bp35、CD27L受体、TNFRSF8、TNFRSF5、CD47、CD52、ICAM-1、LFA-3、L-选择蛋白、Ki-24、MB1、B7、B70、M-CSFR、TNFR-II、IL-7R、OX-40、CD137、CD137L、CD30L、CD40L、FasL、TRAIL、CD257、LIGHT、TRAIL-R1、TRAILR2、TRAIL-R4、TWEAK-R、TNFR、BCMA、B7DC、BTLA、B7-H1、B7-H2、B7-H3、ICOS、VEGFR2、NKG2D、JAG1、GITR、CD4、CCR2、GATA-3、MTORC1、MTORC2、RAPTOR、GATOR、FOXP3、NFAT、IL2R和IL7。可以被靶向的其它示例性基因包含但不限于OCT、G6Pase、Mut、PCCA、PCCB、PCSK9、ALAS1和PAH。可以由T细胞识别并且考虑用于靶向的示范性肿瘤相关抗原包含但不限于MAGE1、MAGE3、MAGE6、BAGE、GAGE、NYESO-1、MART1/Melan A、MC1R、GP100、酪氨酸酶、TRP-1、TRP-2、PSA、CEA、Cyp-B、Her2/Neu、hTERT、MUC1、PRAME、WT1、RAS、CDK-4、MUM-1、KRAS、MSLN和β-连环蛋白。Exemplary genes that can be targeted include, but are not limited to, T cell receptor, B cell receptor, CTLA4, PD1, FOXO1, FOXO3, AKTs, CCR5, CXCR4, LAG3, TIM3, killer immunoglobulin-like receptor, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNFR-II, IL-7R, OX-4 , CD4, CD8, CD9, CD10, CD11, CD12, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK-R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NFAT, IL2R and IL7. Other exemplary genes that can be targeted include, but are not limited to, OCT, G6Pase, Mut, PCCA, PCCB, PCSK9, ALAS1 and PAH. Exemplary tumor-associated antigens that may be recognized by T cells and are contemplated for targeting include, but are not limited to, MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1/Melan A, MC1R, GP100, tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2/Neu, hTERT, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN, and β-catenin.

D.待治疗的受试者D. Subjects to be treated

在一些实施例中，被治疗的受试者是患有癌症、自体免疫疾病、感染性疾病、器官移植、器官衰竭、蛋白质缺乏或其组合的哺乳动物。在一些实施例中，受试者为人类。在一些实施例中，本文所述的方法可使肝细胞翻译某些蛋白质。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送到肝细胞。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送到脾T细胞。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送到脾B细胞。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送到脾单核细胞。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送到骨髓细胞。在一些实施例中，本文所述的方法可以用于将一种或多种DNA、mRNA、sgRNA或siRNA递送至肺细胞。In some embodiments, the subject being treated is a mammal suffering from cancer, autoimmune disease, infectious disease, organ transplantation, organ failure, protein deficiency, or a combination thereof. In some embodiments, the subject is human. In some embodiments, the methods described herein can cause liver cells to translate certain proteins. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to liver cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to spleen T cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to spleen B cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to spleen mononuclear cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to bone marrow cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to lung cells.

应理解，只要本发明仍然可操作，步骤的顺序或执行某个动作的顺序就是不重要的。此外，两个或更多个步骤或动作可以同时进行。Should be understood that, as long as the present invention is still operable, the order of steps or the order in which a certain action is performed is not important. In addition, two or more steps or actions can be performed simultaneously.

虽然本发明已经参考特定的优选实施例进行了具体的展示和描述，但本领域的技术人员应理解，在不偏离由所附权利要求书所界定的本发明的精神和范围的情况下，可以在本文中进行形式和细节的各种变化。While the present invention has been particularly shown and described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the following claims.

示例性实施例Exemplary Embodiments

以下编号的实施例虽然是非限制性的，但是是本公开的某些方面的示例性实施例：1.一种式I化合物：The following numbered examples, although non-limiting, are exemplary embodiments of certain aspects of the present disclosure: 1. A compound of formula I:

或其N-氧化物、或其药学上可接受的盐，其中：or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein:

L¹、L^1'和L^1”中的每一者独立地为不存在的、-C(O)-或-OC(O)-；Each of L¹ , L^1′ and L^1″ is independently absent, —C(O)— or —OC(O)—;

L²、L^2'和L^2”中的每一者独立地为不存在的、任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-；Each of L² , L^2′ and L^2″ is independently absent, an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_m′ -;

每个Cy^A独立地为选自亚苯基和3至12元饱和或部分不饱和的亚碳环基的任选地被取代的环；each Cy^A is independently an optionally substituted ring selected from phenylene and a 3- to 12-membered saturated or partially unsaturated carbocyclylene;

m和m'中的每一者独立地为0、1或2；Each of m and m' is independently 0, 1 or 2;

L³、L^3'和L^3”中的每一者独立地为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-；each of L³ , L^3′, and L^3″ is independently absent, —C(O)—, —C(O)O—, —OC(O)—, —O—, or —OC(O)O—;

R¹、R^1'和R^1”中的每一者独立地为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each of R¹ , R^1′ and R^1″ is independently -(CH₂ )_p -Cy^B , or an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;

每个Cy^B独立地为选自以下的任选地被取代的环：3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧或硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each Cy^B is independently an optionally substituted ring selected from: a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

每个p独立地为0、1、2或3；Each p is independently 0, 1, 2 or 3;

每个L^3a独立地为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each L^3a is independently absent or optionally substituted divalent saturated or unsaturated linear or branched C_1-10 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;

每个R^a独立地为氢或选自以下的任选地被取代的基团：C_6-20脂肪族、3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧或硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each^{Ra is} independently hydrogen or an optionally substituted group selected from the group consisting of a_C6-20 aliphatic, a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

X¹为不存在的、-O-、-S-或-NR-；^X1 is absent, -O-, -S- or -NR-;

X²为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换；^X2 is absent or optionally substituted divalent saturated or unsaturated linear or branched_C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or^-CyC- ;

Cy^C为选自以下的任选地被取代的环：3至7元饱和或部分不饱和的亚碳环、亚苯基、具有1-3个独立地选自氮、氧或硫的杂原子的3至7元饱和或部分不饱和的亚杂环或具有1-3个独立地选自氮、氧或硫的杂原子的5至6元亚杂芳基；Cy^C is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur, or a 5- to 6-membered heteroarylene group having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur;

X³为氢或选自以下的任选地被取代的环：3至7元饱和或部分不饱和的碳环基、苯基、具有1-3个独立地选自氮、氧或硫的杂原子的3至7元饱和或部分不饱和的杂环基以及具有1-3个独立地选自氮、氧或硫的杂原子的5至6元杂芳基；并且^X3 is hydrogen or an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a phenyl, a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur, and a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur; and

每个R独立地为氢或任选地被取代的C_1-6脂肪族基团。Each R is independently hydrogen or an optionally substituted C_1-6 aliphatic group.

2.一种根据实施例1所述的化合物，其中该化合物具有式I-A：2. A compound according to embodiment 1, wherein the compound has formula I-A:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

3.一种根据实施例1或2所述的化合物，其中该化合物具有式I-A-a：3. A compound according to embodiment 1 or 2, wherein the compound has formula I-A-a:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

4.一种根据实施例1或2所述的化合物，其中该化合物具有式I-A-b：4. A compound according to Embodiment 1 or 2, wherein the compound has Formula I-A-b:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

5.一种根据实施例1、2和4中任一项所述的化合物，其中该化合物具有式I-A-c：5. A compound according to any one of embodiments 1, 2 and 4, wherein the compound has Formula I-A-c:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

6.一种根据实施例1或2所述的化合物，其中该化合物具有式I-B：6. A compound according to Embodiment 1 or 2, wherein the compound has Formula I-B:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

7.一种根据实施例1、2和6中任一项所述的化合物，其中该化合物具有式I-B-a：7. A compound according to any one of embodiments 1, 2 and 6, wherein the compound has Formula I-B-a:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

8.一种根据实施例1、2和6中任一项所述的化合物，其中该化合物具有式I-B-b：8. A compound according to any one of embodiments 1, 2 and 6, wherein the compound has Formula I-B-b:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

9.一种根据实施例1、2、6和8中任一项所述的化合物，其中该化合物具有式I-B-c：9. A compound according to any one of embodiments 1, 2, 6 and 8, wherein the compound has Formula I-B-c:

或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

10.一种根据前述实施例中任一项所述的化合物，其中该化合物具有式I-C：10. A compound according to any one of the preceding embodiments, wherein the compound has Formula I-C:

或其N-氧化物、或其药学上可接受的盐。 or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.

11.根据前述实施例中任一项所述的化合物，其中该化合物为式I、I-A、I-A-a、I-A-b、I-A-c、I-B、I-B-a、I-B-b、I-B-c和I-C中任一者的化合物或其药学上可接受的盐。11. A compound according to any of the preceding embodiments, wherein the compound is a compound of any one of Formula I, I-A, I-A-a, I-A-b, I-A-c, I-B, I-B-a, I-B-b, I-B-c and I-C, or a pharmaceutically acceptable salt thereof.

12.根据实施例1-11中任一项所述的化合物，其中L¹为-C(O)-。12. The compound of any one of embodiments 1-11 wherein L¹ is -C(O)-.

13.根据实施例1-12中任一项所述的化合物，其中L^1'为-C(O)-。13. The compound of any one of embodiments 1-12 wherein L^1′ is —C(O)—.

14.根据实施例1-13中任一项所述的化合物，其中L^1”为-C(O)-。14. The compound of any one of embodiments 1-13, wherein L^1″ is -C(O)-.

15.根据实施例1-14中任一项所述的化合物，其中L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。15. The compound of any one of embodiments 1-14 wherein L² is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain.

16.根据实施例1-15中任一项所述的化合物，其中L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。16. The compound of any one of embodiments 1-15 wherein L² is an optionally substituted divalent saturated or unsaturated linear or branched C_1-8 hydrocarbon chain.

17.根据实施例1-16中任一项所述的化合物，其中L²为-CH₂-、-(CH₂)₄-、-(CH₂)₅-或-(CH₂)₆-。17. The compound of any one of embodiments 1-16 wherein L² is -CH₂ -, -(CH₂ )₄ -, -(CH₂ )₅ -, or -(CH₂ )₆ -.

18.根据实施例1-17中任一项所述的化合物，其中L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。18. The compound of any one of embodiments 1-17 wherein L^2' is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain.

19.根据实施例1-18中任一项所述的化合物，其中L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。19. The compound of any one of embodiments 1-18 wherein L^2' is an optionally substituted divalent saturated or unsaturated linear or branched C_1-8 hydrocarbon chain.

20.根据实施例1-19中任一项所述的化合物，其中L^2'为-CH₂-、-(CH₂)₄-、-(CH₂)₅-或-(CH₂)₆-。20. The compound of any one of embodiments 1-19 wherein L^2' is -CH₂ -, -(CH₂ )₄ -, -(CH₂ )₅ -, or -(CH₂ )₆ -.

21.根据实施例1-20中任一项所述的化合物，其中L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。21. The compound of any one of embodiments 1-20, wherein L^2″ is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain.

22.根据实施例1-21中任一项所述的化合物，其中L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-8烃链。22. The compound of any one of embodiments 1-21, wherein L^2″ is an optionally substituted divalent saturated or unsaturated linear or branched C_1-8 hydrocarbon chain.

23.根据实施例1-22中任一项所述的化合物，其中L^2”为-CH₂-、-(CH₂)₄-、-(CH₂)₅-或-(CH₂)₆-。23. The compound of any one of embodiments 1-22, wherein L^2" is -CH₂ -, -(CH₂ )₄ -, -(CH₂ )₅ -, or -(CH₂ )₆ -.

24.根据实施例1-23中任一项所述的化合物，其中L³为不存在的。24. A compound according to any one of embodiments 1-23, wherein L³ is absent.

25.根据实施例1-24中任一项所述的化合物，其中L^3'为不存在的。25. The compound of any one of embodiments 1-24, wherein L^3' is absent.

26.根据实施例1-25中任一项所述的化合物，其中L^3”为不存在的。26. A compound according to any one of embodiments 1-25, wherein L^3" is absent.

27.根据实施例1-23中任一项所述的化合物，其中L³为-C(O)O-。27. The compound of any one of embodiments 1-23, wherein L³ is -C(O)O-.

28.根据实施例1-24和26-27中任一项所述的化合物，其中L^3'为-C(O)O-。28. The compound of any one of embodiments 1-24 and 26-27 wherein L^3' is -C(O)O-.

29.根据实施例1-25和27-28中任一项所述的化合物，其中L^3”为-C(O)O-。29. The compound of any one of embodiments 1-25 and 27-28, wherein L^3" is -C(O)O-.

30.根据实施例1-29中任一项所述的化合物，其中R¹为-(CH₂)_p-Cy^B。30. The compound of any one of embodiments 1-29 wherein R¹ is -(CH₂ )_p -Cy^B .

31.根据实施例1-29中任一项所述的化合物，其中R¹为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。31. The compound of any one of embodiments 1-29 wherein R¹ is an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.

32.根据实施例1-29和31中任一项所述的化合物，其中R¹为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。32. The compound of any one of embodiments 1-29 and 31 wherein R¹ is an optionally substituted saturated or unsaturated linear or branched C_6-12 hydrocarbon chain.

33.根据实施例1-29中任一项所述的化合物，其中R¹为33. A compound according to any one of embodiments 1-29, wherein R¹ is

34.根据实施例1-33中任一项所述的化合物，其中R^1'为-(CH₂)_p-Cy^B。34. The compound of any one of embodiments 1-33 wherein R^1′ is —(CH₂ )_p —Cy^B .

35.根据实施例1-33中任一项所述的化合物，其中R^1'为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。35. The compound of any one of embodiments 1-33 wherein R^1′ is an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.

36.根据实施例1-33和35中任一项所述的化合物，其中R^1'为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。36. The compound of any one of embodiments 1-33 and 35, wherein R^1′ is an optionally substituted saturated or unsaturated linear or branched C_6-12 hydrocarbon chain.

37.根据实施例1-33中任一项所述的化合物，其中R^1'为37. A compound according to any one of embodiments 1-33, wherein R^1' is

38.根据实施例1-37中任一项所述的化合物，其中R^1”为-(CH₂)_p-Cy^B。38. The compound of any one of embodiments 1-37, wherein R^1″ is -(CH₂ )_p -Cy^B .

39.根据实施例1-37中任一项所述的化合物，其中R^1”为任选地被取代的二价饱和或不饱和的直链或支链C_1-20烃链。39. The compound of any one of embodiments 1-37, wherein R^1″ is an optionally substituted divalent saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.

40.根据实施例1-37和39中任一项所述的化合物，其中R^1”为任选地被取代的饱和或不饱和的直链或支链C_6-12烃链。40. The compound of any one of embodiments 1-37 and 39, wherein R^1″ is an optionally substituted saturated or unsaturated linear or branched C_6-12 hydrocarbon chain.

41.根据实施例1-37中任一项所述的化合物，其中R^1”为41. A compound according to any one of embodiments 1-37, wherein R^1" is

42.根据实施例1-41中任一项所述的化合物，其中每个Cy^B为1-金刚烷基。42. The compound of any one of embodiments 1-41 wherein each Cy^B is 1-adamantyl.

43.根据实施例1-42中任一项所述的化合物，其中每个p独立地为0或1。43. The compound of any one of embodiments 1-42, wherein each p is independently 0 or 1.

44.根据实施例1-43中任一项所述的化合物，其中X¹为-O-。44. The compound of any one of embodiments 1-43, wherein X¹ is -O-.

45.根据实施例1-43中任一项所述的化合物，其中X¹为-NR-。45. The compound of any one of embodiments 1-43, wherein X¹ is -NR-.

46.根据实施例1-45中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换。46. A compound according to any one of embodiments 1-45, wherein^X2 is an optionally substituted divalent saturated or unsaturated linear or branched_C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or^-CyC- .

47.根据实施例1-46中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1个亚甲基单元被-NR-置换。47. The compound of any one of embodiments 1-46, wherein X² is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain in which one methylene unit is replaced by -NR-.

48.根据实施例1-47中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_4-8烃链，其中1个亚甲基单元被-NR-置换。48. The compound of any one of embodiments 1-47, wherein X² is an optionally substituted divalent saturated or unsaturated linear or branched C_4-8 hydrocarbon chain in which 1 methylene unit is replaced by -NR-.

49.根据实施例1-48中任一项所述的化合物，其中X³为具有1-3个独立地选自氮、氧或硫的杂原子的任选地被取代的3至7元饱和或部分不饱和的杂环基。49. The compound according to any one of embodiments 1-48, wherein X³ is an optionally substituted 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur.

50.根据实施例1-49中任一项所述的化合物，其中X³为具有1-2个氮原子的任选地被取代的5元饱和或部分不饱和的杂环基。50. The compound according to any one of embodiments 1-49, wherein X³ is an optionally substituted 5-membered saturated or partially unsaturated heterocyclyl having 1-2 nitrogen atoms.

51.根据前述实施例中任一项所述的化合物，其中-X²-X³为51. A compound according to any one of the preceding embodiments, wherein -X² -X³ is

52.根据实施例1-51中任一项所述的化合物，其中R为氢。52. The compound of any one of embodiments 1-51 wherein R is hydrogen.

53.根据实施例1-51中任一项所述的化合物，其中R为任选地被取代的C_1-6脂肪族。53. The compound of any one of embodiments 1-51 wherein R is optionally substituted C_1-6 aliphatic.

54.根据实施例1-51和53中任一项所述的化合物，其中R为-CH₂CH₃。54. The compound of any one of embodiments 1-51 and 53, wherein R is -CH₂ CH₃ .

55.根据实施例1所述的化合物，其中所述化合物选自表1，或其药学上可接受的盐。55. The compound of Embodiment 1, wherein the compound is selected from Table 1, or a pharmaceutically acceptable salt thereof.

56.一种脂质纳米粒子(LNP)制剂，其包含根据实施例1-55中任一项所述的可电离脂质。56. A lipid nanoparticle (LNP) formulation comprising the ionizable lipid according to any one of embodiments 1-55.

57.一种脂质纳米粒子(LNP)制剂，其包含：57. A lipid nanoparticle (LNP) formulation comprising:

根据实施例1-55中任一项所述的可电离脂质；The ionizable lipid according to any one of embodiments 1-55;

磷脂；Phospholipids;

甾醇；和Sterols; and

缀合物-连接子脂质(例如，聚乙二醇脂质)。Conjugate - Linker lipid (eg, polyethylene glycol lipid).

58.根据实施例56所述的LNP制剂，其进一步包含治疗剂和/或预防剂。58. The LNP formulation of embodiment 56, further comprising a therapeutic and/or prophylactic agent.

59.根据实施例58所述的LNP制剂，其中所述治疗剂和/或预防剂为或包括一种或多种核酸。59. The LNP formulation of embodiment 58, wherein the therapeutic and/or prophylactic agent is or includes one or more nucleic acids.

60.根据实施例59所述的LNP制剂，其中所述一种或多种核酸为或包括RNA。60. The LNP formulation of embodiment 59, wherein the one or more nucleic acids is or comprises RNA.

61.根据实施例59所述的LNP制剂，其中所述一种或多种核酸为或包括DNA。61. The LNP formulation of embodiment 59, wherein the one or more nucleic acids is or comprises DNA.

62.根据实施例58-61中任一项所述的LNP制剂，其中所述LNP制剂经调配成将所述治疗剂和/或预防剂递送到靶细胞。62. The LNP formulation of any one of embodiments 58-61, wherein the LNP formulation is formulated to deliver the therapeutic and/or prophylactic agent to a target cell.

63.根据实施例62所述的LNP制剂，其中该靶细胞为或包括脾细胞(例如，脾B细胞、脾T细胞、脾单核细胞)、肝脏细胞(例如，肝细胞(hepatocyte))、骨髓细胞(例如，骨髓单核细胞)、免疫细胞、肾细胞、肌肉细胞、心脏细胞或中枢神经系统中的细胞。63. The LNP formulation of embodiment 62, wherein the target cell is or comprises a spleen cell (e.g., a spleen B cell, a spleen T cell, a spleen monocyte), a liver cell (e.g., a hepatocyte), a bone marrow cell (e.g., a bone marrow mononuclear cell), an immune cell, a kidney cell, a muscle cell, a heart cell, or a cell in the central nervous system.

64.根据实施例63所述的LNP制剂，其中所述靶细胞为或包括造血干细胞(HSC)。64. The LNP formulation of embodiment 63, wherein the target cell is or comprises a hematopoietic stem cell (HSC).

65.一种药物组合物，其包含根据实施例56所述的LNP制剂以及药学上可接受的赋形剂。65. A pharmaceutical composition comprising the LNP formulation according to embodiment 56 and a pharmaceutically acceptable excipient.

66.一种用于向有此需要的受试者施用治疗剂和/或预防剂的方法，该方法包括：向该受试者施用根据实施例56所述的LNP制剂或根据实施例65所述的药物组合物。66. A method for administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising: administering to the subject the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65.

67.一种用于治疗有此需要的受试者的疾病或病症的方法，该方法包括：向该受试者施用根据实施例56所述的LNP制剂或根据实施例65所述的药物组合物，其中该治疗剂和/或预防剂能有效治疗该疾病。67. A method for treating a disease or condition in a subject in need thereof, the method comprising: administering to the subject the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65, wherein the therapeutic and/or prophylactic agent is effective in treating the disease.

68.一种用于延迟和/或遏制有此需要的受试者的疾病或病症进展的方法，该方法包括：向该受试者施用根据实施例56所述的LNP制剂或根据实施例65所述的药物组合物，其中该治疗剂和/或预防剂能有效治疗该疾病。68. A method for delaying and/or arresting the progression of a disease or condition in a subject in need thereof, the method comprising: administering to the subject the LNP formulation according to embodiment 56 or the pharmaceutical composition according to embodiment 65, wherein the therapeutic agent and/or prophylactic agent is effective in treating the disease.

69.一种将治疗剂和/或预防剂递送至来源于受试者的哺乳动物细胞的方法，该方法包括：接触已经施用了根据实施例56所述的LNP制剂或实施例65所述的药物组合物的该受试者的该细胞。69. A method of delivering a therapeutic and/or prophylactic agent to a mammalian cell derived from a subject, the method comprising: contacting the cell of the subject to which the LNP formulation of Example 56 or the pharmaceutical composition of Example 65 has been administered.

70.一种在哺乳动物细胞中产生所关注的多肽的方法，该方法包括：使该细胞与根据实施例56所述的LNP制剂或实施例65所述的药物组合物接触，其中治疗剂和/或预防剂为或包含mRNA，并且其中该mRNA编码所述所关注的多肽，其中该mRNA能够在该细胞中被翻译以产生所述所关注的多肽。70. A method for producing a polypeptide of interest in a mammalian cell, the method comprising: contacting the cell with the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65, wherein the therapeutic and/or prophylactic agent is or comprises mRNA, and wherein the mRNA encodes the polypeptide of interest, wherein the mRNA can be translated in the cell to produce the polypeptide of interest.

71.一种抑制在哺乳动物细胞中产生所关注的多肽的方法，该方法包括：使该细胞与根据实施例56所述的LNP制剂或实施例65所述的药物组合物接触，其中治疗剂和/或预防剂为或包含RNA，其中该RNA能够抑制所述所关注的多肽的产生。71. A method for inhibiting the production of a polypeptide of interest in a mammalian cell, the method comprising: contacting the cell with the LNP formulation according to embodiment 56 or the pharmaceutical composition of embodiment 65, wherein the therapeutic agent and/or prophylactic agent is or comprises RNA, wherein the RNA is capable of inhibiting the production of the polypeptide of interest.

72.一种将治疗剂和/或预防剂特异性地递送至哺乳动物器官的方法，该方法包括：使哺乳动物器官与根据实施例56所述的LNP制剂或实施例65所述的药物组合物接触，其中将该治疗剂和/或预防剂递送至该器官。72. A method of specifically delivering a therapeutic and/or prophylactic agent to a mammalian organ, the method comprising: contacting the mammalian organ with the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65, wherein the therapeutic and/or prophylactic agent is delivered to the organ.

73.根据实施例72所述的方法，其包括：向受试者施用根据实施例56所述的LNP制剂或实施例65所述的药物组合物。73. The method of embodiment 72, comprising administering to a subject the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65.

74.一种通过施用根据实施例56所述的LNP制剂或根据实施例65所述的药物组合物来接种疫苗的方法。74. A method of vaccination by administering the LNP formulation of embodiment 56 or the pharmaceutical composition of embodiment 65.

75.一种在受试者中诱导适应性免疫应答的方法，该方法包括：向受试者施用有效量的组合物，该组合物包含至少一种RNA；其中该组合物包含LNP制剂，该LNP制剂包含根据实施例1所述的化合物或其药学上可接受的盐。75. A method of inducing an adaptive immune response in a subject, the method comprising: administering to the subject an effective amount of a composition comprising at least one RNA; wherein the composition comprises an LNP formulation comprising a compound according to Example 1 or a pharmaceutically acceptable salt thereof.

举例说明Example

本公开举例说明本文所述的组合物、制剂、调配物、纳米粒子和/或纳米材料。本公开还举例说明制备、表征和验证本文所述的组合物、制剂、调配物、纳米粒子和/或纳米材料的方法。The present disclosure exemplifies the compositions, formulations, formulations, nanoparticles and/or nanomaterials described herein. The present disclosure also exemplifies methods of making, characterizing and validating the compositions, formulations, formulations, nanoparticles and/or nanomaterials described herein.

实例1：材料和方法Example 1: Materials and Methods

本实施例提供制备、表征和验证本文所述的组合物、制剂、纳米粒子和/或纳米材料的示范性材料和方法。This example provides exemplary materials and methods for preparing, characterizing, and validating the compositions, formulations, nanoparticles, and/or nanomaterials described herein.

LNP制剂LNP formulation

此外，本实施例提供示范性LNP制剂。In addition, this example provides exemplary LNP formulations.

脂质纳米粒子组分以指定脂质组分摩尔比溶解于100％乙醇中。将核酸(NA)货物溶解于10mM柠檬酸盐、100mM NaCl(pH 4.0)中，使得NA货物的浓度大约为0.22mg/mL。在一些实施例中，NA货物包含以1:10到10:1的功能性NA与条形码的质量比混合的功能性NA和报告DNA条形码两者。如本文所述，NA可为siRNA、反义、表达DNA或mRNA。The lipid nanoparticle components are dissolved in 100% ethanol at a specified lipid component molar ratio. The nucleic acid (NA) cargo is dissolved in 10 mM citrate, 100 mM NaCl (pH 4.0) so that the concentration of the NA cargo is approximately 0.22 mg/mL. In some embodiments, the NA cargo comprises both functional NA and reporter DNA barcodes mixed at a mass ratio of 1:10 to 10:1 of functional NA to barcode. As described herein, the NA can be siRNA, antisense, expressed DNA, or mRNA.

以总脂质与NA质量比为11.7来制备LNP。根据制造商的方案，通过使用PrecisionNanosystems NanoAssemblr Spark或Benchtop系列仪器对脂质和NA溶液进行微流体混合，形成LNP。在使用不同差分流动速率进行混合期间，水溶液与有机溶剂的比保持在大约2:1或3:1。在混合之后，收集LNP，稀释于PBS中(大约1:1v/v)。以20kDa过滤器为背景，在4℃使用PBS透析4至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP制剂经由动态光散射(DLS)表征，以测量大小(例如，直径)和多分散性。此外，经由2-(对甲苯胺基)-6-萘磺酸(TNS)测定来测量LNP亚群的pKa。合并特定直径和多分散范围内的LNP，并且以100kDa透析盒为背景，在4℃进一步对磷酸盐缓冲盐水(PBS)透析1至4小时。在第二次透析之后，使用0.22μM过滤器无菌过滤LNP且存储于4℃以供进一步使用。LNPs are prepared with a total lipid to NA mass ratio of 11.7. According to the manufacturer's protocol, lipid and NA solutions are mixed with microfluidics using PrecisionNanosystems NanoAssemblr Spark or Benchtop series instruments to form LNPs. During mixing using different differential flow rates, the ratio of aqueous solution to organic solvent is maintained at about 2:1 or 3:1. After mixing, LNPs are collected and diluted in PBS (about 1:1v/v). With 20kDa filter as background, PBS is dialyzed for 4 to 24 hours at 4°C for further buffer exchange. After this initial dialysis, each individual LNP preparation is characterized via dynamic light scattering (DLS) to measure size (e.g., diameter) and polydispersity. In addition, the pKa of LNP subpopulations is measured via 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) determination. LNPs within a specific diameter and polydispersity range are merged, and with 100kDa dialysis cassettes as background, phosphate buffered saline (PBS) is further dialyzed for 1 to 4 hours at 4°C. After the second dialysis, the LNPs were sterile filtered using a 0.22 μM filter and stored at 4° C. for further use.

LNP表征LNP characterization

使用高通量动态光散射(DLS)(DynaPro plate reader II,Wyatt)测量DLS-LNP流体动力学直径和多分散指数(PDI)。用1X PBS将LNP稀释到适当浓度且进行分析。DLS-LNP hydrodynamic diameter and polydispersity index (PDI) were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). LNPs were diluted to appropriate concentrations with IX PBS and analyzed.

浓度和包裹功效Concentration and packaging efficacy

按照制造商说明书，通过Qubit microRNA试剂盒(用于siRNA)或HS RNA试剂盒(用于mRNA)测定NA的浓度。通过测量未裂解和裂解的LNP中的核酸浓度来测定包裹效率。The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or the HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring the concentration of nucleic acids in uncleaved and cleaved LNPs.

pKapKa

制备10mM HEPES(Sigma Aldrich)、10mM MES(Sigma Aldrich)、10mM醋酸钠(Sigma)和140nM氯化钠(Sigma Aldrich)的储备溶液，并且使用氯化氢和氢氧化钠将pH调节至约pH 4-10的范围。针对每个pH值重复四次，将140μL pH经过调节的缓冲液添加到96孔板中，然后添加5μL 2-(对甲苯胺基)-6-萘磺酸(60μg/mL)。每孔添加5μLLNP。在轻缓摇动下培育5min之后，使用325nm的激发波长和435nm的发射波长(BioTek Synergy H4 Hybrid)测量荧光。Prepare a stock solution of 10mM HEPES (Sigma Aldrich), 10mM MES (Sigma Aldrich), 10mM sodium acetate (Sigma) and 140nM sodium chloride (Sigma Aldrich), and use hydrogen chloride and sodium hydroxide to adjust the pH to a range of about pH 4-10. Repeat four times for each pH value, add 140μL pH-adjusted buffer to a 96-well plate, then add 5μL 2-(p-toluidine)-6-naphthalenesulfonic acid (60μg/mL). Add 5μL LNPs to each well. After cultivating for 5min under gentle shaking, measure fluorescence using an excitation wavelength of 325nm and an emission wavelength of 435nm (BioTek Synergy H4 Hybrid).

LNP施用LNP administration

将大约8-12周龄的雄性和雌性小鼠用于本实例所述的研究。每只小鼠都被暂时束缚，并且经由尾静脉注射每次实验向最多五只动物经静脉内(IV)施用合并的LNP。也使用年龄匹配的小鼠经由尾静脉注射每次实验向最多三只动物施用媒剂(1X PBS)。在给药后72小时，收集包含肝脏、脾脏、骨髓、肾脏、肺、肌肉和血液的组织进行分析。Male and female mice of approximately 8-12 weeks of age were used for the studies described in this example. Each mouse was temporarily restrained and the combined LNPs were administered intravenously (IV) to up to five animals per experiment via tail vein injection. Age-matched mice were also used to administer vehicle (1X PBS) to up to three animals per experiment via tail vein injection. 72 hours after administration, tissues including liver, spleen, bone marrow, kidney, lung, muscle, and blood were collected for analysis.

流量flow

对肝脏、肾脏、肺和肌肉组织进行机械消化，并且然后使用蛋白酶混合物进行酶促消化，然后通过70uM过滤器以生成单细胞悬浮液。脾组织被机械消化以产生单细胞悬浮液。所有组织均用(氯化铵钾)ACK缓冲液处理以裂解红细胞，并且然后用荧光标记抗体染色以进行流式细胞术和荧光激活细胞分选(FACS)。使用市售抗体。使用BD FACSMelody(BectonDickinson)，经由流式细胞术采集样品以在分选之前生成门。一般来说，门控结构是大小→单峰细胞→活细胞→所关注的细胞。T细胞被定义为CD45+CD3+，单核细胞被定义为CD45+CD11b+，并且B细胞被定义为CD45+CD19+。内皮细胞被定义为CD31+，单核细胞和库普弗细胞被定义为CD45+CD11b+，并且肝脏细胞被定义为CD31-/CD45-。对于siRNA研究，靶基因的下调是门控的。对于mRNA研究，靶基因的上调是门控的。来自给药媒介物的小鼠的组织用于设置用于分选的门。每个具有正确表型的细胞子集的多达100万个细胞被分选到PBS中。在分选之后，经由离心沉淀细胞，并根据制造商的方案使用Quick Extract DNA提取溶液(Lucigen)提取DNA。DNA提取后，将DNA储存于-20℃。The liver, kidney, lung and muscle tissue are mechanically digested, and then enzymatic digestion is performed using a protease mixture, and then passed through a 70uM filter to generate a single cell suspension. Spleen tissue is mechanically digested to produce a single cell suspension. All tissues are treated with (ammonium chloride potassium) ACK buffer to lyse red blood cells, and then stained with fluorescently labeled antibodies to perform flow cytometry and fluorescence activated cell sorting (FACS). Commercially available antibodies are used. Using BD FACSMelody (BectonDickinson), samples are collected via flow cytometry to generate gates before sorting. In general, the gated structure is size → single peak cells → live cells → cells of interest. T cells are defined as CD45+CD3+, monocytes are defined as CD45+CD11b+, and B cells are defined as CD45+CD19+. Endothelial cells are defined as CD31+, monocytes and Kupffer cells are defined as CD45+CD11b+, and liver cells are defined as CD31-/CD45-. For siRNA studies, downregulation of target genes is gated. For mRNA studies, upregulation of target genes is gated. Tissues from mice dosed with vehicle are used to set gates for sorting. Up to 1 million cells of each subset of cells with the correct phenotype are sorted into PBS. After sorting, cells are precipitated via centrifugation and DNA is extracted using Quick Extract DNA extraction solution (Lucigen) according to the manufacturer's protocol. After DNA extraction, DNA is stored at -20 °C.

条形码测序Barcode sequencing

使用QuickExtract(Lucigen)分离DNA(基因组和DNA条形码)，并使用本文所述的Illumina MiniSeq进行测序，将FACS分离样品中的频率DNA条形码计数归一化为注射输入中的频率。将这些数据绘制为“归一化的倍数输入”(数据未示出)。DNA (genomic and DNA barcodes) was isolated using QuickExtract (Lucigen) and sequenced using the Illumina MiniSeq described herein, and the frequency DNA barcode counts in the FACS-isolated samples were normalized to the frequency in the injection input. These data were plotted as "normalized multiple input" (data not shown).

确认confirm

所提供的LNP的结构和功能特征基于本文所述的方案来进行确认。The structural and functional characteristics of the provided LNPs were confirmed based on the protocols described herein.

LNP制备LNP preparation

脂质纳米粒子组分以指定脂质组分摩尔比溶解于100％乙醇中。将核酸(NA)货物溶解于10mM柠檬酸盐、100mM NaCl(pH 4.0)中，使得NA货物的浓度大约为0.22mg/mL。在一些实施例中，NA货物包含以1:10到10:1的功能性NA与条形码的质量比混合的功能性NA和报告DNA条形码两者。以总脂质与NA质量比为11.7来调配LNP。根据制造商的方案，通过使用Precision Nanosystems NanoAssemblr Spark或Benchtop系列仪器对脂质和NA溶液进行微流体混合，形成LNP。在使用差分流动速率进行混合期间，水溶液与有机溶剂的比率保持在2:1或3:1。在混合之后，收集LNP，稀释于PBS中(大约1:1v/v)，并且以20kDa过滤器为背景，在4℃使用PBS透析8至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP调配物经由DLS表征，以测量大小和多分散性，并且经由TNS分析测量LNP亚群的pKa。在透析之后，使用0.22微米的无菌过滤器无菌过滤LNP且存储在4℃下以供进一步使用。The lipid nanoparticle components are dissolved in 100% ethanol at a specified lipid component molar ratio. The nucleic acid (NA) cargo is dissolved in 10mM citrate, 100mM NaCl (pH 4.0) to a concentration of approximately 0.22mg/mL of the NA cargo. In some embodiments, the NA cargo comprises both functional NA and reporter DNA barcodes mixed at a mass ratio of 1:10 to 10:1 of the functional NA to the barcode. LNPs are formulated with a total lipid to NA mass ratio of 11.7. According to the manufacturer's protocol, lipid and NA solutions are microfluidically mixed using Precision Nanosystems NanoAssemblr Spark or Benchtop series instruments to form LNPs. During mixing using differential flow rates, the ratio of aqueous solution to organic solvent is maintained at 2:1 or 3:1. After mixing, LNPs are collected, diluted in PBS (approximately 1:1 v/v), and further buffer exchange is performed at 4°C using PBS for 8 to 24 hours with a 20kDa filter as the background. After this initial dialysis, each individual LNP formulation was characterized via DLS to measure size and polydispersity, and the pKa of LNP subpopulations was measured via TNS analysis.After dialysis, the LNPs were sterile filtered using a 0.22 micron sterile filter and stored at 4°C for further use.

LNP表征LNP characterization

DLSDLS

使用高通量动态光散射(DLS)(DynaPro plate reader II,Wyatt)测量LNP流体动力学直径和多分散指数(PDI)。用1X PBS将LNP稀释到适当浓度且进行分析。LNP hydrodynamic diameter and polydispersity index (PDI) were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt).LNPs were diluted to appropriate concentrations with IX PBS and analyzed.

浓度和包裹功效Concentration and packaging efficacy

按照制造商说明书，通过Qubit microRNA试剂盒(用于siRNA)或HS RNA试剂盒(用于mRNA)测定NA的浓度。通过测量未裂解和裂解的LNP来测定包裹效率。The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

pKapKa

制备10mM HEPES(Sigma Aldrich)、10mM MES(Sigma Aldrich)、10mM醋酸钠(Sigma)和140nM氯化钠(Sigma Aldrich)的储备溶液，并且使用氯化氢和氢氧化钠将pH调节至约pH 4-10的范围。针对每个pH重复四次，将140μL pH经过调节的缓冲液添加到96孔板中，然后添加5μL 2-(对甲苯胺基)-6-萘磺酸(60μg/mL)。每孔添加5μL LNP。在轻缓摇动下培育5min之后，使用325nm的激发波长和435nm的发射波长(BioTek Synergy H4 Hybrid)测量荧光。Prepare the stock solution of 10mM HEPES (Sigma Aldrich), 10mM MES (Sigma Aldrich), 10mM sodium acetate (Sigma) and 140nM sodium chloride (Sigma Aldrich), and use hydrogen chloride and sodium hydroxide to adjust pH to the scope of about pH 4-10. Repeat four times for each pH, add 140 μ L pH adjusted buffer to 96-well plates, then add 5 μ L 2-(p-toluidine)-6-naphthalenesulfonic acid (60 μ g/mL). Every hole adds 5 μ L LNP. After cultivating 5min under gentle shaking, use the excitation wavelength of 325nm and the emission wavelength of 435nm (BioTek Synergy H4 Hybrid) to measure fluorescence.

LNP施用LNP administration

将大约8-12周龄的雄性和雌性小鼠用于本实例所述的研究。每只小鼠都被暂时束缚，经由尾静脉注射每次实验向最多五只动物IV施用混合的LNP。也使用年龄匹配的小鼠经由尾静脉注射每次实验向最多三只动物施用媒剂(1X PBS)。还可以采用另外的施用途径，包含脑室内(ICV)、脑池内(ICM)、鞘内(IT)、肌肉内(IM)、雾化、鼻内(IN)、皮下(SC)、关节内和皮内(ID)。在给药后72小时，收集包含肝脏、脾脏、骨髓和血液的组织进行分析。Male and female mice of about 8-12 weeks of age were used for the research described in this example. Each mouse was temporarily restrained and the mixed LNPs were administered IV to up to five animals per experiment via tail vein injection. Age-matched mice were also used to administer vehicle (1X PBS) to up to three animals per experiment via tail vein injection. Other routes of administration can also be used, including intracerebroventricular (ICV), intracisternal (ICM), intrathecal (IT), intramuscular (IM), atomization, intranasal (IN), subcutaneous (SC), intraarticular and intradermal (ID). 72 hours after administration, tissues including liver, spleen, bone marrow and blood were collected for analysis.

流量flow

对肝脏、肾脏、肺和肌肉(例如，骨骼和心脏)组织进行机械消化，并且然后使用蛋白酶混合物进行酶促消化，然后通过70uM过滤器以生成单细胞悬浮液。脾组织被机械消化以产生单细胞悬浮液。所有组织均用ACK缓冲液处理以裂解红细胞，并且然后用荧光标记抗体染色以进行流式细胞术和荧光激活细胞分选(FACS)。本实例中使用市售抗体。使用BDFACSMelody(Becton Dickinson)，经由流式细胞术采集样品以在分选之前生成门。一般来说，门控结构是大小→单峰细胞→活细胞→所关注的细胞。T细胞被定义为CD45+CD3+，单核细胞被定义为CD45+CD11b+，并且B细胞被定义为CD45+CD19+。内皮细胞被定义为CD31+，单核细胞和库普弗细胞被定义为CD45+CD11b+，并且在肝脏和肌肉中肝细胞和肌细胞分别被定义为CD31-/CD45-。来自给药媒介物的小鼠的组织用于设置用于分选的门。Liver, kidney, lung and muscle (e.g., bone and heart) tissues are mechanically digested, and then enzymatic digestion is performed using a protease mixture, and then passed through a 70uM filter to generate a single cell suspension. Spleen tissue is mechanically digested to produce a single cell suspension. All tissues are treated with ACK buffer to lyse red blood cells, and then stained with fluorescently labeled antibodies to perform flow cytometry and fluorescence activated cell sorting (FACS). Commercially available antibodies are used in this example. Using BDFACS Melody (Becton Dickinson), samples are collected via flow cytometry to generate gates before sorting. In general, the gated structure is size → single peak cell → live cell → cell of interest. T cells are defined as CD45+CD3+, monocytes are defined as CD45+CD11b+, and B cells are defined as CD45+CD19+. Endothelial cells are defined as CD31+, monocytes and Kupffer cells are defined as CD45+CD11b+, and in liver and muscle, hepatocytes and myocytes are defined as CD31-/CD45-, respectively. Tissue from mice dosed with vehicle was used to set gates for sorting.

hEPO表达hEPO expression

对于人EPO(hEPO)蛋白表达，小鼠被暂时束缚并在施用后6小时放血(经由尾静脉)。将血液收集于肝素管中，处理成血浆，并存储于-80℃直到准备使用。根据制造商说明书，使用R&D systems ELISA试剂盒(DuoSet；DY286-05)，使用适当稀释的血浆来测量hEPO蛋白。For human EPO (hEPO) protein expression, mice were temporarily restrained and bled (via tail vein) 6 hours after administration. Blood was collected in heparin tubes, processed into plasma, and stored at -80°C until ready for use. hEPO protein was measured using R&D systems ELISA kit (DuoSet; DY286-05) according to the manufacturer's instructions using appropriately diluted plasma.

耐受性Tolerance

ALT/AST定量ALT/AST quantification

对于大鼠天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)定量，在施用后2、4、6、24、48和72小时暂时束缚大鼠并放血。将血液收集于肝素管中，处理成血浆，并存储于-80℃直到准备使用。根据制造商说明书，使用AST/GOT试剂(ThermoFisher,TR70121)对AST进行定量，并使用ALT/GPT试剂(ThermoFisher,TR71121)对ALT进行定量。For rat aspartate aminotransferase (AST) and alanine aminotransferase (ALT) quantification, rats were temporarily restrained and bled at 2, 4, 6, 24, 48, and 72 hours after administration. Blood was collected in heparin tubes, processed into plasma, and stored at -80°C until ready for use. AST was quantified using AST/GOT reagent (ThermoFisher, TR70121) and ALT was quantified using ALT/GPT reagent (ThermoFisher, TR71121) according to the manufacturer's instructions.

大鼠MCP-1ELISARat MCP-1 ELISA

对于大鼠单核细胞趋化蛋白-1(MCP-1)蛋白表达，大鼠被暂时束缚并在施用后2、4、6、24、48和72小时放血。将血液收集于肝素管中，处理成血浆，并存储于-80℃直到准备使用。根据制造商说明书，使用R&D systems ELISA试剂盒(DuoSet；DY3144-05)，使用适当稀释的血浆来测量MCP-1蛋白。For rat monocyte chemoattractant protein-1 (MCP-1) protein expression, rats were temporarily restrained and bled 2, 4, 6, 24, 48 and 72 hours after administration. Blood was collected in heparin tubes, processed into plasma, and stored at -80°C until ready for use. MCP-1 protein was measured using R&D systems ELISA kit (DuoSet; DY3144-05) according to the manufacturer's instructions using appropriately diluted plasma.

筛选实验Screening experiments

如本文所述，可以在单个筛选实验中同时测试多个LNP(例如，超过300个LNP制剂)。在一些实施例中，在单个小鼠中筛选超过300个LNP。在一些实施例中，在单个小鼠中筛选超过850个LNP(参见图1和图2)。筛选实验用于测量向细胞和组织的mRNA或siRNA递送，如本文所述。As described herein, multiple LNPs (e.g., more than 300 LNP preparations) can be tested simultaneously in a single screening experiment. In certain embodiments, more than 300 LNPs are screened in a single mouse. In certain embodiments, more than 850 LNPs are screened in a single mouse (see Figures 1 and 2). Screening experiments are used to measure mRNA or siRNA delivery to cells and tissues, as described herein.

对于mRNA递送，每种LNP制剂被调配成携带Cre mRNA和条形码，如本文所述。根据本文所述的方法(参见图1)，向LSL-tdTom小鼠(Ai14)(也参见图1)施用每种LNP制剂。参考图1，将各自包含一种或多种组分、条形码序列和Cre mRNA的LNP制剂文库施用至Cre-LoxP报道小鼠中。如本文所述，基于细胞是tdTom-还是tdTom+，使用FACS对小鼠细胞进行分选。然后如本文所述对分选的tdTom+细胞进行测序。For mRNA delivery, each LNP preparation is formulated to carry Cre mRNA and barcode, as described herein. According to the method described herein (see Figure 1), each LNP preparation is applied to LSL-tdTom mice (Ai14) (also see Figure 1). With reference to Figure 1, a library of LNP preparations each comprising one or more components, barcode sequences and Cre mRNA is applied to Cre-LoxP reporter mice. As described herein, based on whether the cell is tdTom- or tdTom+, mouse cells are sorted using FACS. Then the sorted tdTom+ cells are sequenced as described herein.

对于siRNA递送，每种LNP制剂被调配成携带siGFP和条形码，如本文所述。根据本文所述的方法(参见图2)，向GFP小鼠(也参见图2)施用每种LNP制剂。参考图2，将各自包含一种或多种组分、条形码序列和siGFP的LNP制剂文库施用至GFP报道小鼠中。如本文所述，基于GFP表达使用FACS对细胞进行分选。然后如本文所述对分选的细胞进行测序。For siRNA delivery, each LNP preparation is formulated to carry siGFP and barcode, as described herein. According to the method described herein (see Figure 2), each LNP preparation is applied to GFP mice (also see Figure 2). With reference to Figure 2, the LNP preparation library comprising one or more components, barcode sequence and siGFP is applied to GFP reporter mice. As described herein, FACS is used to sort cells based on GFP expression. Then the sorted cells are sequenced as described herein.

LNP制剂使用本文所述的化合物或比较化合物来调配。LNP制剂使用MC3作为对照来调配。进行以下测量：LNP制剂直径、LNP制剂多分散性、对细胞类型和组织类型的任何组合的“归一化递送效率”、LNP制剂pKA(与脂质pKA相关但不相同)、脂质pKA和LNP制剂电离度。也测量了每种LNP制剂池的包裹效率和递送效力。如本文所述执行hEPO表达和Cre表达测量。LNP preparations are formulated using compounds described herein or comparative compounds. LNP preparations are formulated using MC3 as a control. The following measurements are performed: LNP preparation diameter, LNP preparation polydispersity, "normalized delivery efficiency" for any combination of cell type and tissue type, LNP preparation pKA (related to but not identical to lipid pKA), lipid pKA, and LNP preparation ionization. The encapsulation efficiency and delivery efficacy of each LNP preparation pool are also measured. hEPO expression and Cre expression measurements are performed as described herein.

实例2：每次筛选的效力Example 2: Effectiveness of each screening

本实例提供示例性组合物、制剂、纳米粒子和/或纳米材料，以及用于筛选本文所述的此类组合物、制剂、纳米粒子和/或纳米材料的效力的材料和方法。This example provides exemplary compositions, formulations, nanoparticles and/or nanomaterials, as well as materials and methods for screening the efficacy of such compositions, formulations, nanoparticles and/or nanomaterials described herein.

如实例1中所述的示例性LNP筛选可以用于证明每个LNP池是跨许多组织高效的。The exemplary LNP screen as described in Example 1 can be used to demonstrate that each LNP pool is highly potent across many tissues.

实例3：具有向各种细胞类型进行有效递送的示例性LNP制剂Example 3: Exemplary LNP Formulations with Efficient Delivery to Various Cell Types

本实例提供用于向各种细胞类型进行有效递送的示例性LNP组合物、制剂、纳米粒子和/或纳米材料，如本文所述。本实例可以用于证明，在一些实施例中，所提供的脂质显示出向各种细胞类型进行有效递送。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials for effective delivery to various cell types, as described herein. This example can be used to demonstrate that, in some embodiments, provided lipids show effective delivery to various cell types.

基于来源于实例1中的筛选的结果，识别了示例性LNP制剂用于脾递送并且特别是用于B细胞递送。这些所识别到的脂质被调配成LNP制剂并使用本文所述的Cre报道系统进行筛选。每组使用三只Ai14小鼠。有效负载包含0.3mg/kg Cre mRNA。注射后168小时收集数据。将结果与作为对照的MC3-LNP制剂进行比较。Based on the results of the screening derived from Example 1, exemplary LNP formulations were identified for spleen delivery and particularly for B cell delivery. These identified lipids were formulated into LNP formulations and screened using the Cre reporting system described herein. Three Ai14 mice were used per group. The payload contained 0.3 mg/kg Cre mRNA. Data were collected 168 hours after injection. The results were compared with the MC3-LNP formulation as a control.

实施例4：将示范性LNP制剂递送到不同细胞类型Example 4: Delivery of Exemplary LNP Formulations to Different Cell Types

本实施例提供强效递送到如本文所述的各种细胞类型的示范性LNP组合物、制剂、纳米粒子和/或纳米材料。本实例可以用于证明所提供的脂质显示出跨各种细胞类型的有效递送。This example provides an exemplary LNP composition, formulation, nanoparticle and/or nanomaterial that is potently delivered to various cell types as described herein. This example can be used to demonstrate that the lipids provided show effective delivery across various cell types.

选择LNP制剂以使用本文所述的Cre报告基因系统和Ai14小鼠模型来确认功效结果。每个实验中每组使用三只Ai14小鼠。注射后72小时收集数据。本文所述的筛选平台可以识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对特定细胞类型最有效。LNP formulations were selected to confirm efficacy results using the Cre reporter gene system and Ai14 mouse model described herein. Three Ai14 mice were used per group in each experiment. Data were collected 72 hours after injection. The screening platform described herein can identify several highly effective LNP formulations to determine which type of LNP formulation is most effective for a specific cell type.

实例5：示例性LNP制剂递送功能性mRNAExample 5: Exemplary LNP formulations delivering functional mRNA

本实例提供向各种细胞类型递送功能性mRNA的示例性LNP组合物、制剂、纳米粒子和/或纳米材料。本实例可以用于证明所提供的脂质能够在小鼠中递送功能性mRNA。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials for delivering functional mRNA to various cell types. This example can be used to demonstrate that the provided lipids can deliver functional mRNA in mice.

选择示例性LNP制剂来确定每种制剂在小鼠中递送功能性mRNA的能力。LNP制剂各自携带0.15mg/kg hEPO mRNA，并以11.7和19的质量比在2-3只C57BL6小鼠中施用。注射LNP制剂六小时后测量血浆中的hEPO表达。Exemplary LNP formulations were selected to determine the ability of each formulation to deliver functional mRNA in mice. Each LNP formulation carried 0.15 mg/kg hEPO mRNA and was administered to 2-3 C57BL6 mice at a mass ratio of 11.7 and 19. hEPO expression in plasma was measured six hours after injection of the LNP formulation.

实例6：耐受性和功效实验Example 6: Tolerance and efficacy experiments

本实施例提供制备、表征和验证本文所述的组合物、制剂、纳米粒子和/或纳米材料的示范性材料和方法。本实例可以用于证明所提供的脂质能够在小鼠中递送功能性mRNA。This example provides exemplary materials and methods for preparing, characterizing and validating the compositions, formulations, nanoparticles and/or nanomaterials described herein. This example can be used to demonstrate that the provided lipids are capable of delivering functional mRNA in mice.

选择示例性LNP制剂以确定hEPO mRNA递送在大鼠中的耐受性和功效，如本文所述。将每种含有1.0mg/kg hEPO mRNA的LNP制剂注射到Sprague-Dawley大鼠(N＝2)中。对于每种示例性LNP制剂，在注射后24小时从大鼠血浆(U/L)收集丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)。每种LNP制剂注射后6小时从大鼠血浆(ng/mL)收集单核细胞趋化蛋白-1(MCP-1)。使用盐水作为对照。在注射后跨各个时间点(0、2、4、6、24、48、96小时)施用媒剂(对照)和示例性LNP制剂后，从大鼠血浆(ng/mL)收集hEPO。Select exemplary LNP preparations to determine the tolerability and efficacy of hEPO mRNA delivery in rats, as described herein. Each LNP preparation containing 1.0mg/kg hEPO mRNA is injected into Sprague-Dawley rats (N=2). For each exemplary LNP preparation, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are collected from rat plasma (U/L) 24 hours after injection. Monocyte chemoattractant protein-1 (MCP-1) is collected from rat plasma (ng/mL) 6 hours after each LNP preparation injection. Saline is used as a control. After injection, hEPO is collected from rat plasma (ng/mL) across various time points (0, 2, 4, 6, 24, 48, 96 hours).

实施例7：可电离脂质的合成Example 7: Synthesis of ionizable lipids

本实施例提供制备、表征和验证如本文所述的可电离脂质的示范性材料和方法。如以下实例中所述，在某些示例性实例中，根据以下一般程序制备化合物。应当理解，尽管一般方法描绘了某些本公开化合物的合成，但以下一般方法和本领域普通技术人员已知的其它方法可以适用于如本文所描述的所有化合物和这些化合物中的每一者的子类和种类。The present embodiment provides exemplary materials and methods for preparing, characterizing and verifying ionizable lipids as described herein. As described in the following examples, in certain illustrative examples, compounds are prepared according to the following general procedures. It should be understood that although the general method describes the synthesis of certain disclosed compounds, the following general method and other methods known to those of ordinary skill in the art can be applicable to all compounds as described herein and subclasses and species of each of these compounds.

一般注意事项：除非另有说明，否则所有反应均使用无水级溶剂在烧瓶或小瓶中在氮气气氛下随着磁力搅拌进行。无水溶剂购自Sigma-Aldrich并且按原样使用。使用带有预包装硅胶筒的Biotage Selekt或Teledyne-Isco CombiFlash Nextgen300+进行快速柱色谱。使用Merck硅胶60板进行薄层色谱，且使用碘使化合物显色。使用Varian INOVA500MHz或Bruker AVANCE 400MHz波谱仪进行核磁共振(NMR)波谱分析；对于CDCl₃样品参考δ＝0.00ppm处的四甲基硅烷以及对于DMSO样品参考残留溶剂峰(δ＝2.50ppm)，化学位移以百万分之一δ(ppm)进行报告。使用具有QDa检测器(ESI⁺)的Waters Acquity UPLC H-classPlus，使用以下方法中的一种方法进行超高效液相色谱-质谱分析(UPLC-MS)：General Notes: Unless otherwise stated, all reactions were performed in flasks or vials with anhydrous grade solvents under a nitrogen atmosphere with magnetic stirring. Anhydrous solvents were purchased from Sigma-Aldrich and used as is. Flash column chromatography was performed using a Biotage Selekt or Teledyne-Isco CombiFlash Nextgen300+ with a pre-packed silica gel cartridge. Thin layer chromatography was performed using Merck silica gel 60 plates, and iodine was used to develop the compounds. Nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian INOVA500MHz or Bruker AVANCE 400MHz spectrometer; for CDCl₃ samples, reference was made to tetramethylsilane at δ = 0.00ppm and for DMSO samples, reference was made to the residual solvent peak (δ = 2.50ppm), and chemical shifts were reported in parts per million δ (ppm). Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using a Waters Acquity UPLC H-classPlus with a QDa detector (ESI⁺ ) using one of the following methods:

方法A：运行5min，色谱柱：XTERRARP 18(4.6x 50mm)，5μm，流动相：初始50％[0.1％HCOOH在水中]和50％[0.1％HCOOH在(70:30)ACN:THF中]；然后在2.65min内达到2％[0.1％HCOOH在水中]和98％[0.1％HCOOH在(70:30)ACN:THF中]，保持该流动相组合物长达3.75min，并且最后回到初始条件，即；在4.90min内回到50％[0.1％HCOOH在水中]和50％[0.1％HCOOH在(70:30)ACN:THF中]，保持该流动相组合物保持长达5.10min。流速＝1.2mL/min。Method A: Run 5 min, column: XTERRARP 18 (4.6 x 50 mm), 5 μm, mobile phase: initial 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF]; then reach 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] in 2.65 min, keep this mobile phase composition for up to 3.75 min, and finally return to initial conditions, i.e.; return to 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] in 4.90 min, keep this mobile phase composition for up to 5.10 min. Flow rate = 1.2 mL/min.

方法B：运行12min，色谱柱：XTERRA RP 18(4.6x 50mm)，5μm(流动相：初始80％[0.1％HCOOH在水中]和20％[0.1％HCOOH在(70:30)ACN:THF中]；保持该初始条件0.75min；然后在3.0min内达到65％[0.1％HCOOH在水中]和35％[0.1％HCOOH在(70:30)ACN:THF中]，然后在6.0min内达到2％[0.1％HCOOH在水中]和98％[0.1％HCOOH在(70:30)ACN:THF中]，保持该流动相组合物长达9.0min，并且最后回到初始条件，即；在11.00min回到80％[0.1％HCOOH在水中]和20％[0.1％HCOOH在(70:30)ACN:THF中]，保持该流动相组合物保持长达12.10min。流速＝1.2ml/minMethod B: Run 12 min, column: XTERRA RP 18 (4.6 x 50 mm), 5 μm (mobile phase: initial 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF]; maintain this initial condition for 0.75 min; then reach 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH in (70:30) ACN:THF] in 3.0 min, then reach 2% [0.1% HCOOH in (70:30) ACN:THF] in 6.0 min) OH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF], keep this mobile phase composition for 9.0 min, and finally return to the initial conditions, i.e.; return to 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in (70:30) ACN:THF] at 11.00 min, keep this mobile phase composition for 12.10 min. Flow rate = 1.2 ml/min

缩写列表List of abbreviations

ACN：乙腈ACN: Acetonitrile

d：二重峰d: Doublet

DCC：N,N'-二环己基碳二亚胺DCC: N,N'-dicyclohexylcarbodiimide

DCM：二氯甲烷DCM: dichloromethane

DIPEA：N,N-二异丙基乙胺DIPEA: N,N-diisopropylethylamine

DMAP：4-(二甲基氨基)吡啶DMAP: 4-(dimethylamino)pyridine

DMSO：二甲基亚砜DMSO: dimethyl sulfoxide

EDC：N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐EDC: N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride

Equiv：等同物Equiv: equivalent

Et：乙基Et: Ethyl

i-Pr：异丙基i-Pr: isopropyl

m：多重峰m: multiplet

Me：甲基Me：Methyl

p：五重峰p: Quintet

q：四重峰q: quartet

Rt：滞留时间Rt: residence time

s：单峰s: Single peak

t：三重峰t: triplet

TEA：三乙胺TEA: triethylamine

THF：四氢呋喃THF: Tetrahydrofuran

实例7-1：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Example 7-1: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester

步骤1：(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸Step 1: (Z)-6-(Non-3-en-1-yloxy)-6-oxohexanoic acid

一般程序A：General Procedure A:

在0℃，向己二酸(5.1g，35mmol，5当量)在DCM:THF(1:1)(20mL)中的搅拌溶液中添加EDC(1.7g，9.1mmol，1.3当量)、DIPEA(2.7g，21mmol，3.0当量)和DMAP(170mg，1.4mmol，0.2当量)。将反应混合物搅拌5min，并且然后添加(Z)-壬-3-烯-1-醇(1.0g，7.0mmol，1.0当量)。将反应混合物在25℃搅拌12h。完成后，将反应混合物在减压下浓缩，用水(30mL)稀释并用DCM(3×50mL)萃取。将有机层用盐水(20mL)洗涤，并且经无水Na₂SO₄干燥，过滤并在减压下浓缩。由此获得的粗品材料通过CombiFlash柱色谱纯化，用0-10％EtOAc-己烷洗脱，以得到(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸(1.3g，69％)，为无色油状物。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.7Hz,3H),1.16–1.38(m,6H),1.46–1.56(m,4H),2.00(q,J＝7.1Hz,2H),2.20(t,J＝6.6Hz,2H),2.24–2.35(m,4H),4.00(t,J＝6.7Hz,2H),5.27–5.39(m,1H),5.40–5.51(m,1H),12.00(s,1H)。To a stirred solution of adipic acid (5.1 g, 35 mmol, 5 eq.) in DCM:THF (1:1) (20 mL) at 0°C was added EDC (1.7 g, 9.1 mmol, 1.3 eq.), DIPEA (2.7 g, 21 mmol, 3.0 eq.) and DMAP (170 mg, 1.4 mmol, 0.2 eq.). The reaction mixture was stirred for 5 min, and then (Z)-non-3-en-1-ol (1.0 g, 7.0 mmol, 1.0 eq.) was added. The reaction mixture was stirred at 25°C for 12 h. Upon completion, the reaction mixture was concentrated under reduced pressure, diluted with water (30 mL) and extracted with DCM (3×50 mL). The organic layer was washed with brine (20 mL) and dried over anhydrous Na₂ SO₄ , filtered and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 0-10% EtOAc-hexanes to give (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid (1.3 g, 69%) as a colorless oil.¹ H NMR (400MHz, DMSO-d₆ ) δ0.86(t,J＝6.7Hz,3H),1.16–1.38(m,6H),1.46–1.56(m,4H),2.00(q,J＝7.1Hz,2H),2.20(t,J＝6.6Hz,2H),2.24–2.35(m,4H), 4.00(t,J＝6.7Hz,2H),5.27–5.39(m,1H),5.40–5.51(m,1H),12.00(s,1H).

步骤2：O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipic acid ester

一般程序B：General Procedure B:

向(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸(406mg，1.5mmol，3.0当量)在DMF(2mL)中的搅拌溶液中添加EDC(479mg，2.5mmol，5.0当量)、DIPEA(388mg，3.0mmol，6.0当量)和DMAP(12mg，0.1mmol，0.2当量)。将反应混合物在0℃搅拌5min，并且然后添加2,2-双(羟甲基)丙烷-1,3-二醇(68mg，0.5mmol，1.0当量)并在25℃搅拌12h。完成后，将反应混合物用水(20mL)稀释并用EtOAc(3×30mL)萃取。将有机层进一步用冷水(2×20mL)洗涤并经无水Na₂SO₄干燥，过滤并在减压下浓缩。由此获得的粗品材料通过CombiFlash柱色谱纯化，用9-10％EtOAc-己烷洗脱，以得到O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(100mg，23％)，为无色油状物。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.8Hz,9H),1.21–1.41(m,21H),1.58–1.71(m,12H),1.97–2.07(m,6H),2.26–2.43(m,16H),3.50(s,2H),4.01–4.16(m,12H),5.26–5.38(m,3H),5.44–5.55(m,3H)。步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(实例7-1)To a stirred solution of (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid (406 mg, 1.5 mmol, 3.0 equiv) in DMF (2 mL) was added EDC (479 mg, 2.5 mmol, 5.0 equiv), DIPEA (388 mg, 3.0 mmol, 6.0 equiv) and DMAP (12 mg, 0.1 mmol, 0.2 equiv). The reaction mixture was stirred at 0 °C for 5 min, and then 2,2-bis(hydroxymethyl)propane-1,3-diol (68 mg, 0.5 mmol, 1.0 equiv) was added and stirred at 25 °C for 12 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3×30 mL). The organic layer was further washed with cold water (2×20 mL) and dried over anhydrous Na₂ SO₄ , filtered and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 9-10% EtOAc-hexanes to give O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester (100 mg, 23%) as a colorless oil.¹ H NMR (400MHz, chloroform-d) δ0.88(t,J＝6.8Hz,9H),1.21–1.41(m,21H),1.58–1.71(m,12H),1.97–2.07(m,6H),2.26–2.43(m,16H),3.50(s,2H),4.01–4.1 6(m,12H),5.26–5.38(m,3H),5.44–5.55(m,3H). Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester (Example 7-1)

一般程序C：General Procedure C:

向O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(100mg，0.11mmol，1当量)在二氯甲烷(3mL)中的搅拌溶液中添加吡啶(18mg，0.22mmol，2.0当量)、氯甲酸4-硝基苯酯(45mg，0.22mmol，2.0当量)和DMAP(3mg，0.02mmol，0.2当量)。将反应混合物在25℃搅拌2h。然后加入3-(二乙氨基)丙烷-1-醇(44mg，0.34mmol，3.0当量)和DIPEA(43mg，0.34mmol，3.0当量)并在25℃搅拌12h。完成后，将反应混合物用二氯甲烷(30mL)稀释，用1M碳酸钠(3x 10mL)洗涤，经无水Na₂SO₄干燥并在减压下浓缩。由此获得的粗品材料通过快速柱色谱纯化，用5％MeOH的DCM溶液洗脱，以得到O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(53mg，45％)，为无色油状物。UPLC-MS(方法A)：Rt 2.13min，m/z计算值[M+H]：1050.7，实测值1052.0。To a stirred solution of O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate (100 mg, 0.11 mmol, 1 eq) in dichloromethane (3 mL) was added pyridine (18 mg, 0.22 mmol, 2.0 eq), 4-nitrophenyl chloroformate (45 mg, 0.22 mmol, 2.0 eq) and DMAP (3 mg, 0.02 mmol, 0.2 eq). The reaction mixture was stirred at 25 °C for 2 h. Then 3-(Diethylamino)propan-1-ol (44 mg, 0.34 mmol, 3.0 eq.) and DIPEA (43 mg, 0.34 mmol, 3.0 eq.) were added and stirred for 12 h at 25 °C. After completion, the reaction mixture was diluted with dichloromethane (30 mL), washed with 1 M sodium carbonate (3 x 10 mL), dried over_{anhydrousNa2SO4} and concentrated under reduced pressure. The crude material thus obtained was purified by flash column chromatography eluting with 5% MeOH in DCM to give O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate (53 mg, 45%) as a colorless oil. UPLC-MS (Method A): Rt 2.13 min, m/z calcd [M+H]: 1050.7, found 1052.0.

实例7-2：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯Example 7-2: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate

步骤1：6-(壬氧基)-6-氧代己酸Step 1: 6-(Nonyloxy)-6-oxohexanoic acid

根据一般程序A制备，用壬烷-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.2g，65％。¹HNMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.6Hz,3H),1.14–1.37(m,12H),1.44–1.61(m,6H),2.20(t,J＝6.8Hz,2H),2.28(t,J＝6.9Hz,2H),3.99(t,J＝6.6Hz,2H),12.00(s,1H)。Prepared according to General Procedure A, substituting nonan-1-ol for (Z)-non-3-en-1-ol. Isolated 1.2 g, 65%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J = 6.6 Hz, 3H), 1.14-1.37 (m, 12H), 1.44-1.61 (m, 6H), 2.20 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 6.9 Hz, 2H), 3.99 (t, J = 6.6 Hz, 2H), 12.00 (s, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipate

根据一般程序B制备，用6-(壬氧基)-6-氧代己酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出102mg，21％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.2Hz,9H),1.16–1.39(m,35H),1.44–1.74(m,20H),2.25–2.43(m,12H),3.50(s,2H),4.04(t,J＝6.6Hz,6H),4.10(s,6H)。Prepared according to General Procedure B, substituting 6-(nonyloxy)-6-oxohexanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 102 mg, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.2 Hz, 9H), 1.16-1.39 (m, 35H), 1.44-1.74 (m, 20H), 2.25-2.43 (m, 12H), 3.50 (s, 2H), 4.04 (t, J = 6.6 Hz, 6H), 4.10 (s, 6H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯(实例7-2)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate (Example 7-2)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出39mg，56％。UPLC-MS(方法A)：Rt 2.36min，m/z计算值[M+H]：1056.8，实测值1058.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 39 mg, 56%. UPLC-MS (Method A): Rt 2.36 min, m/z calcd [M+H]: 1056.8, found 1058.0.

实例7-3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Example 7-3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

步骤1：(Z)-6-(辛-5-烯-1-基氧基)-6-氧代己酸Step 1: (Z)-6-(Oct-5-en-1-yloxy)-6-oxohexanoic acid

根据一般程序A制备，用(Z)-辛-5-烯-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.4g，68％。¹H NMR(400MHz,DMSO-d₆)δ0.90(t,J＝7.4Hz,3H),1.27–1.40(m,2H),1.42–1.66(m,6H),2.00(d,J＝6.7Hz,4H),2.20(t,J＝7.1Hz,2H),2.28(t,J＝6.8Hz,2H),3.99(t,J＝6.2Hz,2H),5.18–5.47(m,2H),12.01(s,1H)。Prepared according to General Procedure A, substituting (Z)-oct-5-en-1-ol for (Z)-non-3-en-1-ol. Isolated 1.4 g, 68%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.90 (t, J=7.4 Hz, 3H), 1.27-1.40 (m, 2H), 1.42-1.66 (m, 6H), 2.00 (d, J=6.7 Hz, 4H), 2.20 (t, J=7.1 Hz, 2H), 2.28 (t, J=6.8 Hz, 2H), 3.99 (t, J=6.2 Hz, 2H), 5.18-5.47 (m, 2H), 12.01 (s, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

根据一般程序B制备，用(Z)-6-(辛-5-烯-1-基氧基)-6-氧代己酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出102mg，21％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,9H),1.33–1.46(m,6H),1.56–1.72(m,21H),1.96–2.11(m,12H),2.24–2.42(m,12H),3.50(s,2H),4.02–4.12(m,12H),5.24–5.42(m,6H)。Prepared according to General Procedure B, substituting (Z)-6-(oct-5-en-1-yloxy)-6-oxohexanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 102 mg, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (t, J=7.5 Hz, 9H), 1.33-1.46 (m, 6H), 1.56-1.72 (m, 21H), 1.96-2.11 (m, 12H), 2.24-2.42 (m, 12H), 3.50 (s, 2H), 4.02-4.12 (m, 12H), 5.24-5.42 (m, 6H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯(实例7-3)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester (Example 7-3)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-3-烯-1-基)二己二酸酯。分离出54mg，65％。UPLC-MS(方法A)：Rt2.07min，m/z计算值[M+H]：1008.7，实测值1010.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diadipic acid for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-oct-3-en-1-yl)diadipic acid. Isolated 54 mg, 65%. UPLC-MS (Method A): Rt 2.07 min, m/z calcd [M+H]: 1008.7, found 1010.0.

实例7-4：二((Z)-壬-3-烯-1-基)O,O'-(2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二己二酸酯Example 7-4: Di((Z)-non-3-en-1-yl)O,O'-(2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)diadipic acid ester

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出39mg，56％。UPLC-MS(方法A)：Rt 2.18min，m/z计算值[M+H]：1033.7，实测值1035.1。Prepared according to General Procedure C, substituting 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 39 mg, 56%. UPLC-MS (Method A): Rt 2.18 min, m/z calcd [M+H]: 1033.7, found 1035.1.

实例7-5：二壬基O,O'-(2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二己二酸酯Example 7-5: Dinonyl O,O'-(2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)diadipate

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O,O'-(2-(羟甲基)-2-(((6-(壬氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出53mg，59％。UPLC-MS(方法A)：Rt 2.31min，m/z计算值[M+H]：1038.7，实测值1040.1。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol and O,O'-(2-(hydroxymethyl)-2-(((6-(nonyloxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipoate. Isolated 53 mg, 59%. UPLC-MS (Method A): Rt 2.31 min, m/z calcd [M+H]: 1038.7, found 1040.1.

实例7-6：二((Z)-辛-5-烯-1-基)O,O'-(2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二己二酸酯Example 7-6: Di((Z)-oct-5-en-1-yl)O,O'-(2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)diadipic acid ester

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O,O'-(2-(羟甲基)-2-(((6-(((Z)-辛-5-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出37mg，47％。UPLC-MS(方法A)：Rt 2.04min，m/z计算值[M+H]：991.6，实测值993.0。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol and O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-oct-5-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diadipic acid for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipic acid. Isolated 37 mg, 47%. UPLC-MS (Method A): Rt 2.04 min, m/z calcd. [M+H]: 991.6, found 993.0.

实例7-7：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Example 7-7: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

步骤1：(Z)-7-(壬-3-烯-1-基氧基)-7-氧代庚酸Step 1: (Z)-7-(Non-3-en-1-yloxy)-7-oxoheptanoic acid

根据一般程序A制备，用庚二酸取代己二酸。分离出1.3g，65％。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.6Hz,3H),1.21–1.36(m,8H),1.42–1.57(m,4H),2.00(q,J＝7.1Hz,2H),2.18(t,J＝7.3Hz,2H),2.21–2.36(m,4H),4.00(t,J＝6.7Hz,2H),5.29–5.38(m,1H),5.46(q,J＝8.1Hz,1H),11.96(s,1H)。Prepared according to General Procedure A, substituting pimelic acid for adipic acid. Isolated 1.3 g, 65%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J = 6.6 Hz, 3H), 1.21-1.36 (m, 8H), 1.42-1.57 (m, 4H), 2.00 (q, J = 7.1 Hz, 2H), 2.18 (t, J = 7.3 Hz, 2H), 2.21-2.36 (m, 4H), 4.00 (t, J = 6.7 Hz, 2H), 5.29-5.38 (m, 1H), 5.46 (q, J = 8.1 Hz, 1H), 11.96 (s, 1H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

根据一般程序B制备，用(Z)-7-(壬-3-烯-1-基氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出152mg，23％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.6Hz,9H),1.17–1.46(m,24H),1.52–1.72(m,14H),2.02(d,J＝8.0Hz,6H),2.24–2.42(m,17H),3.49(s,2H),4.01–4.14(m,12H),5.24–5.41(m,3H),5.43–5.53(m,3H)。Prepared according to General Procedure B, substituting (Z)-7-(non-3-en-1-yloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 152 mg, 23%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (t, J = 6.6 Hz, 9H), 1.17-1.46 (m, 24H), 1.52-1.72 (m, 14H), 2.02 (d, J = 8.0 Hz, 6H), 2.24-2.42 (m, 17H), 3.49 (s, 2H), 4.01-4.14 (m, 12H), 5.24-5.41 (m, 3H), 5.43-5.53 (m, 3H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)(实例7-7)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) (Example 7-7)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出26mg，54％。UPLC-MS(方法A)：Rt 2.26min，m/z计算值[M+H]：1092.8，实测值1094.0。Prepared according to General Procedure C, substituting O'1,O1-(2-(hydroxymethyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 26 mg, 54%. UPLC-MS (Method A): Rt 2.26 min, m/z calcd [M+H]: 1092.8, found 1094.0.

实例7-8：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Example 7-8: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

步骤1：7-(壬氧基)-7-氧代庚酸Step 1: 7-(Nonyloxy)-7-oxoheptanoic acid

根据一般程序A制备，用庚二酸取代己二酸，并用壬-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.4g，69％。¹H NMR(400MHz,DMSO-d₆)δ0.85(t,J＝6.6Hz,3H),1.22–1.32(m,14H),1.42–1.58(m,6H),2.18(t,J＝7.3Hz,2H),2.27(t,J＝7.4Hz,2H),3.99(t,J＝6.5Hz,2H),11.97(s,1H)。Prepared according to General Procedure A, substituting pimelic acid for adipic acid and nonan-1-ol for (Z)-non-3-en-1-ol. Isolated 1.4 g, 69%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.85 (t, J = 6.6 Hz, 3H), 1.22-1.32 (m, 14H), 1.42-1.58 (m, 6H), 2.18 (t, J = 7.3 Hz, 2H), 2.27 (t, J = 7.4 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 11.97 (s, 1H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

根据一般程序B制备，用7-(壬氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出142mg，21％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,9H),1.18–1.42(m,40H),1.56–1.70(m,21H),2.25–2.37(m,12H),3.50(s,2H),4.04(t,J＝6.8Hz,6H),4.10(s,6H)。Prepared according to General Procedure B, substituting 7-(nonyloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 142 mg, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 9H), 1.18-1.42 (m, 40H), 1.56-1.70 (m, 21H), 2.25-2.37 (m, 12H), 3.50 (s, 2H), 4.04 (t, J = 6.8 Hz, 6H), 4.10 (s, 6H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)(实例7-8)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate) (Example 7-8)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出16mg，37％。UPLC-MS(方法A)：Rt 2.42min，m/z计算值[M+H]：1098.8，实测值1100.0。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O'1,O1-(2-(hydroxymethyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate). Isolated 16 mg, 37%. UPLC-MS (Method A): Rt 2.42 min, m/z calcd [M+H]: 1098.8, found 1100.0.

实例7-9：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Example 7-9: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

步骤1：(Z)-7-(辛-5-烯-1-基氧基)-7-氧代庚酸Step 1: (Z)-7-(Oct-5-en-1-yloxy)-7-oxoheptanoic acid

根据一般程序A制备，用庚二酸取代己二酸，并用(Z)-辛-5-烯-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.3g，66％。¹H NMR(400MHz,DMSO-d₆)δ0.91(t,J＝7.5Hz,3H),1.21–1.40(m,4H),1.42–1.64(m,6H),2.01(q,J＝7.9Hz,4H),2.18(t,J＝7.3Hz,2H),2.27(t,J＝7.3Hz,2H),4.00(t,J＝6.5Hz,2H),5.08–5.56(m,2H),11.99(s,1H)。Prepared according to General Procedure A, substituting pimelic acid for adipic acid and (Z)-oct-5-en-1-ol for (Z)-non-3-en-1-ol. Isolated 1.3 g, 66%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.91 (t, J=7.5 Hz, 3H), 1.21-1.40 (m, 4H), 1.42-1.64 (m, 6H), 2.01 (q, J=7.9 Hz, 4H), 2.18 (t, J=7.3 Hz, 2H), 2.27 (t, J=7.3 Hz, 2H), 4.00 (t, J=6.5 Hz, 2H), 5.08-5.56 (m, 2H), 11.99 (s, 1H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

根据一般程序B制备，用(Z)-7-(辛-5-烯-1-基氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出132mg，20％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,9H),1.28–1.46(m,9H),1.57–1.69(m,20H),1.96–2.10(m,13H),2.25–2.37(m,13H),3.50(s,2H),4.01–4.12(m,12H),5.17–5.50(m,6H)。Prepared according to General Procedure B, substituting (Z)-7-(oct-5-en-1-yloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 132 mg, 20%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (t, J=7.5 Hz, 9H), 1.28-1.46 (m, 9H), 1.57-1.69 (m, 20H), 1.96-2.10 (m, 13H), 2.25-2.37 (m, 13H), 3.50 (s, 2H), 4.01-4.12 (m, 12H), 5.17-5.50 (m, 6H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)(实例7-9)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) (Example 7-9)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出40mg，49％。UPLC-MS(方法A)：Rt 2.14min，m/z计算值[M+H]：1050.7，实测值1052.0。Prepared according to General Procedure C, substituting O'1,O1-(2-(hydroxymethyl)-2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 40 mg, 49%. UPLC-MS (Method A): Rt 2.14 min, m/z calcd [M+H]: 1050.7, found 1052.0.

实例7-10：7,7'-二((Z)-壬-3-烯-1-基)O'1,O1-(2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(庚二酸酯)Example 7-10: 7,7'-di((Z)-non-3-en-1-yl)O'1,O1-(2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(pimelate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出36mg，51％。UPLC-MS(方法A)：Rt 2.23min，m/z计算值[M+H]：1075.7，实测值1077.2。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol and substituting 0'1,01-(2-(hydroxymethyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) for 0,0'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 36 mg, 51%. UPLC-MS (Method A): Rt 2.23 min, m/z calcd. [M+H]: 1075.7, found 1077.2.

实例7-11：7,7'-二壬基O'1,O1-(2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(庚二酸酯)Example 7-11: 7,7'-Dinonyl O'1,O1-(2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(pimelate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出47mg，51％。UPLC-MS(方法A)：Rt 2.37min，m/z计算值[M+H]：1081.8，实测值1083.1。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol and substituting O'1,O1-(2-(hydroxymethyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 47 mg, 51%. UPLC-MS (Method A): Rt 2.37 min, m/z calcd [M+H]: 1081.8, found 1083.1.

实例7-12：7,7'-二((Z)-辛-5-烯-1-基)O'1,O1-(2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(庚二酸酯)Example 7-12: 7,7'-di((Z)-oct-5-en-1-yl)O'1,O1-(2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(pimelate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-辛-5-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出65mg，56％。UPLC-MS(方法A)：Rt 2.05min，m/z计算值[M+H]：1033.7，实测值1035.1。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol and O'1,O1-(2-(hydroxymethyl)-2-(((7-(((Z)-oct-5-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 65 mg, 56%. UPLC-MS (Method A): Rt 2.05 min, m/z calcd. [M+H]: 1033.7, found 1035.1.

实例7-13：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Example 7-13: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate)

步骤1：(Z)-8-(壬-3-烯-1-基氧基)-8-氧代辛酸Step 1: (Z)-8-(Non-3-en-1-yloxy)-8-oxooctanoic acid

根据一般程序A制备，用辛二酸取代己二酸。分离出550mg，52％。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.7Hz,3H),1.19–1.37(m,11H),1.40–1.57(m,4H),2.00(q,J＝7.0Hz,2H),2.18(t,J＝7.4Hz,2H),2.21–2.36(m,4H),4.00(t,J＝6.7Hz,2H),5.27–5.38(m,1H),5.40–5.52(m,1H)。Prepared according to General Procedure A, suberic acid substituted for adipic acid. Isolated 550 mg, 52%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J = 6.7 Hz, 3H), 1.19-1.37 (m, 11H), 1.40-1.57 (m, 4H), 2.00 (q, J = 7.0 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.21-2.36 (m, 4H), 4.00 (t, J = 6.7 Hz, 2H), 5.27-5.38 (m, 1H), 5.40-5.52 (m, 1H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate)

根据一般程序B制备，用(Z)-8-(壬-3-烯-1-基氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出70mg，19％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.7Hz,9H),1.21–1.38(m,34H),1.49–1.70(m,16H),1.97–2.09(m,6H),2.22–2.42(m,19H),3.48(s,2H),3.97–4.20(m,6H),5.26–5.39(m,2H),5.42–5.55(m,2H)。Prepared according to General Procedure B, substituting (Z)-8-(non-3-en-1-yloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 70 mg, 19%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (t, J = 6.7 Hz, 9H), 1.21-1.38 (m, 34H), 1.49-1.70 (m, 16H), 1.97-2.09 (m, 6H), 2.22-2.42 (m, 19H), 3.48 (s, 2H), 3.97-4.20 (m, 6H), 5.26-5.39 (m, 2H), 5.42-5.55 (m, 2H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)(实例7-13)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) (Example 7-13)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出18mg，39％。UPLC-MS(方法A)：Rt 2.33min，m/z计算值[M+H]：1133.8，实测值1135.2。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 18 mg, 39%. UPLC-MS (Method A): Rt 2.33 min, m/z calcd [M+H]: 1133.8, found 1135.2.

实例7-14：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(壬氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Example 7-14: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(nonyloxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate)

步骤1：8-(壬氧基)-8-氧代辛酸Step 1: 8-(Nonyloxy)-8-oxooctanoic acid

根据一般程序A制备，用辛二酸取代己二酸，并用壬-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.4g，67％。¹H NMR(400MHz,DMSO-d₆)δ0.85(t,J＝6.4Hz,3H),1.22–1.32(m,16H),1.41–1.61(m,6H),2.18(t,J＝7.3Hz,2H),2.26(t,J＝7.2Hz,2H),3.99(t,J＝6.6Hz,2H),11.95(s,1H)。Prepared according to General Procedure A, suberic acid for adipic acid and nonan-1-ol for (Z)-non-3-en-1-ol. Isolated 1.4 g, 67%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.85 (t, J = 6.4 Hz, 3H), 1.22-1.32 (m, 16H), 1.41-1.61 (m, 6H), 2.18 (t, J = 7.3 Hz, 2H), 2.26 (t, J = 7.2 Hz, 2H), 3.99 (t, J = 6.6 Hz, 2H), 11.95 (s, 1H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((8-(壬氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((8-(nonyloxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate)

根据一般程序B制备，用8-(壬氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出190mg，20％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.4Hz,9H),1.15–1.41(m,48H),1.47–1.70(m,19H),2.22–2.37(m,12H),3.49(s,2H),4.04(t,J＝6.8Hz,6H),4.10(s,6H)。Prepared according to General Procedure B, substituting 8-(nonyloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 190 mg, 20%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.4 Hz, 9H), 1.15-1.41 (m, 48H), 1.47-1.70 (m, 19H), 2.22-2.37 (m, 12H), 3.49 (s, 2H), 4.04 (t, J = 6.8 Hz, 6H), 4.10 (s, 6H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(壬氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)(实例7-14)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(nonyloxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate) (Example 7-14)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出25mg，43％。UPLC-MS(方法A)：Rt 2.47min，m/z计算值[M+H]：1139.8，实测值1141.2。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 25 mg, 43%. UPLC-MS (Method A): Rt 2.47 min, m/z calcd [M+H]: 1139.8, found 1141.2.

实例7-15：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(((Z)-辛-5-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Example 7-15: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(((Z)-oct-5-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

步骤1：(Z)-8-(辛-5-烯-1-基氧基)-8-氧代辛酸Step 1: (Z)-8-(Oct-5-en-1-yloxy)-8-oxooctanoic acid

根据一般程序A制备，用辛二酸取代己二酸，并用(Z)-辛-5-烯-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.9g，66％。¹H NMR(400MHz,DMSO-d₆)δ0.91(t,J＝7.4Hz,3H),1.13–1.30(m,5H),1.28–1.40(m,2H),1.43–1.62(m,6H),1.92–2.08(m,4H),2.18(t,J＝7.3Hz,2H),2.27(t,J＝7.3Hz,2H),4.00(t,J＝6.6Hz,2H),5.18–5.50(m,2H)。Prepared according to General Procedure A, suberic acid for adipic acid and (Z)-oct-5-en-1-ol for (Z)-non-3-en-1-ol. Isolated 1.9 g, 66%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.91 (t, J = 7.4 Hz, 3H), 1.13-1.30 (m, 5H), 1.28-1.40 (m, 2H), 1.43-1.62 (m, 6H), 1.92-2.08 (m, 4H), 2.18 (t, J = 7.3 Hz, 2H), 2.27 (t, J = 7.3 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 5.18-5.50 (m, 2H).

步骤2：O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-辛-5-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Step 2: O'1,O1-(2-(Hydroxymethyl)-2-(((8-(((Z)-oct-5-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

根据一般程序B制备，用(Z)-8-(辛-5-烯-1-基氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出140mg，20％。¹H NMR(400MHz,氯仿-d)δ0.94(t,J＝7.6Hz,9H),1.21–1.46(m,19H),1.56–1.68(m,18H),1.95–2.13(m,12H),2.21–2.37(m,12H),3.49(s,2H),4.05(t,J＝6.7Hz,6H),4.10(s,6H),5.10–5.55(m,6H)。Prepared according to General Procedure B, substituting (Z)-8-(oct-5-en-1-yloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 140 mg, 20%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.94 (t, J = 7.6 Hz, 9H), 1.21-1.46 (m, 19H), 1.56-1.68 (m, 18H), 1.95-2.13 (m, 12H), 2.21-2.37 (m, 12H), 3.49 (s, 2H), 4.05 (t, J = 6.7 Hz, 6H), 4.10 (s, 6H), 5.10-5.55 (m, 6H).

步骤3：O'1,O1-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((8-(((Z)-辛-5-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)(实例7-15)Step 3: O'1,O1-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((8-(((Z)-oct-5-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) (Example 7-15)

根据一般程序C制备，用O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-辛-5-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出54mg，57％。UPLC-MS(方法A)：Rt 2.22min，m/z计算值[M+H]：1091.8，实测值1093.1。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((8-(((Z)-oct-5-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 54 mg, 57%. UPLC-MS (Method A): Rt 2.22 min, m/z calcd [M+H]: 1091.8, found 1093.1.

实例7-16：8,8'-二((Z)-辛-3-烯-1-基)O'1,O1-(2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(辛二酸酯)Example 7-16: 8,8'-di((Z)-oct-3-en-1-yl)O'1,O1-(2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(suberate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出83mg，63％。UPLC-MS(方法A)：Rt 2.31min，m/z计算值[M+H]：1116.8，实测值1118.1。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol and O'1,O1-(2-(hydroxymethyl)-2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 83 mg, 63%. UPLC-MS (Method A): Rt 2.31 min, m/z calcd. [M+H]: 1116.8, found 1118.1.

实例7-17：8,8'-二((Z)-辛-3-烯-1-基)O'1,O1-(2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(辛二酸酯)Example 7-17: 8,8'-di((Z)-oct-3-en-1-yl)O'1,O1-(2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(suberate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((8-(壬氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出21mg，46％。UPLC-MS(方法A)：Rt 2.43min，m/z计算值[M+H]：1122.8，实测值1124.2。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol and O'1,O1-(2-(hydroxymethyl)-2-(((8-(nonyloxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 21 mg, 46%. UPLC-MS (Method A): Rt 2.43 min, m/z calcd [M+H]: 1122.8, found 1124.2.

实例7-18：8,8'-二((Z)-辛-3-烯-1-基)O'1,O1-(2-(((8-(((Z)-壬-3-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二(辛二酸酯)Example 7-18: 8,8'-di((Z)-oct-3-en-1-yl)O'1,O1-(2-(((8-(((Z)-non-3-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di(suberate)

根据一般程序C制备，用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇，并用O'1,O1-(2-(羟甲基)-2-(((8-(((Z)-辛-5-烯-1-基)氧基)-8-氧代辛酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出91mg，54％。UPLC-MS(方法A)：Rt 2.18min，m/z计算值[M+H]：1074.8，实测值1076.0。Prepared according to General Procedure C substituting 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol and O'1,O1-(2-(hydroxymethyl)-2-(((8-(((Z)-oct-5-en-1-yl)oxy)-8-oxooctanoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 91 mg, 54%. UPLC-MS (Method A): Rt 2.18 min, m/z calcd. [M+H]: 1074.8, found 1076.0.

实例7-19：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Example 7-19: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester

一般程序D：General Procedure D:

在0℃，向(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸(320mg，1.2mmol，2.0当量)在DMF(2mL)中的搅拌溶液中添加EDC(350mg，0.6mmol，3.0当量)、DIPEA(310mg，2.4mmol，4.0当量)和DMAP(15mg，0.12mmol，0.2当量)。将反应混合物搅拌5min，并且然后添加2,2-双(羟甲基)丙烷-1,3-二醇(82mg，0.6mmol，1.0当量)。将反应混合物在25℃搅拌2h。完成后，将反应混合物用水(30mL)稀释并用乙酸乙酯(3x 40mL)萃取。将有机层用盐水洗涤并经无水Na₂SO₄干燥，在减压下浓缩。由此获得的粗品残余物通过CombiFlash柱色谱纯化，用0-16％乙酸乙酯-己烷洗脱，以得到O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(80mg，21％)，为无色油状物。¹HNMR(400MHz,氯仿-d)δ0.88(t,J＝6.9Hz,6H),1.18–1.46(m,12H),1.59–1.72(m,8H),2.02(d,J＝7.4Hz,4H),2.29–2.39(m,12H),2.63–2.81(m,2H),3.57(s,4H),4.06(t,J＝6.9Hz,4H),4.13(s,4H),5.23–5.41(m,2H),5.42–5.58(m,2H)。To a stirred solution of (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid (320 mg, 1.2 mmol, 2.0 equiv) in DMF (2 mL) at 0°C was added EDC (350 mg, 0.6 mmol, 3.0 equiv), DIPEA (310 mg, 2.4 mmol, 4.0 equiv) and DMAP (15 mg, 0.12 mmol, 0.2 equiv). The reaction mixture was stirred for 5 min, and then 2,2-bis(hydroxymethyl)propane-1,3-diol (82 mg, 0.6 mmol, 1.0 equiv) was added. The reaction mixture was stirred at 25°C for 2 h. Upon completion, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 x 40 mL). The organic layer was washed with brine and dried over_{anhydrousNa2SO4} and concentrated under reduced pressure. The crude residue thus obtained was purified by CombiFlash column chromatography eluting with 0-16% ethyl acetate-hexane to give O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate (80 mg, 21%) as a colorless oil.¹ HNMR(400MHz, chloroform-d)δ0.88(t,J＝6.9Hz,6H),1.18–1.46(m,12H),1.59–1.72(m,8H),2.02(d,J＝7.4Hz,4H),2.29–2.39(m,12H),2.63–2.81(m,2H),3. 57(s,4H),4.06(t,J＝6.9Hz,4H),4.13(s,4H),5.23–5.41(m,2H),5.42–5.58(m,2H).

步骤2：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Step 2: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate

一般程序E：General Procedure E:

在0℃，向1-金刚烷乙酸(67mg，0.34mmol，0.8当量)在DCM:THF(1:1)(4mL)中的搅拌溶液中添加EDC(120mg，0.64mmol，1.5当量)、DIPEA(110mg，0.86mmol，2.0当量)和DMAP(11mg，0.086mmol，0.2当量)。将反应混合物在0℃搅拌5min，并且然后添加O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(280mg，0.43mmol，1.0当量)。将反应混合物在25℃搅拌12h。完成后，将反应混合物用水(30mL)稀释并用DCM(3×50mL)萃取。将有机层用盐水洗涤并经无水Na₂SO₄干燥，在减压下浓缩。由此获得的粗品材料通过CombiFlash柱色谱纯化，用20-28％EtOAc-己烷洗脱，以得到O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(140mg,40％)，为无色油状物。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.8Hz,6H),1.21–1.41(m,18H),1.58(s,6H),1.60–1.74(m,9H),1.94–1.98(m,3H),2.02(q,J＝7.1Hz,4H),2.08(s,2H),2.27–2.41(m,11H),2.59(t,J＝6.7Hz,1H),3.51(d,J＝6.6Hz,2H),4.01–4.10(m,6H),4.11(s,4H),5.26–5.38(m,2H),5.40–5.59(m,2H)。To a stirred solution of 1-adamantaneacetic acid (67 mg, 0.34 mmol, 0.8 eq.) in DCM:THF (1:1) (4 mL) at 0°C was added EDC (120 mg, 0.64 mmol, 1.5 eq.), DIPEA (110 mg, 0.86 mmol, 2.0 eq.) and DMAP (11 mg, 0.086 mmol, 0.2 eq.). The reaction mixture was stirred at 0°C for 5 min, and then O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate (280 mg, 0.43 mmol, 1.0 eq.) was added. The reaction mixture was stirred at 25°C for 12 h. Upon completion, the reaction mixture was diluted with water (30 mL) and extracted with DCM (3×50 mL). The organic layer was washed with brine and dried over anhydrous Na₂ SO₄ and concentrated under reduced pressure. The crude material thus obtained was purified by CombiFlash column chromatography eluting with 20-28% EtOAc-hexanes to afford O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester (140 mg, 40%) as a colorless oil.¹ H NMR (400MHz, chloroform-d) δ0.88(t,J＝6.8Hz,6H),1.21–1.41(m,18H),1.58(s,6H),1.60–1.74(m,9H),1.94–1.98(m,3H),2.02(q,J＝7.1Hz,4H),2.08(s,2 H),2.27–2.41(m,11H),2.59(t,J＝6.7Hz,1H),3.51(d,J＝6.6Hz,2H),4.01–4.10(m,6H),4.11(s,4H),5.26–5.38(m,2H),5.40–5.59(m,2H).

步骤3：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯(实例7-19)Step 3: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester (Example 7-19)

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出69mg，37％。UPLC-MS(方法A)：Rt 2.06min，m/z计算值[M+H]：974.7，实测值975.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate for O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 69 mg, 37%. UPLC-MS (Method A): Rt 2.06 min, m/z calcd [M+H]: 974.7, found 975.0.

实例7-20：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯Example 7-20: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二壬基二己二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl) dinonyl diadipoate

根据一般程序D制备，用6-(壬氧基)-6-氧代己酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出210mg，22％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.19–1.39(m,24H),1.54–1.71(m,12H),2.29–2.34(m,4H),2.35–2.40(m,4H),2.67–2.77(m,2H),3.57(d,J＝5.0Hz,4H),4.05(t,J＝6.8Hz,4H),4.13(s,4H)。Prepared according to General Procedure D, substituting 6-(nonyloxy)-6-oxohexanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 210 mg, 22%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.19-1.39 (m, 24H), 1.54-1.71 (m, 12H), 2.29-2.34 (m, 4H), 2.35-2.40 (m, 4H), 2.67-2.77 (m, 2H), 3.57 (d, J = 5.0 Hz, 4H), 4.05 (t, J = 6.8 Hz, 4H), 4.13 (s, 4H).

步骤2：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二壬基二己二酸酯Step 2: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dinonyl diadipate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二壬基二己二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出100mg，42％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.17–1.40(m,20H),1.55–1.73(m,37H),1.96(s,3H),2.08(s,2H),2.26–2.40(m,2H),3.51(d,J＝6.7Hz,2H),4.00–4.09(m,5H),4.11(s,3H)。Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)dinonyl diadipoate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipoate. Isolated 100 mg, 42%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.17-1.40 (m, 20H), 1.55-1.73 (m, 37H), 1.96 (s, 3H), 2.08 (s, 2H), 2.26-2.40 (m, 2H), 3.51 (d, J = 6.7 Hz, 2H), 4.00-4.09 (m, 5H), 4.11 (s, 3H).

步骤3：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯(实例7-20)Step 3: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate (Example 7-20)

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二壬基二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出61mg，57％。UPLC-MS(方法A)：Rt 2.18min，m/z计算值[M+H]：978.7，实测值979.1。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dinonyl diadipate. Isolated 61 mg, 57%. UPLC-MS (Method A): Rt 2.18 min, m/z calcd [M+H]: 978.7, found 979.1.

实例7-21：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Example 7-21: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

根据一般程序D制备，用(Z)-6-(辛-5-烯-1-基氧基)-6-氧代己酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出280mg，21％。¹H NMR(400MHz,氯仿-d)δ0.94(t,J＝7.5Hz,6H),1.33–1.45(m,6H),1.48–1.74(m,13H),1.93–2.12(m,8H),2.27–2.41(m,8H),3.57(s,4H),4.06(t,J＝6.7Hz,4H),4.13(s,3H),5.08–5.57(m,4H)。Prepared according to General Procedure D, substituting (Z)-6-(oct-5-en-1-yloxy)-6-oxohexanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 280 mg, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.94 (t, J = 7.5 Hz, 6H), 1.33-1.45 (m, 6H), 1.48-1.74 (m, 13H), 1.93-2.12 (m, 8H), 2.27-2.41 (m, 8H), 3.57 (s, 4H), 4.06 (t, J = 6.7 Hz, 4H), 4.13 (s, 3H), 5.08-5.57 (m, 4H).

步骤2：O,O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Step 2: O,O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出140mg，40％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.7Hz,6H),1.33–1.46(m,4H),1.55–1.80(m,28H),1.91–2.16(m,8H),2.23–2.44(m,9H),3.51(d,J＝6.6Hz,2H),4.03–4.13(m,11H),5.17–5.54(m,4H)。Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diadipic acid ester for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipic acid ester. Isolated 140 mg, 40%.¹ H NMR (400MHz, chloroform-d) δ0.95 (t, J = 7.7Hz, 6H), 1.33–1.46 (m, 4H), 1.55–1.80 (m, 28H), 1.91–2.16 (m, 8H), 2.23–2.44 (m, 9H), 3.51 (d, J = 6.6Hz, 2H), 4.0 3–4.13(m,11H),5.17–5.54(m,4H).

步骤3：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯(实例7-21)Step 3: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester (Example 7-21)

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出31mg，58％。UPLC-MS(方法A)：Rt 2.02min，m/z计算值[M+H]：946.7，实测值947.1。Prepared according to General Procedure C, substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate for O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diadipate. Isolated 31 mg, 58%. UPLC-MS (Method A): Rt 2.02 min, m/z calcd [M+H]: 946.7, found 947.1.

实例7-22：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯Example 7-22: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipic acid ester

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出26mg，37％。UPLC-MS(方法A)：Rt 2.04min，m/z计算值[M+H]：957.7，实测值958.2。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate for O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 26 mg, 37%. UPLC-MS (Method A): Rt 2.04 min, m/z calcd. [M+H]: 957.7, found 958.2.

实例7-23：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二壬基二己二酸酯Example 7-23: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)dinonyl diadipoate

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二壬基二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出42mg，58％。UPLC-MS(方法A)：Rt 2.17min，m/z计算值[M+H]：961.7，实测值962.2。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate for O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dinonyl diadipate and 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol. Isolated 42 mg, 58%. UPLC-MS (Method A): Rt 2.17 min, m/z calcd [M+H]: 961.7, found 962.2.

实例7-24：O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯Example 7-24: O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diadipic acid ester

根据一般程序C制备，用O,O'-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二己二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出50mg，61％。UPLC-MS(方法A)：Rt 1.99min，m/z计算值[M+H]：929.6，实测值930.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate for O,O'-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 50 mg, 61%. UPLC-MS (Method A): Rt 1.99 min, m/z calcd. [M+H]: 929.6, found 930.0.

实例7-25：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Example 7-25: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

根据一般程序D制备，用(Z)-7-(壬-3-烯-1-基氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出345mg，21％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.8Hz,6H),1.18–1.45(m,16H),1.58–1.71(m,8H),2.0(q,J＝7.2Hz,4H),2.25–2.41(m,12H),2.74(t,J＝6.4Hz,2H),3.57(d,J＝6.3Hz,4H),4.05(t,J＝6.9Hz,4H),4.13(s,4H),5.24-5.42(m,2H),5.43–5.55(m,2H)。Prepared according to General Procedure D, substituting (Z)-7-(non-3-en-1-yloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 345 mg, 21%.^{1. 57} (d,J＝6.3Hz,4H),4.05(t,J＝6.9Hz,4H),4.13(s,4H),5.24-5.42(m,2H),5.43–5.55(m,2H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出280mg，51％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.7Hz,6H),1.21–1.41(m,17H),1.47–1.79(m,21H),1.90–2.08(m,9H),2.24–2.41(m,11H),3.50(d,J＝6.8Hz,2H),4.01–4.13(m,10H),5.26–5.40(m,2H),5.43–5.53(m,2H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)di(pimelate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 280 mg, 51%.¹ H NMR (400MHz, chloroform-d) δ0.88(t,J＝6.7Hz,6H),1.21–1.41(m,17H),1.47–1.79(m,21H),1.90–2.08(m,9H),2.24–2.41(m,11H),3.50(d,J＝6.8Hz,2H),4 .01–4.13(m,10H),5.26–5.40(m,2H),5.43–5.53(m,2H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)(实例7-25)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) (Example 7-25)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出73mg，61％。UPLC-MS(方法A)：Rt 2.18min，m/z计算值[M+H]：1002.7，实测值1004.1。Prepared according to General Procedure C, substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 73 mg, 61%. UPLC-MS (Method A): Rt 2.18 min, m/z calcd [M+H]: 1002.7, found 1004.1.

实例7-26：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Example 7-26: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

根据一般程序D制备，用7-(壬氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出610mg，22％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.5Hz,6H),1.14–1.46(m,30H),1.51–1.73(m,8H),2.25–2.39(m,8H),2.78(s,2H),2.87(s,1H),2.94(s,1H),3.53–3.62(m,4H),4.04(t,J＝6.8Hz,4H),4.13(s,4H)。Prepared according to General Procedure D, substituting 7-(nonyloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 610 mg, 22%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.5 Hz, 6H), 1.14-1.46 (m, 30H), 1.51-1.73 (m, 8H), 2.25-2.39 (m, 8H), 2.78 (s, 2H), 2.87 (s, 1H), 2.94 (s, 1H), 3.53-3.62 (m, 4H), 4.04 (t, J = 6.8 Hz, 4H), 4.13 (s, 4H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出320mg，42％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.24–1.41(m,29H),1.46–1.75(m,22H),1.96(s,3H),2.08(s,2H),2.25–2.37(m,8H),3.50(s,2H),3.69–3.78(m,1H),4.00–4.13(m,10H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyldi(pimelate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 320 mg, 42%.¹ H NMR (400MHz, chloroform-d) δ0.87(t,J＝6.7Hz,6H),1.24–1.41(m,29H),1.46–1.75(m,22H),1.96(s,3H),2.08(s,2H),2.25–2.37(m,8H),3.50(s,2H),3.6 9–3.78(m,1H),4.00–4.13(m,10H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)(实例7-26)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate) (Example 7-26)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出21mg，39％。UPLC-MS(方法A)：Rt 2.30min，m/z计算值[M+H]：1006.7，实测值1008.2。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate). Isolated 21 mg, 39%. UPLC-MS (Method A): Rt 2.30 min, m/z calcd [M+H]: 1006.7, found 1008.2.

实例7-27：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Example 7-27: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

根据一般程序D制备，用(Z)-7-(辛-5-烯-1-基氧基)-7-氧代庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出108mg，23％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,6H),1.31–1.44(m,8H),1.55–1.71(m,12H),2.03(h,J＝7.2Hz,8H),2.25–2.40(m,8H),2.69–2.79(m,2H),3.57(d,J＝5.6Hz,4H),4.05(t,J＝6.7Hz,4H),4.13(s,4H),5.24–5.43(m,4H)。Prepared according to General Procedure D, substituting (Z)-7-(oct-5-en-1-yloxy)-7-oxoheptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 108 mg, 23%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (t, J = 7.5 Hz, 6H), 1.31-1.44 (m, 8H), 1.55-1.71 (m, 12H), 2.03 (h, J = 7.2 Hz, 8H), 2.25-2.40 (m, 8H), 2.69-2.79 (m, 2H), 3.57 (d, J = 5.6 Hz, 4H), 4.05 (t, J = 6.7 Hz, 4H), 4.13 (s, 4H), 5.24-5.43 (m, 4H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出180mg，48％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,6H),1.28–1.46(m,8H),1.55–1.75(m,27H),1.92–2.11(m,11H),2.25–2.37(m,8H),3.50(s,2H),4.00–4.15(m,10H),5.24–5.43(m,4H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 180 mg, 48%.¹ H NMR (400MHz, chloroform-d) δ0.95 (t, J = 7.5Hz, 6H), 1.28–1.46 (m, 8H), 1.55–1.75 (m, 27H), 1.92–2.11 (m, 11H), 2.25–2.37 (m, 8H), 3.50 (s, 2H), 4.00–4.15 ( m,10H),5.24–5.43(m,4H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)(实例7-27)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) (Example 7-27)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出31mg，42％。UPLC-MS(方法A)：Rt 2.30min，m/z计算值[M+H]：974.7，实测值976.1。Prepared according to General Procedure C, substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 31 mg, 42%. UPLC-MS (Method A): Rt 2.30 min, m/z calcd [M+H]: 974.7, found 976.1.

实例7-28：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Example 7-28: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出41mg，45％。UPLC-MS(方法A)：Rt 2.15min，m/z计算值[M+H]：985.7，实测值987.0。Prepared according to General Procedure C, substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 41 mg, 45%. UPLC-MS (Method A): Rt 2.15 min, m/z calcd. [M+H]: 985.7, found 987.0.

实例7-29：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Example 7-29: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出29mg，53％。UPLC-MS(方法A)：Rt 2.28min，m/z计算值[M+H]：989.7，实测值991.2。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 29 mg, 53%. UPLC-MS (Method A): Rt 2.28 min, m/z calcd. [M+H]: 989.7, found 991.2.

实例7-30：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)Example 7-30: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-辛-5-烯-1-基)二(庚二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出47mg，45％。UPLC-MS(方法A)：Rt 2.06min，m/z计算值[M+H]：957.7，实测值959.0。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-oct-5-en-1-yl)di(pimelate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 47 mg, 45%. UPLC-MS (Method A): Rt 2.06 min, m/z calcd. [M+H]: 957.7, found 959.0.

实例7-31：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Example 7-31: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(octanedioate)

根据一般程序D制备，用(Z)-8-(壬-3-烯-1-基氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出220mg，22％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.8Hz,6H),1.23–1.40(m,22H),1.57–1.68(m,6H),2.02(q,J＝7.2Hz,4H),2.24–2.41(m,12H),2.73(t,J＝6.4Hz,2H),3.57(d,J＝6.5Hz,4H),4.05(t,J＝6.9Hz,4H),4.13(s,4H),5.25–5.39(m,2H),5.43–5.58(m,2H)。Prepared according to General Procedure D, substituting (Z)-8-(non-3-en-1-yloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 220 mg, 22%.¹ H NMR (400MHz, chloroform-d) δ0.88(t,J＝6.8Hz,6H),1.23–1.40(m,22H),1.57–1.68(m,6H),2.02(q,J＝7.2Hz,4H),2.24–2.41(m,12H),2.73(t,J＝6.4Hz,2H),3 .57(d,J＝6.5Hz,4H),4.05(t,J＝6.9Hz,4H),4.13(s,4H),5.25–5.39(m,2H),5.43–5.58(m,2H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出280mg，51％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.5Hz,6H),1.19–1.43(m,25H),1.55–1.76(m,21H),1.87–2.14(m,8H),2.23-2.45(m,10H),3.50(d,J＝6.7Hz,2H),4.01–4.13(m,8H),5.28–5.41(m,2H),5.42–5.56(m,2H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)di(suberate). Isolated 280 mg, 51%.¹ H NMR (400MHz, chloroform-d) δ0.88(t,J＝6.5Hz,6H),1.19–1.43(m,25H),1.55–1.76(m,21H),1.87–2.14(m,8H),2.23-2.45(m,10H),3.50(d,J＝6.7Hz,2H),4 .01–4.13(m,8H),5.28–5.41(m,2H),5.42–5.56(m,2H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)(实例7-31)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) (Example 7-31)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出31mg，52％。UPLC-MS(方法A)：Rt 2.24min，m/z计算值[M+H]：1030.7，实测值1032.1。Prepared according to General Procedure C, substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 31 mg, 52%. UPLC-MS (Method A): Rt 2.24 min, m/z calcd [M+H]: 1030.7, found 1032.1.

实例7-32：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Example 7-32: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)8,8'-dinonyl di(octanedioate)

根据一般程序D制备，用8-(壬氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出370mg，21％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.18–1.42(m,28H),1.50–1.70(m,16H),2.24–2.38(m,8H),2.72(s,2H),3.57(s,4H),4.04(t,J＝6.7Hz,4H),4.13(s,4H)。Prepared according to General Procedure D, substituting 8-(nonyloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 370 mg, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.18-1.42 (m, 28H), 1.50-1.70 (m, 16H), 2.24-2.38 (m, 8H), 2.72 (s, 2H), 3.57 (s, 4H), 4.04 (t, J = 6.7 Hz, 4H), 4.13 (s, 4H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-dinonyl di(suberate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸)酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出290mg，50％。¹H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.17–1.41(m,37H),1.55–1.65(m,17H),1.69(d,J＝12.5Hz,3H),1.96(s,3H),2.24–2.36(m,8H),3.50(s,2H),4.00–4.15(m,12H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)8,8'-dinonyldi(suberate). Isolated 290 mg, 50%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.17-1.41 (m, 37H), 1.55-1.65 (m, 17H), 1.69 (d, J = 12.5 Hz, 3H), 1.96 (s, 3H), 2.24-2.36 (m, 8H), 3.50 (s, 2H), 4.00-4.15 (m, 12H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)(实例7-32)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate) (Example 7-32)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出50mg，61％。UPLC-MS(方法A)：Rt 2.33min，m/z计算值[M+H]：1034.7，实测值1036.1。Prepared according to General Procedure C substituting O'1,O1-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-dinonyl di(suberate). Isolated 50 mg, 61%. UPLC-MS (Method A): Rt 2.33 min, m/z calcd [M+H]: 1034.7, found 1036.1.

实例7-33：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Example 7-33: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

步骤1：O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Step 1: O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

根据一般程序D制备，用(Z)-8-(辛-5-烯-1-基氧基)-8-氧代辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出324mg，20％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,6H),1.29–1.46(m,12H),1.56–1.68(m,12H),1.96–2.11(m,8H),2.24-2.38(m,8H),2.70(t,J＝6.5Hz,2H),3.57(d,J＝6.5Hz,4H),4.06(t,J＝6.7Hz,4H),4.13(s,4H),5.21–5.47(m,4H)。Prepared according to General Procedure D, substituting (Z)-8-(oct-5-en-1-yloxy)-8-oxooctanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 324 mg, 20%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (t, J = 7.5 Hz, 6H), 1.29-1.46 (m, 12H), 1.56-1.68 (m, 12H), 1.96-2.11 (m, 8H), 2.24-2.38 (m, 8H), 2.70 (t, J = 6.5 Hz, 2H), 3.57 (d, J = 6.5 Hz, 4H), 4.06 (t, J = 6.7 Hz, 4H), 4.13 (s, 4H), 5.21-5.47 (m, 4H).

步骤2：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Step 2: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

根据一般程序E制备，用O'1,O1-(2,2-双(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出204mg，52％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,6H),1.22–1.45(m,12H),1.46–1.79(m,27H),1.90–2.14(m,12H),2.24–2.36(m,8H),2.55(s,1H),3.49(d,J＝6.7Hz,2H),4.01–4.13(m,8H),5.23–5.46(m,4H)。Prepared according to General Procedure E, substituting O'1,O1-(2,2-bis(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 204 mg, 52%.¹ H NMR (400MHz, chloroform-d) δ0.95(t,J＝7.5Hz,6H),1.22–1.45(m,12H),1.46–1.79(m,27H),1.90–2.14(m,12H),2.24–2.36(m,8H),2.55(s,1H),3.49(d,J =6.7Hz,2H),4.01–4.13(m,8H),5.23–5.46(m,4H).

步骤3：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)(实例7-33)Step 3: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) (Example 7-33)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出73mg，61％。UPLC-MS(方法A)：Rt 2.09min，m/z计算值[M+H]：1002.7，实测值1004.0。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 73 mg, 61%. UPLC-MS (Method A): Rt 2.09 min, m/z calcd [M+H]: 1002.7, found 1004.0.

实例7-34：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)Example 7-34: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-壬-3-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出87mg，62％。UPLC-MS(方法A)：Rt 2.16min，m/z计算值[M+H]：1013.7，实测值1015.0。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-non-3-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 87 mg, 62%. UPLC-MS (Method A): Rt 2.16 min, m/z calcd. [M+H]: 1013.7, found 1015.0.

实例7-35：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)Example 7-35: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)8,8'-dinonyl di(suberate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二壬基二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出148mg，70％。UPLC-MS(方法B)：Rt 5.47min，m/z计算值[M+H]：1017.7，实测值1019.2。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-dinonyl di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 148 mg, 70%. UPLC-MS (Method B): Rt 5.47 min, m/z calcd. [M+H]: 1017.7, found 1019.2.

实例7-36：O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)Example 7-36: O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate)

根据一般程序C制备，用O'1,O1-(2-((2-((3r,5r,7r)-金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)8,8'-二((Z)-辛-5-烯-1-基)二(辛二酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出50mg，55％。UPLC-MS(方法A)：Rt 2.13min，m/z计算值[M+H]：985.7，实测值987.0。Prepared according to General Procedure C substituting O'1,O1-(2-((2-((3r,5r,7r)-adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)8,8'-di((Z)-oct-5-en-1-yl)di(suberate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 50 mg, 55%. UPLC-MS (Method A): Rt 2.13 min, m/z calcd. [M+H]: 985.7, found 987.0.

实例7-37：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(((Z)-壬-3-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Example 7-37: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(((Z)-non-3-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

步骤1：(Z)-5-(壬-3-烯-1-基氧基)-5-氧代戊酸Step 1: (Z)-5-(Non-3-en-1-yloxy)-5-oxopentanoic acid

根据一般程序A制备，用戊二酸取代己二酸。分离出2.1g，54％。¹H NMR(400MHz,氯仿-d)δ0.86(t,J＝6.7Hz,3H),1.16–1.47(m,6H),1.87–2.06(m,4H),2.30–2.46(m,6H),4.06(t,J＝6.9Hz,2H),5.25–5.37(m,1H),5.43–5.54(m,1H),12.07(s,1H)。Prepared according to General Procedure A, substituting glutaric acid for adipic acid. Isolated 2.1 g, 54%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.86 (t, J = 6.7 Hz, 3H), 1.16-1.47 (m, 6H), 1.87-2.06 (m, 4H), 2.30-2.46 (m, 6H), 4.06 (t, J = 6.9 Hz, 2H), 5.25-5.37 (m, 1H), 5.43-5.54 (m, 1H), 12.07 (s, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((5-(((Z)-壬-3-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((5-(((Z)-non-3-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

根据一般程序B制备，用(Z)-5-(壬-3-烯-1-基氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出250mg，20％。1H NMR(400MHz,DMSO-d₆)δ0.85(t,J＝7.0Hz,9H),1.19–1.36(m,21H),1.69–1.81(m,6H),2.00(q,J＝6.7Hz,6H),2.25–2.39(m,18H),3.99(t,J＝6.7Hz,6H),4.07(s,6H),5.26–5.37(m,3H),5.39–5.51(m,3H)。Prepared according to General Procedure B, substituting (Z)-5-(non-3-en-1-yloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 250 mg, 20%. 1H NMR (400 MHz, DMSO-_d6 ) δ 0.85 (t, J = 7.0 Hz, 9H), 1.19-1.36 (m, 21H), 1.69-1.81 (m, 6H), 2.00 (q, J = 6.7 Hz, 6H), 2.25-2.39 (m, 18H), 3.99 (t, J = 6.7 Hz, 6H), 4.07 (s, 6H), 5.26-5.37 (m, 3H), 5.39-5.51 (m, 3H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(((Z)-壬-3-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯(实例7-37)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(((Z)-non-3-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate (Example 7-37)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((5-(((Z)-壬-3-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出55mg，48％。UPLC-MS(方法A)：Rt2.09min，m/z计算值[M+H]：1008.7，实测值1009.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((5-(((Z)-non-3-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diglutarate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 55 mg, 48%. UPLC-MS (Method A): Rt 2.09 min, m/z calcd [M+H]: 1008.7, found 1009.0.

实例7-38：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(壬氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯Example 7-38: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(nonyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate

步骤1：5-(壬氧基)-5-氧代戊酸Step 1: 5-(Nonyloxy)-5-oxopentanoic acid

根据一般程序A制备，用戊二酸取代己二酸，并用壬-1-醇取代(Z)-壬-3-烯-1-醇。分离出2.6g，67％。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝7.0Hz,3H),1.18–1.35(m,12H),1.54(q,J＝6.8Hz,2H),1.66–1.79(m,2H),2.24(t,J＝7.3Hz,2H),2.31(t,J＝7.4Hz,2H),4.00(t,J＝6.6Hz,2H),12.07(s,1H)。Prepared according to General Procedure A, substituting glutaric acid for adipic acid and nonan-1-ol for (Z)-non-3-en-1-ol. Isolated 2.6 g, 67%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J = 7.0 Hz, 3H), 1.18-1.35 (m, 12H), 1.54 (q, J = 6.8 Hz, 2H), 1.66-1.79 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 12.07 (s, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((5-(壬氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((5-(nonyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl) dinonyl diglutarate

根据一般程序B制备，用5-(壬氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出290mg，23％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.6Hz,9H),1.09–1.45(m,36H),1.54–1.65(m,6H),1.86–2.02(m,6H),2.31–2.44(m,12H),3.51(d,J＝6.4Hz,2H),4.01–4.12(m,12H)。Prepared according to General Procedure B, substituting 5-(nonyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 290 mg, 23%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.6 Hz, 9H), 1.09-1.45 (m, 36H), 1.54-1.65 (m, 6H), 1.86-2.02 (m, 6H), 2.31-2.44 (m, 12H), 3.51 (d, J = 6.4 Hz, 2H), 4.01-4.12 (m, 12H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(壬氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯(实例7-38)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(nonyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate (Example 7-38)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((5-(壬氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出75mg，54％。UPLC-MS(方法A)：Rt 2.27min，m/z计算值[M+H]：1014.7，实测值1015.1。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate for O,O'-(2-(hydroxymethyl)-2-(((5-(nonyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate. Isolated 75 mg, 54%. UPLC-MS (Method A): Rt 2.27 min, m/z calcd [M+H]: 1014.7, found 1015.1.

实例7-39：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(((Z)-辛-5-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯Example 7-39: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(((Z)-oct-5-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate

步骤1：(Z)-5-(辛-5-烯-1-基氧基)-5-氧代戊酸Step 1: (Z)-5-(oct-5-en-1-yloxy)-5-oxopentanoic acid

根据一般程序A制备，用戊二酸取代己二酸，并用(Z)-辛-5-烯-1-醇取代(Z)-壬-3-烯-1-醇。分离出2.3g，58％。¹H NMR(400MHz,DMSO-d₆)δ0.91(t,J＝7.4Hz,3H),1.28–1.41(m,2H),1.49–1.64(m,2H),1.66–1.81(m,2H),1.93–2.06(m,4H),2.23(t,J＝7.2Hz,2H),2.32(t,J＝7.3Hz,2H),4.01(t,J＝6.4Hz,2H),5.23–5.39(m,2H),11.95–12.19(m,1H)。Prepared according to General Procedure A, substituting glutaric acid for adipic acid and (Z)-oct-5-en-1-ol for (Z)-non-3-en-1-ol. Isolated 2.3 g, 58%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.91 (t, J = 7.4 Hz, 3H), 1.28-1.41 (m, 2H), 1.49-1.64 (m, 2H), 1.66-1.81 (m, 2H), 1.93-2.06 (m, 4H), 2.23 (t, J = 7.2 Hz, 2H), 2.32 (t, J = 7.3 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 5.23-5.39 (m, 2H), 11.95-12.19 (m, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((5-(((Z)-辛-5-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((5-(((Z)-oct-5-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate

根据一般程序B制备，用(Z)-5-(辛-5-烯-1-基氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出235mg，20％。¹H NMR(400MHz,氯仿-d)δ0.95(t,J＝7.5Hz,9H),1.31–1.47(m,6H),1.56–1.70(m,6H),1.86–2.16(m,18H),2.31–2.44(m,12H),2.57–2.66(m,1H),3.51(d,J＝6.8Hz,2H),4.02–4.13(m,12H),5.12–5.56(m,6H)。Prepared according to General Procedure B, substituting (Z)-5-(oct-5-en-1-yloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 235 mg, 20%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.95 (t, J = 7.5 Hz, 9H), 1.31-1.47 (m, 6H), 1.56-1.70 (m, 6H), 1.86-2.16 (m, 18H), 2.31-2.44 (m, 12H), 2.57-2.66 (m, 1H), 3.51 (d, J = 6.8 Hz, 2H), 4.02-4.13 (m, 12H), 5.12-5.56 (m, 6H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(((Z)-辛-5-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯(实例7-39)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(((Z)-oct-5-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate (Example 7-39)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((5-(((Z)-辛-5-烯-1-基)氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出43mg，36％。UPLC-MS(方法A)：Rt1.95min，m/z计算值[M+H]：966.6，实测值967.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((5-(((Z)-oct-5-en-1-yl)oxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diglutarate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 43 mg, 36%. UPLC-MS (Method A): Rt 1.95 min, m/z calcd [M+H]: 966.6, found 967.0.

实例7-40：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二庚基二戊二酸酯Example 7-40: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)diheptyl diglutarate

步骤1：5-(庚氧基)-5-氧代戊酸Step 1: 5-(Heptyloxy)-5-oxopentanoic acid

根据一般程序A制备，用戊二酸取代己二酸，并用庚烷-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.95g，55％。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.5Hz,3H),1.21–1.30(m,8H),1.51–1.59(m,2H),1.66–1.79(m,2H),2.24(t,J＝7.3Hz,2H),2.32(t,J＝7.4Hz,2H),4.00(t,J＝6.6Hz,2H),12.08(s,1H)。Prepared according to General Procedure A, substituting glutaric acid for adipic acid and heptane-1-ol for (Z)-non-3-en-1-ol. Isolated 1.95 g, 55%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J=6.5 Hz, 3H), 1.21-1.30 (m, 8H), 1.51-1.59 (m, 2H), 1.66-1.79 (m, 2H), 2.24 (t, J=7.3 Hz, 2H), 2.32 (t, J=7.4 Hz, 2H), 4.00 (t, J=6.6 Hz, 2H), 12.08 (s, 1H).

步骤2：二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯Step 2: Diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate

根据一般程序B制备，用5-(庚氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出170mg，15％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.3Hz,9H),1.16–1.42(m,24H),1.56–1.69(m,6H),1.87–2.00(m,6H),2.31–2.44(m,12H),3.50(d,J＝6.0Hz,2H),4.05(t,J＝6.8Hz,6H),4.10(s,6H)。Prepared according to General Procedure B, substituting 5-(heptyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 170 mg, 15%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.3 Hz, 9H), 1.16-1.42 (m, 24H), 1.56-1.69 (m, 6H), 1.87-2.00 (m, 6H), 2.31-2.44 (m, 12H), 3.50 (d, J = 6.0 Hz, 2H), 4.05 (t, J = 6.8 Hz, 6H), 4.10 (s, 6H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二庚基二戊二酸酯(实例7-40)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)diheptyl diglutarate (Example 7-40)

根据一般程序C制备，用二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出27mg，37％。UPLC-MS(方法A)：Rt 2.03min，m/z计算值[M+H]：930.6，实测值930.9。Prepared according to General Procedure C substituting diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 27 mg, 37%. UPLC-MS (Method A): Rt 2.03 min, m/z calcd [M+H]: 930.6, found 930.9.

实例7-41：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯Example 7-41: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(octyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate

步骤1：5-(辛氧基)-5-氧代戊酸Step 1: 5-(Octyloxy)-5-oxopentanoic acid

根据一般程序A制备，用戊二酸取代己二酸，并用辛烷-1-醇取代(Z)-壬-3-烯-1-醇。分离出1.9g，50％。1H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.9Hz,3H),1.22–1.33(m,8H),1.34–1.44(m,1H),1.50–1.61(m,2H),1.66–1.79(m,2H),2.23(t,J＝7.3Hz,2H),2.31(t,J＝7.4Hz,2H),3.36(t,J＝6.4Hz,1H),4.00(t,J＝6.6Hz,2H),12.08(s,1H)。Prepared according to General Procedure A, substituting glutaric acid for adipic acid and octan-1-ol for (Z)-non-3-en-1-ol. Isolated 1.9 g, 50%. 1H NMR (400 MHz, DMSO-_d6 ) δ 0.86 (t, J = 6.9 Hz, 3H), 1.22-1.33 (m, 8H), 1.34-1.44 (m, 1H), 1.50-1.61 (m, 2H), 1.66-1.79 (m, 2H), 2.23 (t, J = 7.3 Hz, 2H), 2.31 (t, J = 7.4 Hz, 2H), 3.36 (t, J = 6.4 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 12.08 (s, 1H).

步骤2：O,O'-(2-(羟甲基)-2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯Step 2: O,O'-(2-(Hydroxymethyl)-2-(((5-(octyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate

根据一般程序B制备，用5-(辛氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出44mg，20％。¹H NMR(400MHz,DMSO-d₆)δ0.86(t,J＝6.5Hz,3H),1.14–1.40(m,10H),1.47–1.61(m,2H),1.64–1.85(m,2H),2.24(t,J＝7.3Hz,2H),2.31(t,J＝7.4Hz,2H),4.00(t,J＝6.6Hz,2H),12.07(s,1H)。Prepared according to General Procedure B, substituting 5-(octyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 44 mg, 20%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.86 (t, J=6.5 Hz, 3H), 1.14-1.40 (m, 10H), 1.47-1.61 (m, 2H), 1.64-1.85 (m, 2H), 2.24 (t, J=7.3 Hz, 2H), 2.31 (t, J=7.4 Hz, 2H), 4.00 (t, J=6.6 Hz, 2H), 12.07 (s, 1H).

步骤3：O,O'-(2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)-2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯(实例7-41)Step 3: O,O'-(2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((5-(octyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate (Example 7-41)

根据一般程序C制备，用O,O'-(2-(羟甲基)-2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出44mg，37％。UPLC-MS(方法A)：Rt 2.12min，m/z计算值[M+H]：972.7，实测值973.1。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((5-(octyloxy)-5-oxopentanoyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 44 mg, 37%. UPLC-MS (Method A): Rt 2.12 min, m/z calcd [M+H]: 972.7, found 973.1.

实例7-42：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Example 7-42: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

根据一般程序D制备，用(Z)-5-(壬-3-烯-1-基氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出90mg，25％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝7.2Hz,6H),1.13–1.45(m,12H),1.88–2.10(m,8H),2.31–2.47(m,12H),2.58–2.74(m,2H),3.58(d,J＝6.2Hz,4H),4.06(t,J＝7.1Hz,4H),4.13(s,4H),5.18–5.68(m,4H)。Prepared according to General Procedure D, substituting (Z)-5-(non-3-en-1-yloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 90 mg, 25%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (t, J = 7.2 Hz, 6H), 1.13-1.45 (m, 12H), 1.88-2.10 (m, 8H), 2.31-2.47 (m, 12H), 2.58-2.74 (m, 2H), 3.58 (d, J = 6.2 Hz, 4H), 4.06 (t, J = 7.1 Hz, 4H), 4.13 (s, 4H), 5.18-5.68 (m, 4H).

步骤2：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Step 2: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出85mg，42％。1H NMR(400MHz,氯仿-d)δ0.88(t,J＝6.8Hz,6H),1.22–1.39(m,17H),1.55–1.75(m,15H),1.90-2.07(m,8H),2.31–2.44(m,12H),3.51(d,J＝6.8Hz,2H),4.02–4.15(m,8H),5.15–5.67(m,4H)。Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diglutarate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 85 mg, 42%. 4 .02–4.15(m,8H),5.15–5.67(m,4H).

步骤3：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯(实例7-42)Step 3: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate (Example 7-42)

根据一般程序C制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出21mg，20％。UPLC-MS(方法A)：Rt 2.11min，m/z计算值[M+H]：946.6，实测值947.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate. Isolated 21 mg, 20%. UPLC-MS (Method A): Rt 2.11 min, m/z calcd [M+H]: 946.6, found 947.0.

实例7-43：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯Example 7-43: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diglutarate

根据一般程序D制备，用5-(壬氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出110mg，24％。1H NMR(400MHz,氯仿-d)δ0.83–0.92(m,6H),1.17–1.41(m,24H),1.56–1.69(m,4H),1.85–2.05(m,4H),2.29–2.53(m,8H),3.58(s,4H),4.05(t,J＝7.6Hz,4H),4.13(s,4H)。Prepared according to General Procedure D, substituting 5-(nonyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 110 mg, 24%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.83–0.92 (m, 6H), 1.17–1.41 (m, 24H), 1.56–1.69 (m, 4H), 1.85–2.05 (m, 4H), 2.29–2.53 (m, 8H), 3.58 (s, 4H), 4.05 (t, J=7.6 Hz, 4H), 4.13 (s, 4H).

步骤2：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二壬基二戊二酸酯Step 2: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dinonyl diglutarate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二戊二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出52mg，42％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.18–1.38(m,26H),1.53(s,6H),1.58–1.74(m,8H),1.89–2.00(m,7H),2.08(s,2H),2.31–2.44(m,8H),2.57(t,J＝6.9Hz,1H),3.51(d,J＝6.8Hz,2H),4.01–4.13(m,10H)。Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diglutarate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 52 mg, 42%. 1H NMR (400MHz, chloroform-d) δ0.87(t,J＝6.7Hz,6H),1.18–1.38(m,26H),1.53(s,6H),1.58–1.74(m,8H),1.89–2.00(m,7H),2.08(s,2H),2.31–2.44(m,8H), 2.57(t,J＝6.9Hz,1H), 3.51(d,J＝6.8Hz,2H), 4.01–4.13(m,10H).

步骤3：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二壬基二戊二酸酯(实例7-43)Step 3: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dinonyl diglutarate (Example 7-43)

根据一般程序C制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二壬基二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出20mg，32％。UPLC-MS(方法A)：Rt 2.19min，m/z计算值[M+H]：950.7，实测值951.1。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dinonyl diglutarate. Isolated 20 mg, 32%. UPLC-MS (Method A): Rt 2.19 min, m/z calcd [M+H]: 950.7, found 951.1.

实例7-44：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯Example 7-44: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate

根据一般程序D制备，用(Z)-5-(辛-5-烯-1-基氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出160mg，19％。1H NMR(400MHz,氯仿-d)δ0.94(t,J＝7.5Hz,6H),1.33–1.46(m,4H),1.57–1.69(m,6H),1.89–2.12(m,10H),2.32–2.48(m,8H),2.58–2.81(m,2H),3.58(s,4H),4.06(t,J＝6.7Hz,4H),4.12(s,4H),5.23–5.44(m,4H)。Prepared according to General Procedure D, substituting (Z)-5-(oct-5-en-1-yloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 160 mg, 19%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.94 (t, J = 7.5 Hz, 6H), 1.33-1.46 (m, 4H), 1.57-1.69 (m, 6H), 1.89-2.12 (m, 10H), 2.32-2.48 (m, 8H), 2.58-2.81 (m, 2H), 3.58 (s, 4H), 4.06 (t, J = 6.7 Hz, 4H), 4.12 (s, 4H), 5.23-5.44 (m, 4H).

步骤2：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯Step 2: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出90mg，51％。1H NMR(400MHz,氯仿-d)δ0.94(t,J＝7.5Hz,6H),1.33–1.46(m,5H),1.48–1.75(m,18H),1.87–2.13(m,16H),2.30–2.44(m,7H),3.51(d,J＝6.2Hz,2H),4.02–4.13(m,10H),4.95–5.76(m,4H)。Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-oct-5-en-1-yl)diglutarate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 90 mg, 51%. 1H NMR (400 MHz, CHLOROFORM-d) δ0.94 (t, J=7.5 Hz, 6H), 1.33–1.46 (m, 5H), 1.48–1.75 (m, 18H), 1.87–2.13 (m, 16H), 2.30–2.44 (m, 7H), 3.51 (d, J=6.2 Hz, 2H), 4.02–4.13 (m, 10H), 4.95–5.76 (m, 4H).

步骤3：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯(实例7-44)Step 3: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate (Example 7-44)

根据一般程序C制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二((Z)-辛-5-烯-1-基)二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出64mg，35％。UPLC-MS(方法A)：Rt 2.01min，m/z计算值[M+H]：918.6，实测值919.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)di((Z)-oct-5-en-1-yl)diglutarate. Isolated 64 mg, 35%. UPLC-MS (Method A): Rt 2.01 min, m/z calcd [M+H]: 918.6, found 919.0.

实例7-45：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二庚基二戊二酸酯Example 7-45: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)diheptyl diglutarate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二庚基二戊二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)diheptyl diglutarate

根据一般程序D制备，用5-(庚氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出160mg，20％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.8Hz,6H),1.19–1.39(m,17H),1.50–1.67(m,4H),1.89–2.01(m,4H),2.32–2.46(m,8H),3.58(s,4H),3.62(t,J＝6.6Hz,1H),4.05(t,J＝6.8Hz,4H),4.12(s,4H)。Prepared according to General Procedure D, substituting 5-(heptyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 160 mg, 20%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.8 Hz, 6H), 1.19-1.39 (m, 17H), 1.50-1.67 (m, 4H), 1.89-2.01 (m, 4H), 2.32-2.46 (m, 8H), 3.58 (s, 4H), 3.62 (t, J = 6.6 Hz, 1H), 4.05 (t, J = 6.8 Hz, 4H), 4.12 (s, 4H).

步骤2：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二庚基二戊二酸酯Step 2: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diheptyl diglutarate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二庚基二戊二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出65mg，43％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.7Hz,6H),1.21–1.38(m,18H),1.55–1.65(m,12H),1.69(d,J＝12.0Hz,3H),1.89–1.99(m,6H),2.08(s,2H),2.31–2.44(m,8H),2.59(t,J＝6.9Hz,1H),3.51(d,J＝6.9Hz,2H),4.01–4.09(m,6H),4.10(s,4H)。步骤3：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二庚基二戊二酸酯(实例7-45)Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)diheptyl diglutarate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 65 mg, 43%. 1H NMR (400MHz, chloroform-d) δ0.87(t,J＝6.7Hz,6H),1.21–1.38(m,18H),1.55–1.65(m,12H),1.69(d,J＝12.0Hz,3H),1.89–1.99(m,6H),2.08(s,2H),2.31–2. 44(m,8H),2.59(t,J＝6.9Hz,1H),3.51(d,J＝6.9Hz,2H),4.01–4.09(m,6H),4.10(s,4H). Step 3: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)diheptyl diglutarate (Example 7-45)

根据一般程序C制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二庚基二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出45mg，62％。UPLC-MS(方法A)：Rt 2.00min，m/z计算值[M+H]：894.6，实测值895.1。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diheptyl diglutarate. Isolated 45 mg, 62%. UPLC-MS (Method A): Rt 2.00 min, m/z calcd [M+H]: 894.6, found 895.1.

实例7-46：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯Example 7-46: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate

步骤1：O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二辛基二戊二酸酯Step 1: O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate

根据一般程序D制备，用5-(辛氧基)-5-氧代戊酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出165mg，21％。1H NMR(400MHz,氯仿-d)δ0.87(t,J＝6.4Hz,6H),1.16–1.40(m,18H),1.90–2.02(m,6H),2.30–2.51(m,8H),3.64(s,10H),4.05(t,J＝6.7Hz,4H),4.21(s,4H)。Prepared according to General Procedure D, substituting 5-(octyloxy)-5-oxopentanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 165 mg, 21%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.4 Hz, 6H), 1.16-1.40 (m, 18H), 1.90-2.02 (m, 6H), 2.30-2.51 (m, 8H), 3.64 (s, 10H), 4.05 (t, J = 6.7 Hz, 4H), 4.21 (s, 4H).

步骤2：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二辛基二戊二酸酯Step 2: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate

根据一般程序E制备，用O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二辛基二戊二酸酯取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出90mg，38％。1H NMR(400MHz,氯仿-d)δ0.88(s,6H),1.19–1.38(m,28H),1.45–1.77(m,10H),1.87–2.01(m,6H),2.08(s,2H),2.29–2.47(m,6H),3.51(s,2H),4.02–4.13(m,10H)Prepared according to General Procedure E, substituting O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 90 mg, 38%. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (s, 6H), 1.19–1.38 (m, 28H), 1.45–1.77 (m, 10H), 1.87–2.01 (m, 6H), 2.08 (s, 2H), 2.29–2.47 (m, 6H), 3.51 (s, 2H), 4.02–4.13 (m, 10H)

步骤3：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯(实例7-46)Step 3: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate (Example 7-46)

根据一般程序C制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二辛基二戊二酸酯取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出17mg，31％。UPLC-MS(方法A)：Rt 2.14min，m/z计算值[M+H]：922.6，实测值923.0。Prepared according to General Procedure C substituting O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)diadipate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate. Isolated 17 mg, 31%. UPLC-MS (Method A): Rt 2.14 min, m/z calcd [M+H]: 922.6, found 923.0.

实例7-47：二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二戊二酸酯Example 7-47: Diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)diglutarate

一般程序F：General Procedure F:

向4-(吡咯烷-1-基)丁酸(1.0当量)在DCM(3mL/0.07mmol)中的搅拌溶液中添加EDC(1.3当量)、DMAP(0.2当量)和二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯(1.0当量)和无水DIPEA(3.0当量)。将反应混合物在25℃搅拌16h。完成后，将反应混合物用DCM(25mL)稀释并用饱和NaHCO₃(3x25 mL)萃取。有机层用水(2x25 mL)和盐水(2x15 mL)洗涤。将有机部分经无水Na₂SO₄干燥，过滤并在减压下浓缩。将粗品材料通过CombiFlash柱色谱纯化，用10％MeOH-DCM洗脱，以得到二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二戊二酸酯(41mg，58％)，为无色液体。UPLC-MS(方法A)：Rt1.98min，m/z计算值[M+H]：912.6，实测值913.0。To a stirred solution of 4-(pyrrolidin-1-yl)butanoic acid (1.0 equiv) in DCM (3 mL/0.07 mmol) was added EDC (1.3 equiv), DMAP (0.2 equiv) and diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate (1.0 equiv) and anhydrous DIPEA (3.0 equiv). The reaction mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with DCM (25 mL) and extracted with saturated NaHCO₃ (3x25 mL). The organic layer was washed with water (2x25 mL) and brine (2x15 mL). The organic portion was dried over anhydrous Na₂ SO₄ , filtered and concentrated under reduced pressure. The crude material was purified by CombiFlash column chromatography eluting with 10% MeOH-DCM to give diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)diglutarate (41 mg, 58%) as a colorless liquid. UPLC-MS (Method A): Rt 1.98 min, m/z calcd [M+H]: 912.6, found 913.0.

实例7-48：二辛基O,O'-(2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二戊二酸酯Example 7-48: Dioctyl O,O'-(2-(((5-(octyloxy)-5-oxopentanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)diglutarate

根据一般程序F制备，用O,O'-(2-(羟甲基)-2-(((5-(辛氧基)-5-氧代戊酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出43mg，35％。UPLC-MS(方法B)：Rt 5.27min，m/z计算值[M+H]：954.6，实测值955.2。Prepared according to General Procedure F substituting O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate for diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate. Isolated 43 mg, 35%. UPLC-MS (Method B): Rt 5.27 min, m/z calcd [M+H]: 954.6, found 955.2.

实例7-49：O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二辛基二戊二酸酯Example 7-49: O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)dioctyl diglutarate

根据一般程序F制备，用O,O'-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二辛基二戊二酸酯取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出33mg，59％。UPLC-MS(方法A)：Rt 2.15min，m/z计算值[M+H]：904.6，实测值905.0。Prepared according to General Procedure F substituting diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate with O,O'-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)dioctyl diglutarate. Isolated 33 mg, 59%. UPLC-MS (Method A): Rt 2.15 min, m/z calcd [M+H]: 904.6, found 905.0.

实例7-50：二((Z)-壬-3-烯-1-基)O,O'-(2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二己二酸酯Example 7-50: Di((Z)-non-3-en-1-yl)O,O'-(2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)diadipic acid ester

根据一般程序F制备，用O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出33mg，31％。UPLC-MS(方法B)：Rt 5.29min，m/z计算值[M+H]：1032.7，实测值1033.1。Prepared according to General Procedure F substituting O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate with O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 33 mg, 31%. UPLC-MS (Method B): Rt 5.29 min, m/z calcd [M+H]: 1032.7, found 1033.1.

实例7-51：7,7'-二((Z)-壬-3-烯-1-基)O'1,O1-(2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二(庚二酸酯)Example 7-51: 7,7'-di((Z)-non-3-en-1-yl)O'1,O1-(2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)di(pimelate)

根据一般程序F制备，用O'1,O1-(2-(羟甲基)-2-(((7-(((Z)-壬-3-烯-1-基)氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出50mg，84％。UPLC-MS(方法A)：Rt 2.25min，m/z计算值[M+H]：1075.5，实测值1075.8。Prepared according to General Procedure F substituting diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate with O'1,O1-(2-(hydroxymethyl)-2-(((7-(((Z)-non-3-en-1-yl)oxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate). Isolated 50 mg, 84%. UPLC-MS (Method A): Rt 2.25 min, m/z calcd [M+H]: 1075.5, found 1075.8.

实例7-52：7,7'-二壬基O'1,O1-(2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)二(庚二酸酯)Example 7-52: 7,7'-Dinonyl O'1,O1-(2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)di(pimelate)

根据一般程序F制备，用O'1,O1-(2-(羟甲基)-2-(((7-(壬氧基)-7-氧代庚酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出43mg，80％。UPLC-MS(方法A)：Rt 2.39min，m/z计算值[M+H]：1080.8，实测值1080.8。Prepared according to General Procedure F substituting O'1,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate with O'1,O1-(2-(hydroxymethyl)-2-(((7-(nonyloxy)-7-oxoheptanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate). Isolated 43 mg, 80%. UPLC-MS (Method A): Rt 2.39 min, m/z calcd [M+H]: 1080.8, found 1080.8.

实例7-53：O'1,O1-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)Example 7-53: O'1,O1-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate)

根据一般程序F制备，用O'1,O1-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二((Z)-壬-3-烯-1-基)二(庚二酸酯)取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出27mg，46％。UPLC-MS(方法A)：Rt 2.25min，m/z计算值[M+H]：984.7，实测值984.8。Prepared according to General Procedure F, substituting diheptyl O,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl) diglutarate with O'1,O1-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-di((Z)-non-3-en-1-yl)di(pimelate). Isolated 27 mg, 46%. UPLC-MS (Method A): Rt 2.25 min, m/z calcd [M+H]: 984.7, found 984.8.

实例7-54：O'1,O1-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(((4-(吡咯烷-1-基)丁酰基)氧基)甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)Example 7-54: O'1,O1-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate)

根据一般程序F制备，用O'1,O1-(2-((2-(金刚烷-1-基)乙酰氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)7,7'-二壬基二(庚二酸酯)取代二庚基O,O'-(2-(((5-(庚氧基)-5-氧代戊酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)二戊二酸酯。分离出17mg，57％。UPLC-MS(方法A)：Rt 2.25min，m/z计算值[M+H]：988.7，实测值988.8。Prepared according to General Procedure F, substituting O'1,O'-(2-(((5-(heptyloxy)-5-oxopentanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)diglutarate with O'1,O1-(2-((2-(adamantan-1-yl)acetoxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)7,7'-dinonyl di(pimelate). Isolated 17 mg, 57%. UPLC-MS (Method A): Rt 2.25 min, m/z calcd [M+H]: 988.7, found 988.8.

实例7-55：Example 7-55:

步骤1：6-羟基己基2-丁基辛酸酯Step 1: 6-Hydroxyhexyl 2-butyl octanoate

一般程序G：General Procedure G:

向2-丁基辛酸(1当量)在DCM(10.0mL)中的搅拌溶液中添加EDC(1.3当量)、DMAP(0.1当量)和DIPEA(1.5当量)。将反应混合物在25℃搅拌10min，并且然后添加己烷-1,6-二醇(3.0当量)。将反应混合物在25℃进一步搅拌16h。将水(50mL)添加到反应物料中并用DCM(2X 100mL)萃取。有机层经无水Na₂SO₄干燥，在减压下蒸发，以得到粗品化合物。由此获得的粗品化合物通过组合快速色谱使用10％乙酸乙酯在己烷中纯化，以得到6-羟基己基2-丁基辛酸酯(1.0g，67％)，为无色油状物。¹H NMR(400MHz,氯仿-d)δ0.80–0.90(m,6H),1.10–1.70(m,25H),2.26–2.33(m,1H),3.61–3.66(m,2H),4.06(t,J＝6.6Hz,2H)。To a stirred solution of 2-butyloctanoic acid (1 eq.) in DCM (10.0 mL), EDC (1.3 eq.), DMAP (0.1 eq.) and DIPEA (1.5 eq.) were added. The reaction mixture was stirred at 25 °C for 10 min, and then hexane-1,6-diol (3.0 eq.) was added. The reaction mixture was further stirred at 25 °C for 16 h. Water (50 mL) was added to the reaction mass and extracted with DCM (2X 100 mL). The organic layer was dried over anhydrous Na₂ SO₄ and evaporated under reduced pressure to obtain a crude compound. The crude compound thus obtained was purified by combined flash chromatography using 10% ethyl acetate in hexane to obtain 6-hydroxyhexyl 2-butyloctanoate (1.0 g, 67%) as a colorless oil.¹ H NMR (400MHz, chloroform-d) δ0.80–0.90 (m, 6H), 1.10–1.70 (m, 25H), 2.26–2.33 (m, 1H), 3.61–3.66 (m, 2H), 4.06 (t, J = 6.6Hz, 2H).

步骤2：6-((2-丁基辛酰基)氧基)己酸Step 2: 6-((2-butyloctanoyl)oxy)hexanoic acid

一般程序H：General Procedure H:

在冰冷条件下，向6-羟基己基2-丁基辛酸酯(1.0当量)在丙酮(10mL)中的搅拌溶液中滴加琼斯试剂(2M溶液，1.2当量)。去除冷却浴，并继续搅拌2h。完成后，添加异丙醇(3mL)并过滤混合物。将滤液减压浓缩。将粗品物料用水稀释并将水层的pH调节至6并将混合物用5％MeOH的DCM溶液(3x 50mL)萃取。合并的二氯甲烷萃取物经硫酸钠干燥并过滤。将滤液在减压下浓缩，以得到6-((2-丁基辛酰基)氧基)己酸(700mg，67％)，为浅绿色油状物。Under ice-cold conditions, Jones reagent (2M solution, 1.2 equivalents) was added dropwise to a stirred solution of 6-hydroxyhexyl 2-butyl octanoate (1.0 equivalents) in acetone (10mL). The cooling bath was removed and stirring was continued for 2h. After completion, isopropanol (3mL) was added and the mixture was filtered. The filtrate was concentrated under reduced pressure. The crude material was diluted with water and the pH of the water layer was adjusted to 6 and the mixture was extracted with a 5% MeOH solution in DCM (3x 50mL). The combined dichloromethane extracts were dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 6-((2-butyloctanoyl)oxy)hexanoic acid (700mg, 67%), which was a light green oil.

步骤3：((2,2-双(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)Step 3: ((2,2-bis(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate)

根据一般程序D制备，用6-((2-丁基辛酰基)氧基)己酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出160mg，20％。¹H NMR:(400MHz,氯仿-d)δ0.78–0.87(m,12H),1.13–1.70(m,40H),2.26–2.37(m,6H),2.66–2.69(m,2H),2.87(s,2H),2.94(s,2H),3.56–3.57(m,4H),4.04–4.07(m,4H),4.13(s,4H)。Prepared according to General Procedure D, substituting 6-((2-butyloctanoyl)oxy)hexanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 160 mg, 20%.¹ H NMR: (400 MHz, CHLOROFORM-d) δ 0.78–0.87 (m, 12H), 1.13–1.70 (m, 40H), 2.26–2.37 (m, 6H), 2.66–2.69 (m, 2H), 2.87 (s, 2H), 2.94 (s, 2H), 3.56–3.57 (m, 4H), 4.04–4.07 (m, 4H), 4.13 (s, 4H).

步骤4：4,4-双(((Z)-辛-5-烯-1-基)氧基)丁腈Step 4: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butyronitrile

向含有对甲苯磺酸吡啶鎓盐(0.12g，0.48mmol，0.05当量)的小瓶中添加4,4-二乙氧基丁腈(1.5g，9.5mmol，1当量)和顺式-5-辛烯-1-醇(3.7g，29mmol，3当量)。将小瓶盖紧，并将所得混合物在105℃加热72h。此后，将混合物冷却至室温。粗品材料通过快速柱色谱纯化(100g二氧化硅，0至100％二氯甲烷的己烷溶液，历时20分钟)。分离出4,4-双(((Z)-辛-5-烯-1-基)氧基)丁腈(1.14g，37％)，为无色油状物。¹H NMR(500MHz,氯仿-d)δ5.43–5.27(m,4H),4.56(t,J＝5.3Hz,1H),3.61(dt,J＝9.3,6.6Hz,2H),3.44(dt,J＝9.3,6.6Hz,2H),2.42(t,J＝7.4Hz,2H),2.12–1.91(m,9H),1.66–1.54(m,5H),1.49–1.36(m,4H),0.96(t,J＝7.6Hz,6H)。4,4-diethoxybutyronitrile (1.5 g, 9.5 mmol, 1 eq) and cis-5-octen-1-ol (3.7 g, 29 mmol, 3 eq) were added to a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 eq). The vial was capped tightly and the resulting mixture was heated at 105 ° C for 72 h. Thereafter, the mixture was cooled to room temperature. The crude material was purified by flash column chromatography (100 g silica, 0 to 100% dichloromethane in hexane over 20 minutes). 4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butyronitrile (1.14 g, 37%) was isolated as a colorless oil.¹ H NMR (500MHz, chloroform-d) δ5.43–5.27(m,4H),4.56(t,J＝5.3Hz,1H),3.61(dt,J＝9.3,6.6Hz,2H),3.44(dt,J＝9.3,6.6Hz,2H),2.42(t,J＝7.4Hz,2H),2.12–1 .91(m,9H),1.66–1.54(m,5H),1.49–1.36(m,4H),0.96(t,J＝7.6Hz,6H).

步骤5：4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酸Step 5: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid

向含有4,4-双(((Z)-辛-5-烯-1-基)氧基)丁腈(1.14g，3.54mmol，1当量)的小瓶中添加氢氧化钾(0.60g，10.6mmol，3当量)，然后乙醇(3.5mL)和水(3.5mL)。将小瓶盖紧，并将反应混合物加热18h至110℃。此后，将混合物冷却至室温。将混合物用乙酸乙酯(20mL)稀释，并通过添加1M HCl将pH调节至～5。分离所得双相混合物，并将水相用乙酸乙酯(2x20mL)再萃取两次。合并有机萃取物，经硫酸钠干燥，过滤并浓缩，以得到4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酸(1.16g，96％产率)，为白色粘稠固体。¹H NMR(400MHz,氯仿-d)δ5.41–5.24(m,4H),4.45(t,J＝5.6Hz,1H),3.51(dt,J＝9.0,6.7Hz,2H),3.39(dt,J＝9.0,6.7Hz,2H),2.17(t,J＝7.6Hz,2H),2.08–1.98(m,8H),1.81(q,J＝7.3Hz,2H),1.59–1.52(m,4H),1.44–1.32(m,4H),0.94(t,J＝7.5Hz,6H)。To a vial containing 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyronitrile (1.14 g, 3.54 mmol, 1 eq) was added potassium hydroxide (0.60 g, 10.6 mmol, 3 eq) followed by ethanol (3.5 mL) and water (3.5 mL). The vial was capped tightly and the reaction mixture was heated to 110 °C for 18 h. Thereafter, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL) and the pH was adjusted to ~5 by adding 1 M HCl. The resulting biphasic mixture was separated and the aqueous phase was extracted twice more with ethyl acetate (2x20 mL). The organic extracts were combined, dried over sodium sulfate, filtered and concentrated to give 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (1.16 g, 96% yield) as a white viscous solid.¹ H NMR (400MHz, chloroform-d) δ5.41–5.24(m,4H),4.45(t,J＝5.6Hz,1H),3.51(dt,J＝9.0,6.7Hz,2H),3.39(dt,J＝9.0,6.7Hz,2H),2.17(t,J＝7.6Hz,2H),2.08–1 .98(m,8H),1.81(q,J＝7.3Hz,2H),1.59–1.52(m,4H),1.44–1.32(m,4H),0.94(t,J＝7.5Hz,6H).

步骤6：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)Step 6: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate)

根据一般程序E制备，用((2,2-双(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酸取代1-金刚烷乙酸。分离出90mg，34％。¹H NMR:(400MHz,氯仿-d)δ0.85–0.96(m,14H),1.24–1.64(m,38H),1.91–2.58(m,16H),3.36–3.56(m,5H),4.03–4.09(m,8H),4.45–4.47(m,1H),5.28–5.36(m,4H)。Prepared according to General Procedure E, substituting ((2,2-bis(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and substituting 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for 1-adamantaneacetic acid. Isolated 90 mg, 34%.¹ H NMR: (400 MHz, CHLOROFORM-d) δ 0.85–0.96 (m, 14H), 1.24–1.64 (m, 38H), 1.91–2.58 (m, 16H), 3.36–3.56 (m, 5H), 4.03–4.09 (m, 8H), 4.45–4.47 (m, 1H), 5.28–5.36 (m, 4H).

步骤7：实例7-55Step 7: Example 7-55

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出62mg，56％。¹H NMR(400MHz,氯仿-d)δ0.85–1.65(m,81H),1.87–1.60(m,22H),3.36–3.58(m,4H),4.03–4.17(m,13H),4.45–4.47(m,1H),5.26–5.39(m,4H)。UPLC-MS(方法A)：Rt 2.32min，m/z计算值[M+H]：1208.9，实测值1209.3。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 62 mg, 56%.¹ H NMR (400 MHz, chloroform-d) δ 0.85–1.65 (m, 81H), 1.87–1.60 (m, 22H), 3.36–3.58 (m, 4H), 4.03–4.17 (m, 13H), 4.45–4.47 (m, 1H), 5.26–5.39 (m, 4H). UPLC-MS (Method A): Rt 2.32 min, m/z calculated value [M+H]: 1208.9, found value 1209.3.

实例7-56：Example 7-56:

步骤1：7-羟基庚基2-丁基辛酸酯Step 1: 7-Hydroxyheptyl 2-butyl octanoate

根据一般程序G制备，用庚烷-1,7-二醇取代己烷-1,6-二醇。分离出1.1g，70％。¹HNMR(400MHz,氯仿-d)δ0.85–0.88(m,6H),1.15–1.70(m,24H),2.26–2.33(m,1H),3.63(t,J＝6.5Hz,2H),4.05(t,J＝6.7Hz,2H)。Prepared according to General Procedure G, substituting heptane-1,7-diol for hexane-1,6-diol. Isolated 1.1 g, 70%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.85-0.88 (m, 6H), 1.15-1.70 (m, 24H), 2.26-2.33 (m, 1H), 3.63 (t, J = 6.5 Hz, 2H), 4.05 (t, J = 6.7 Hz, 2H).

步骤2：7-((2-丁基辛酰基)氧基)庚酸Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid

根据一般程序H制备，用7-羟基庚基2-丁基辛酸酯取代6-羟基己基2-丁基辛酸酯。分离出800mg，66％。¹H NMR(400MHz,DMSO-d₆)δ0.82–0.85(m,6H),1.19–1.60(m,24H),2.16–2.31(m,3H),4.00(t,J＝6.3Hz,2H),11.96(s,1H)。Prepared according to General Procedure H, substituting 7-hydroxyheptyl 2-butyl octanoate for 6-hydroxyhexyl 2-butyl octanoate. Isolated 800 mg, 66%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.82-0.85 (m, 6H), 1.19-1.60 (m, 24H), 2.16-2.31 (m, 3H), 4.00 (t, J=6.3 Hz, 2H), 11.96 (s, 1H).

步骤3：((2,2-双(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)Step 3: ((2,2-bis(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate)

根据一般程序D制备，用7-((2-丁基辛酰基)氧基)庚酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出170mg，19％。¹H NMR(400MHz,氯仿-d)δ0.87–0.91(m,12H),1.23–1.64(m,47H),2.30–2.39(m,6H),2.76(t,J＝6.4Hz,2H),2.89–2.97(m,1H),3.58–3.60(m,4H),4.06–4.16(m,8H)。Prepared according to General Procedure D, substituting 7-((2-butyloctanoyl)oxy)heptanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 170 mg, 19%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.87-0.91 (m, 12H), 1.23-1.64 (m, 47H), 2.30-2.39 (m, 6H), 2.76 (t, J=6.4 Hz, 2H), 2.89-2.97 (m, 1H), 3.58-3.60 (m, 4H), 4.06-4.16 (m, 8H).

步骤4：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)Step 4: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate)

根据一般程序E制备，用((2,2-双(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酸取代1-金刚烷乙酸。分离出85mg，33％。¹H NMR:(400MHz,氯仿-d)δ0.84–0.96(m,17H),1.13–1.71(m,46H),1.91–2.59(m,18H),3.36–3.56(m,6H),4.03–4.09(m,9H),4.45–4.47(m,1H),5.26–5.34(m,4H)。Prepared according to General Procedure E, substituting ((2,2-bis(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for 1-adamantaneacetic acid. Isolated 85 mg, 33%.¹ H NMR: (400 MHz, CHLOROFORM-d) δ 0.84–0.96 (m, 17H), 1.13–1.71 (m, 46H), 1.91–2.59 (m, 18H), 3.36–3.56 (m, 6H), 4.03–4.09 (m, 9H), 4.45–4.47 (m, 1H), 5.26–5.34 (m, 4H).

步骤5：实例7-56Step 5: Example 7-56

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-辛酸丁酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出60mg，54％。UPLC-MS(方法A)：Rt 2.36min，m/z计算值[M+H]：1236.9，实测值1237.3。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octanoic acid butyl ester) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 60 mg, 54%. UPLC-MS (Method A): Rt 2.36 min, m/z calcd [M+H]: 1236.9, found 1237.3.

实例7-57：Example 7-57:

步骤1：2-丁基辛酸8-羟基辛酯Step 1: 2-Butyloctanoate 8-Hydroxyoctyl ester

根据一般程序G制备，用庚烷-1,7-二醇取代己烷-1,6-二醇。分离出1.0g，61％。¹HNMR(400MHz,氯仿-d)δ0.85–0.86(m,6H),1.19–1.70(m,27H),2.27–2.29(m,1H),3.60–3.65(m,2H),4.05(t,J＝6.6Hz,2H)。Prepared according to General Procedure G, substituting heptane-1,7-diol for hexane-1,6-diol. Isolated 1.0 g, 61%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.85-0.86 (m, 6H), 1.19-1.70 (m, 27H), 2.27-2.29 (m, 1H), 3.60-3.65 (m, 2H), 4.05 (t, J=6.6 Hz, 2H).

步骤2：8-((2-丁基辛酰基)氧基)辛酸Step 2: 8-((2-butyloctanoyl)oxy)octanoic acid

根据一般程序H制备，用8-羟基辛基2-丁基辛酸酯取代6-羟基己基2-丁基辛酸酯。分离出680mg，66％。¹H NMR(400MHz,DMSO-d₆)δ0.82–0.85(m,6H),1.21–1.60(m,25H),2.16–2.27(m,4H),4.01(t,J＝6.2Hz,2H),11.95(s,1H)。Prepared according to General Procedure H, substituting 8-hydroxyoctyl 2-butyl octanoate for 6-hydroxyhexyl 2-butyl octanoate. Isolated 680 mg, 66%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 0.82-0.85 (m, 6H), 1.21-1.60 (m, 25H), 2.16-2.27 (m, 4H), 4.01 (t, J=6.2 Hz, 2H), 11.95 (s, 1H).

步骤3：2,2-双(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)Step 3: 2,2-bis(hydroxymethyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate)

根据一般程序D制备，用8-((2-丁基辛酰基)氧基)辛酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出200mg，18％。¹H NMR(400MHz,氯仿-d)δ0.84–0.88(m,12H),1.20–1.62(m,52H),2.27–2.35(m,6H),2.70–2.73(m,2H),3.55–3.57(m,4H),4.03–4.13(m,8H)。Prepared according to General Procedure D, substituting 8-((2-butyloctanoyl)oxy)octanoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 200 mg, 18%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.84–0.88 (m, 12H), 1.20–1.62 (m, 52H), 2.27–2.35 (m, 6H), 2.70–2.73 (m, 2H), 3.55–3.57 (m, 4H), 4.03–4.13 (m, 8H).

步骤4：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)Step 4: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate)

根据一般程序E制备，用2,2-双(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酸取代1-金刚烷乙酸。分离出160mg，33％。¹HNMR(400MHz,氯仿-d)δ0.84–0.96(m,17H),1.13–1.71(m,46H),1.91–2.59(m,18H),3.36–3.56(m,6H),4.03–4.09(m,9H),4.45–4.47(m,1H),5.26–5.34(m,4H)。Prepared according to General Procedure E, substituting 2,2-bis(hydroxymethyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for 1-adamantaneacetic acid. Isolated 160 mg, 33%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.84–0.96 (m, 17H), 1.13–1.71 (m, 46H), 1.91–2.59 (m, 18H), 3.36–3.56 (m, 6H), 4.03–4.09 (m, 9H), 4.45–4.47 (m, 1H), 5.26–5.34 (m, 4H).

步骤5：实例7-57Step 5: Example 7-57

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出60mg，56％。UPLC-MS(方法A)：Rt 2.40min，m/z计算值[M+H]：1265.0，实测值1265.4。Prepared according to General Procedure C, substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl bis(8-((2-butyloctanoyl)oxy)octanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate. Isolated 60 mg, 56%. UPLC-MS (Method A): Rt 2.40 min, m/z calcd [M+H]: 1265.0, found 1265.4.

实例7-58：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(((((1-乙基哌啶-3-基)甲氧基)羰基)氧基)甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)Example 7-58: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate)

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用(1-乙基哌啶-3-基)甲醇取代3-(二乙氨基)丙烷-1-醇。分离出62mg，56％。UPLC-MS(方法A)：Rt 2.30min，m/z计算值[M+H]：1220.9，实测值1221.2。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and (1-ethylpiperidin-3-yl)methanol for 3-(diethylamino)propan-1-ol. Isolated 62 mg, 56%. UPLC-MS (Method A): Rt 2.30 min, m/z calcd. [M+H]: 1220.9, found 1221.2.

实例7-59：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(((((1-乙基哌啶-3-基)甲氧基)羰基)氧基)甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)Example 7-59: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate)

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用(1-乙基哌啶-3-基)甲醇取代3-(二乙氨基)丙烷-1-醇。分离出79mg，60％。UPLC-MS(方法A)：Rt 2.38min，m/z计算值[M+H]：1248.9，实测值1249.3。Prepared according to General Procedure C substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and (1-ethylpiperidin-3-yl)methanol for 3-(diethylamino)propan-1-ol. Isolated 79 mg, 60%. UPLC-MS (Method A): Rt 2.38 min, m/z calcd. [M+H]: 1248.9, found 1249.3.

实例7-60：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(((((1-乙基哌啶-3-基)甲氧基)羰基)氧基)甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)Example 7-60: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用(1-乙基哌啶-3-基)甲醇取代3-(二乙氨基)丙烷-1-醇。分离出52mg，53％。UPLC-MS(方法A)：Rt 2.40min，m/z计算值[M+H]：1277.0，实测值1277.3。Prepared according to General Procedure C, substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl bis(8-((2-butyloctanoyl)oxy)octanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and (1-ethylpiperidin-3-yl)methanol for 3-(diethylamino)propan-1-ol. Isolated 52 mg, 53%. UPLC-MS (Method A): Rt 2.40 min, m/z calcd. [M+H]: 1277.0, found 1277.3.

实例7-61：Example 7-61:

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用3-(乙基(甲基)氨基)丙烷-1-醇取代3-(二乙基氨基)丙烷-1-醇。分离出111mg，70％。UPLC-MS(方法A)：Rt 2.24min，m/z计算值[M+H]：1194.9，实测值1195.2。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 3-(ethyl(methyl)amino)propan-1-ol for 3-(diethylamino)propan-1-ol. Isolated 111 mg, 70%. UPLC-MS (Method A): Rt 2.24 min, m/z calcd. [M+H]: 1194.9, found 1195.2.

实例7-62：Example 7-62:

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用3-(乙基(甲基)氨基)丙烷-1-醇取代3-(二乙基氨基)丙烷-1-醇。分离出81mg，60％。UPLC-MS(方法A)：Rt 2.33min，m/z计算值[M+H]：1222.9，实测值1223.3。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 3-(ethyl(methyl)amino)propan-1-ol for 3-(diethylamino)propan-1-ol. Isolated 81 mg, 60%. UPLC-MS (Method A): Rt 2.33 min, m/z calcd. [M+H]: 1222.9, found 1223.3.

实例7-63：Example 7-63:

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用3-(乙基(甲基)氨基)丙烷-1-醇取代3-(二乙氨基)丙烷-1-醇。分离出88mg，65％。UPLC-MS(方法A)：Rt 2.40min，m/z计算值[M+H]：1251.0，实测值1251.3。Prepared according to General Procedure C, substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl bis(8-((2-butyloctanoyl)oxy)octanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 3-(ethyl(methyl)amino)propan-1-ol for 3-(diethylamino)propan-1-ol. Isolated 88 mg, 65%. UPLC-MS (Method A): Rt 2.40 min, m/z calcd. [M+H]: 1251.0, found 1251.3.

实例7-64：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)Example 7-64: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate)

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(6-氧代己烷-6,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出89mg，60％。UPLC-MS(方法A)：Rt 2.23min，m/z计算值[M+H]：1191.9，实测值1192.2。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 89 mg, 60%. UPLC-MS (Method A): Rt 2.23 min, m/z calcd. [M+H]: 1191.9, found 1192.2.

实例7-65：((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)Example 7-65: ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate)

根据一般程序C制备，用((2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基)双(氧基))双(7-氧代庚烷-7,1-二基)双(2-丁基辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出103mg，70％。UPLC-MS(方法A)：Rt 2.33min，m/z计算值[M+H]：1219.9，实测值1220.3。Prepared according to General Procedure C, substituting ((2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-butyloctanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethan-1-amine for 3-(diethylamino)propan-1-ol. Isolated 103 mg, 70%. UPLC-MS (Method A): Rt 2.33 min, m/z calcd. [M+H]: 1219.9, found 1220.3.

实例7-66：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)Example 7-66: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基双(8-((2-丁基辛酰基)氧基)辛酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出100mg，70％。UPLC-MS(方法A)：Rt 2.36min，m/z计算值[M+H]：1248.0，实测值1248.4。Prepared according to General Procedure C, substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diylbis(8-((2-butyloctanoyl)oxy)octanoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 100 mg, 70%. UPLC-MS (Method A): Rt 2.36 min, m/z calcd. [M+H]: 1248.0, found 1248.4.

实例7-67：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八烷-9,12-二烯酸酯)Example 7-67: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecane-9,12-dienoate)

步骤1：2,2-双(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八烷-9,12-二烯酸酯)Step 1: 2,2-bis(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecane-9,12-dienoate)

根据一般程序D制备，用(9Z,12Z)-十八-9,12-二烯酸取代(Z)-6-(壬-3-烯-1-基氧基)-6-氧代己酸。分离出2.4g，21％。¹H NMR(400MHz,氯仿-d)δ0.88(t,J＝7.0Hz,6H),1.20–1.46(m,27H),1.57–1.75(m,5H),1.97–2.16(m,8H),2.33(t,J＝7.6Hz,4H),2.67(t,J＝6.2Hz,2H),2.76(t,J＝6.3Hz,4H),3.56(d,J＝5.8Hz,4H),4.07–4.32(m,4H),5.27–5.43(m,8H)。Prepared according to General Procedure D, substituting (9Z,12Z)-octadecadienoic acid for (Z)-6-(non-3-en-1-yloxy)-6-oxohexanoic acid. Isolated 2.4 g, 21%.¹ H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (t, J = 7.0 Hz, 6H), 1.20-1.46 (m, 27H), 1.57-1.75 (m, 5H), 1.97-2.16 (m, 8H), 2.33 (t, J = 7.6 Hz, 4H), 2.67 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 6.3 Hz, 4H), 3.56 (d, J = 5.8 Hz, 4H), 4.07-4.32 (m, 4H), 5.27-5.43 (m, 8H).

步骤2：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)Step 2: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecadienoate)

根据一般程序E制备，用2,2-双(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八碳-9,12-二烯酸酯)取代O,O'-(2,2-双(羟甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用4,4-双(((Z)-辛基-5-烯-1-基)氧基)丁酸取代1-金刚烷乙酸。分离出500mg，30％。¹H NMR(400MHz,氯仿-d)δ0.84–0.99(m,12H),1.22–1.45(m,33H),1.56–1.71(m,7H),1.92(q,J＝7.2Hz,2H),1.96–2.09(m,16H),2.30(t,J＝7.6Hz,4H),2.40(t,J＝7.4Hz,2H),2.50–2.64(m,1H),2.76(t,J＝6.2Hz,4H),3.33–3.44(m,2H),3.45–3.63(m,4H),4.09(s,6H),4.47(t,J＝5.3Hz,1H),5.24–5.43(m,12H)。Prepared according to General Procedure E, substituting 2,2-bis(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) for O,O'-(2,2-bis(hydroxymethyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 4,4-bis(((Z)-octyl-5-en-1-yl)oxy)butanoic acid for 1-adamantanacetic acid. Isolated 500 mg, 30%.² .40(t,J＝7.4Hz,2H),2.50–2.64(m,1H),2.76(t,J＝6.2Hz,4H),3.33–3.44(m,2H),3.45–3.63(m,4H),4.09(s,6H),4.47(t,J＝5.3Hz,1H),5.24–5.43 (m,12H).

步骤3：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((3-(二乙氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)(实例7-67)Step 3: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) (Example 7-67)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯。分离出64mg，62％。UPLC-MS(方法A)：Rt2.41min，m/z计算值[M+H]：1140.9，实测值1141.2。Prepared according to General Procedure C substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)di((Z)-non-3-en-1-yl)diadipate. Isolated 64 mg, 62%. UPLC-MS (Method A): Rt 2.41 min, m/z calcd [M+H]: 1140.9, found 1141.2.

实例7-68：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(((((1-乙基哌啶-3-基)甲氧基)羰基)氧基)甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)Example 7-68: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用(1-乙基哌啶-3-基)甲醇取代3-(二乙氨基)丙烷-1-醇。分离出97mg，65％。UPLC-MS(方法A)：Rt 2.45min，m/z计算值[M+H]：1152.9，实测值1153.1。Prepared according to General Procedure C substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and (1-ethylpiperidin-3-yl)methanol for 3-(diethylamino)propan-1-ol. Isolated 97 mg, 65%. UPLC-MS (Method A): Rt 2.45 min, m/z calcd. [M+H]: 1152.9, found 1153.1.

实例7-69：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((3-(乙基(甲基)氨基)丙氧基)羰基)氧基)甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)Example 7-69: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用3-(乙基(甲基)氨基)丙烷-1-醇取代3-(二乙基氨基)丙烷-1-醇。分离出79mg，61％。UPLC-MS(方法A)：Rt 2.45min，m/z计算值[M+H]：1126.9，实测值1127.1。Prepared according to General Procedure C substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 3-(ethyl(methyl)amino)propan-1-ol for 3-(diethylamino)propan-1-ol. Isolated 79 mg, 61%. UPLC-MS (Method A): Rt 2.45 min, m/z calcd. [M+H]: 1126.9, found 1127.1.

实例7-70：2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-((((2-(吡咯烷-1-基)乙基)氨基甲酰基)氧基)甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)Example 7-70: 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-((((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)

根据一般程序C制备，用2-(((4,4-双(((Z)-辛-5-烯-1-基)氧基)丁酰基)氧基)甲基)-2-(羟甲基)丙烷-1,3-二基(9Z,9'Z,12Z,12'Z)-双(十八-9,12-二烯酸酯)取代O,O'-(2-(羟甲基)-2-(((6-(((Z)-壬-3-烯-1-基)氧基)-6-氧代己酰基)氧基)甲基)丙烷-1,3-二基)二((Z)-壬-3-烯-1-基)二己二酸酯，并用2-(吡咯烷-1-基)乙烷-1-胺取代3-(二乙氨基)丙烷-1-醇。分离出106mg，70％。UPLC-MS(方法A)：Rt 2.43min，m/z计算值[M+H]：1123.9，实测值1124.1。Prepared according to General Procedure C substituting 2-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) for O,O'-(2-(hydroxymethyl)-2-(((6-(((Z)-non-3-en-1-yl)oxy)-6-oxohexanoyl)oxy)methyl)propane-1,3-diyl)bis((Z)-non-3-en-1-yl)diadipate and 2-(pyrrolidin-1-yl)ethane-1-amine for 3-(diethylamino)propan-1-ol. Isolated 106 mg, 70%. UPLC-MS (Method A): Rt 2.43 min, m/z calcd. [M+H]: 1123.9, found 1124.1.

实例8：LNP制剂跨各种细胞类型的mRNA递送Example 8: LNP formulations for mRNA delivery across various cell types

本实例提供向各种细胞类型递送功能性mRNA的示例性LNP组合物、制剂、纳米粒子和/或纳米材料。本文所述的筛选平台可以识别用于跨各种细胞类型递送功能性mRNA的几种高效LNP制剂。本实例可以用于证明所提供的脂质能够在小鼠中递送功能性mRNA且/或提高编码报告基因的mRNA向某些细胞类型的递送(例如，1.5-2倍提高)。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials for delivering functional mRNA to various cell types. The screening platform described herein can identify several efficient LNP formulations for delivering functional mRNA across various cell types. This example can be used to demonstrate that the lipids provided can deliver functional mRNA in mice and/or improve the delivery of mRNA encoding reporter genes to certain cell types (e.g., 1.5-2 times improvement).

选择LNP制剂以确定每种制剂在小鼠中跨各种胞类型递送功能性mRNA的能力。LNP制剂各自含有1.0mg/kg报告基因mRNA，如本文所述。本文所述的流式测定用于测量注射LNP制剂后24小时的报告基因表面表达。The LNP formulations were selected to determine the ability of each formulation to deliver functional mRNA across various cell types in mice. Each LNP formulation contained 1.0 mg/kg reporter mRNA, as described herein. The flow assay described herein was used to measure the reporter surface expression 24 hours after the LNP formulation was injected.

示例性LNP制剂可以跨各种HSC细胞类型(Lin-、Sca-1+、LSK细胞、LT-HSC(CD34-Flk2-)和LT-HSC(CD150+CD48-))、骨髓(BM)细胞类型(活细胞、T细胞、B细胞、CD11b、巨噬细胞、NK细胞、CD11c)、脾细胞类型(T细胞、B细胞、CD11b、巨噬细胞、NK细胞、CD11c)和肝脏细胞类型(CD31、肝细胞、CD45、CD11b、库普弗细胞、NK细胞、T细胞、B细胞)将编码报告基因的mRNA功能性递送至小鼠，其中与盐水对照(媒剂)相比，每种LNP制剂含有1.0mg/kg报告基因mRNA。Exemplary LNP formulations can functionally deliver mRNA encoding a reporter gene to mice across various HSC cell types (Lin-, Sca-1+, LSK cells, LT-HSC (CD34-Flk2-), and LT-HSC (CD150+CD48-)), bone marrow (BM) cell types (viable cells, T cells, B cells, CD11b, macrophages, NK cells, CD11c), spleen cell types (T cells, B cells, CD11b, macrophages, NK cells, CD11c), and liver cell types (CD31, hepatocytes, CD45, CD11b, Kupffer cells, NK cells, T cells, B cells), wherein each LNP formulation contains 1.0 mg/kg of the reporter gene mRNA compared to a saline control (vehicle).

本实例可以用于证明所提供的脂质能够在小鼠中递送功能性mRNA且/或提高编码报告基因的mRNA向某些细胞类型的递送(例如，1.5-2倍提高)。This example can be used to demonstrate that the provided lipids are capable of delivering functional mRNA in mice and/or improving the delivery of mRNA encoding a reporter gene to certain cell types (eg, 1.5-2 fold improvement).

实例9：LNP制剂的至脾细胞、肝脏细胞和骨髓细胞的mRNA递送Example 9: mRNA delivery to spleen cells, liver cells and bone marrow cells by LNP formulation

本实施例提供强效递送到如本文所述的各种细胞类型的示范性LNP组合物、制剂、纳米粒子和/或纳米材料。本文所述的筛选平台可以识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对特定细胞类型最有效。This embodiment provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials that are potently delivered to various cell types as described herein. The screening platform described herein can identify several highly effective LNP formulations to determine which type of LNP formulation is most effective for a specific cell type.

本实例可以用于证明可电离脂质在小鼠中跨各种细胞类型有效地递送mRNA。本实例也可以用于证明所提供的脂质显示出跨各种细胞类型(包括骨髓树突状细胞、骨髓T细胞、脾B细胞、脾树突状细胞和LSK(Lin-Sca-1+c-Kit+)细胞、HSPC和小鼠LT-HSC)的有效递送。This example can be used to demonstrate that ionizable lipids effectively deliver mRNA across various cell types in mice. This example can also be used to demonstrate that the provided lipids show effective delivery across various cell types (including bone marrow dendritic cells, bone marrow T cells, spleen B cells, spleen dendritic cells and LSK (Lin-Sca-1+c-Kit+) cells, HSPCs and mouse LT-HSCs).

选择LNP制剂以使用本文所述的报告基因系统和C57BL6小鼠模型确认功效结果。每个实验中每组使用三只C57BL6小鼠。注射后24小时收集数据。The LNP formulation was selected to confirm efficacy results using the reporter gene system and C57BL6 mouse model described herein. Three C57BL6 mice were used per group in each experiment. Data were collected 24 hours after injection.

热图可以用于显示含有不同可电离脂质的各种LNP制剂的平均中值荧光，该平均中值荧光被归一化为含有跨各种细胞类型(骨髓树突状细胞、脾B细胞和脾树突状细胞)的示例性脂质的LNP制剂的平均中值荧光。每种LNP制剂含有1.0 mg/kg报告基因mRNA。Heatmaps can be used to display the average median fluorescence of various LNP formulations containing different ionizable lipids, which are normalized to the average median fluorescence of LNP formulations containing exemplary lipids across various cell types (bone marrow dendritic cells, splenic B cells, and splenic dendritic cells). Each LNP formulation contained 1.0 mg/kg reporter mRNA.

热图可以用于显示含有不同可电离脂质的各种LNP制剂的归一化平均％报告基因表达或平均标准化中值荧光。平均％报告基因和平均中值荧光值分别被归一化为含有递送至T细胞(骨髓细胞)或LSK(Lin-Sca-1+c-Kit+)细胞的示例性脂质的LNP制剂的％报告基因表达或平均中值荧光值。Heat map can be used to display the normalized average % reporter gene expression or average standardized median fluorescence of various LNP formulations containing different ionizable lipids. The average % reporter gene and average median fluorescence values are normalized to the % reporter gene expression or average median fluorescence values of LNP formulations containing exemplary lipids delivered to T cells (bone marrow cells) or LSK (Lin-Sca-1+c-Kit+) cells, respectively.

实例10：LNP制剂的至Balb/C、C57BL6J小鼠品系和人源化(NSG-CD34+)免疫小鼠的Example 10: Transfection of LNP Formulations into Balb/C, C57BL6J Mouse Strains and Humanized (NSG-CD34+) Immunized MicemRNA递送mRNA delivery

本实例提供向各种细胞类型进行有效mRNA递送的示例性LNP组合物、制剂、纳米粒子和/或纳米材料，如本文所述。本文所述的筛选平台可以用于识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对特定细胞类型最有效。本实例可以用于证明所提供的脂质显示出跨各种细胞类型(包括骨髓活细胞、骨髓CD34+细胞、骨髓(LSK细胞)、人LT-HSC、血细胞(huCD45+)和血液细胞(muCD45+))的有效递送。This example provides exemplary LNP compositions, preparations, nanoparticles and/or nanomaterials for effective mRNA delivery to various cell types, as described herein. The screening platform described herein can be used to identify several efficient LNP preparations to determine which type of LNP preparation is most effective for a specific cell type. This example can be used to demonstrate that the lipids provided show effective delivery across various cell types, including live bone marrow cells, bone marrow CD34+ cells, bone marrow (LSK cells), human LT-HSC, blood cells (huCD45+) and blood cells (muCD45+).

选择LNP制剂以使用报告基因系统和人源化(NSG-CD34+)小鼠模型或两种小鼠品系(Balb/C、C57BL6J)小鼠来确认功效结果，如本文所述。每种LNP制剂含有1.0 mg/kg报告基因mRNA。LNP formulations were selected to confirm efficacy results using a reporter gene system and a humanized (NSG-CD34+) mouse model or two mouse strains (Balb/C, C57BL6J) mice, as described herein. Each LNP formulation contained 1.0 mg/kg reporter gene mRNA.

在两种小鼠品系(Balb/C、C57BL6J小鼠)中测试了本文所述的示例性LNP制剂向血液细胞、骨髓(活细胞)和骨髓(LSK细胞)的递送。The exemplary LNP formulations described herein were tested for delivery to blood cells, bone marrow (live cells), and bone marrow (LSK cells) in two mouse strains (Balb/C, C57BL6J mice).

每个实验中每组使用三只Balb/C小鼠和/或C57BL6J和/或NSG-CD34+小鼠。注射后24小时收集数据。Three Balb/C mice and/or C57BL6J and/or NSG-CD34+ mice were used per group in each experiment. Data were collected 24 hours after injection.

实例11：用于将肌内mRNA递送至引流淋巴结的LNP制剂Example 11: LNP Formulations for Intramuscular mRNA Delivery to Draining Lymph Nodes

本实例提供了将mRNA递送至引流淋巴结的示例性LNP组合物、制剂、纳米粒子和/或纳米材料(例如，以供用作mRNA疫苗)。本文所述的筛选平台可以用于识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对于至引流淋巴结的mRNA递送最有效(例如，以供用作mRNA疫苗)。因此，本实例可以用于证明所提供的脂质显示出至引流淋巴结的有效递送。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials for delivering mRNA to draining lymph nodes (e.g., for use as an mRNA vaccine). The screening platform described herein can be used to identify several highly effective LNP formulations to determine which type of LNP formulation is most effective for mRNA delivery to draining lymph nodes (e.g., for use as an mRNA vaccine). Therefore, this example can be used to demonstrate that the provided lipids show effective delivery to draining lymph nodes.

选择LNP制剂以确定每种制剂在Balb/C小鼠中肌内递送功能性mRNA的能力。每种LNP制剂含有10μg Trilink Fluc mRNA。注射后六小时，分离皮肤引流淋巴结(dLN)并使用本文所述的标准发光测定来测量发光。LNP formulations were selected to determine the ability of each formulation to deliver functional mRNA intramuscularly in Balb/C mice. Each LNP formulation contained 10 μg of Trilink Fluc mRNA. Six hours after injection, skin draining lymph nodes (dLNs) were isolated and luminescence was measured using a standard luminescence assay described herein.

实例12：利用LNP制剂在脾脏、肝脏和骨髓中进行碱基编辑Example 12: Base Editing in Spleen, Liver, and Bone Marrow Using LNP Formulations

本实例提供了在各种细胞类型中赋予基因编辑(例如，使用碱基编辑器)的示例性LNP组合物、制剂、纳米粒子和/或纳米材料。本文所述的筛选平台可以用于识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对于体内各种细胞类型的碱基编辑最有效(例如，可以在小鼠中进行细胞的碱基编辑)。本实例可以用于证明所提供的脂质可以用于在各种细胞类型(包括骨髓、脾脏和肝脏)中执行碱基编辑和/或改善LNP制剂的至特定细胞类型的递送。本实例也可以用于证明含有示例性化合物的LNP制剂中所含的mRNA在递送期间在LNP中更加稳定。This example provides exemplary LNP compositions, preparations, nanoparticles and/or nanomaterials that confer gene editing (e.g., using base editors) in various cell types. The screening platform described herein can be used to identify several efficient LNP preparations to determine which type of LNP preparation is most effective for base editing of various cell types in vivo (e.g., base editing of cells can be performed in mice). This example can be used to prove that the lipids provided can be used to perform base editing and/or improve the delivery of LNP preparations to specific cell types in various cell types (including bone marrow, spleen and liver). This example can also be used to prove that the mRNA contained in the LNP preparation containing exemplary compounds is more stable in LNP during delivery.

方法method

选择示例性LNP制剂以确定每种制剂在Balb/C小鼠模型中执行碱基编辑的能力，如本文所述。Exemplary LNP formulations were selected to determine the ability of each formulation to perform base editing in the Balb/C mouse model, as described herein.

将脂质纳米粒子组分以指定的脂质组分摩尔比溶解在100％乙醇中。将编码腺嘌呤碱基编辑器的mRNA和经化学修饰的sgRNA以1:1的质量比溶解在10mM柠檬酸盐、100mMNaCl(pH 4.0)中，导致NA货物的浓度为大约0.22mg/mL。调配LNP制剂，其中摩尔比为50％可电离脂质:38.5％胆固醇:1.5％PEG2000-DMG:10％DSPC，总脂质与NA质量比为11.7至33。根据制造商的方案，通过使用Precision Nanosystems NanoAssemblr Spark或Benchtop系列仪器对脂质和NA溶液进行微流体混合，形成LNP制剂。在混合期间使用不同的流速维持3:1的水与有机溶剂比。在混合之后，收集LNP制剂，稀释于PBS中(大约2:1v/v)，并且以20kDa过滤器为背景，在4℃使用PBS透析8至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP制剂经由动态光散射(DLS)表征，以测量大小和多分散性。LNP制剂亚群的pKa经由TNS测定进行测量。在透析之后，使用0.22微米的无菌过滤器无菌过滤LNP制剂并存储于4℃以供进一步使用。在一些实施例中，可以根据制造商的方案使用100kDa Amicon过滤器来浓缩LNP制剂。The lipid nanoparticle components were dissolved in 100% ethanol at a specified lipid component molar ratio. The mRNA encoding the adenine base editor and the chemically modified sgRNA were dissolved in 10mM citrate, 100mM NaCl (pH 4.0) at a mass ratio of 1: 1, resulting in a NA cargo concentration of approximately 0.22mg/mL. LNP formulations were formulated with a molar ratio of 50% ionizable lipids: 38.5% cholesterol: 1.5% PEG2000-DMG: 10% DSPC, and a total lipid to NA mass ratio of 11.7 to 33. According to the manufacturer's protocol, the lipid and NA solutions were microfluidically mixed using a Precision Nanosystems NanoAssemblr Spark or Benchtop series instrument to form LNP formulations. A 3: 1 water to organic solvent ratio was maintained using different flow rates during mixing. After mixing, the LNP preparations are collected, diluted in PBS (about 2: 1v/v), and with a 20kDa filter as background, 4°C is used for dialysis of PBS for 8 to 24 hours for further buffer exchange. After this initial dialysis, each individual LNP preparation is characterized via dynamic light scattering (DLS), to measure size and polydispersity. The pKa of the LNP preparation subpopulation is measured via TNS determination. After dialysis, the LNP preparations are aseptically filtered using a 0.22 micron sterile filter and stored at 4°C for further use. In certain embodiments, the LNP preparations can be concentrated using a 100kDa Amicon filter according to the manufacturer's protocol.

LNP表征LNP characterization

使用高通量动态光散射(DLS)(DynaPro plate reader II,Wyatt)测量DLS-LNP制剂流体动力学直径和多分散指数(PDI)。用1X PBS将LNP制剂稀释到适当浓度且进行分析。The hydrodynamic diameter and polydispersity index (PDI) of DLS-LNP preparations were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). The LNP preparations were diluted to appropriate concentrations with IX PBS and analyzed.

浓度和包裹功效Concentration and packaging efficacy

按照制造商说明书，通过Qubit microRNA试剂盒(用于siRNA)或HS RNA试剂盒(用于mRNA)测定NA的浓度。通过测量未裂解和裂解的LNP来测定包裹效率。The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

LNP施用LNP administration

大约8-12周龄的雄性Balb/C小鼠用于本文所述的实验。将每只小鼠暂时束缚，并经由尾静脉注射IV施用LNP制剂。也使用年龄匹配的小鼠经由尾静脉注射施用媒剂(1XPBS)作为对照。给药后四到六天，收集包括肝脏、脾脏和骨髓的组织。根据制造商说明书，使用Nextera DNAFlex Library Prep Kit(Illumina)(使用96孔板Nextera索引引物(Illumina))来对基因组DNA进行分离和片段化以及接头连接。文库大小和浓度由片段分析仪(Agilent)确认，并发送至Novogene用于使用Illumina HiSeq进行全基因组测序。Male Balb/C mice of about 8-12 weeks of age are used for experiments described herein. Each mouse is temporarily restrained and LNP preparations are administered via tail vein injection IV. Age-matched mice are also used to administer vehicle (1XPBS) via tail vein injection as a control. Four to six days after administration, tissues including liver, spleen and bone marrow are collected. According to manufacturer's instructions, Nextera DNAFlex Library Prep Kit (Illumina) (using 96-well plate Nextera index primers (Illumina)) is used to separate genomic DNA and fragmentation and joint connection. Library size and concentration are confirmed by fragment analyzer (Agilent) and sent to Novogene for whole genome sequencing using Illumina HiSeq.

碱基编辑Base editing

对于碱基编辑，将编码腺嘌呤碱基编辑器(ABE)的mRNA和靶向ALAS1的向导RNA(sgRNA)以1:1(质量比)共同包裹在如本文所述的LNP制剂中。通过尾静脉注射将LNP制剂以3.0mg/kg总RNA施用至Balb/c小鼠。给药后4天，将小鼠安乐死并收获肝脏、脾脏和骨髓。碱基编辑是通过使用提取的基因组DNA在ALAS1靶位点处执行靶向深度测序分析来确定的。For base editing, mRNA encoding adenine base editor (ABE) and guide RNA (sgRNA) targeting ALAS1 were co-encapsulated in a LNP formulation as described herein at 1:1 (mass ratio). The LNP formulation was administered to Balb/c mice at 3.0 mg/kg total RNA by tail vein injection. Four days after administration, the mice were euthanized and the liver, spleen, and bone marrow were harvested. Base editing was determined by performing targeted deep sequencing analysis at the ALAS1 target site using extracted genomic DNA.

实例13：将示范性LNP制剂递送至小鼠HSPCExample 13: Delivery of Exemplary LNP Formulations to Mouse HSPCs

本实施例提供强效递送到如本文所述的各种细胞类型的示范性LNP组合物、制剂、纳米粒子和/或纳米材料。本实例可以用于证明所提供的脂质在特定细胞类型(诸如转染的HSPC)中表现出剂量依赖性增加。This example provides potent delivery to various cell types as described herein, exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials. This example can be used to demonstrate that the lipids provided show a dose-dependent increase in specific cell types (such as transfected HSPCs).

选择LNP制剂以使用本文所述的Cre报告基因系统和Ai14小鼠模型以三种不同剂量(0.1mg/kg、0.3mg/kg、1.0mg/kg)来确认功效结果。The LNP formulations were selected to confirm efficacy results at three different doses (0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg) using the Cre reporter system and Ai14 mouse model described herein.

实例14：将示范性LNP制剂递送至小鼠HSPCExample 14: Delivery of Exemplary LNP Formulations to Mouse HSPCs

本实施例提供强效递送到如本文所述的各种细胞类型的示范性LNP组合物、制剂、纳米粒子和/或纳米材料。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials for potent delivery to various cell types as described herein.

选择LNP制剂，以使用本文所述的Cre报告基因系统和Ai14小鼠模型通过在多个时间点(例如，以0.3mg/kg Cre mRNA的剂量进行的LNP递送后2、8和16周或在以1.0mg/kg CremRNA的剂量进行的LNP递送后2、8和16周)处监测HSPC中％tdTomato+的耐久性来确认功效结果。LNP formulations were selected to confirm efficacy results using the Cre reporter system and Ai14 mouse model described herein by monitoring the durability of % tdTomato+ in HSPCs at multiple time points (e.g., 2, 8, and 16 weeks after LNP delivery at a dose of 0.3 mg/kg Cre mRNA or 2, 8, and 16 weeks after LNP delivery at a dose of 1.0 mg/kg Cre mRNA).

在观察转染的HSPC的耐久性时，分析该转染的HSPC，以通过测量在第16周时间点处施用0.3mg/kg和1.0mg/kg Cre mRNA的小鼠队列中外周血中单核细胞、B细胞、T细胞和红细胞的％tdTomato+丰度来确定其重建各种免疫和造血谱系的能力。In looking at the durability of the transfected HSPCs, the transfected HSPCs were analyzed to determine their ability to reconstitute various immune and hematopoietic lineages by measuring the % tdTomato+ abundance of monocytes, B cells, T cells, and erythrocytes in the peripheral blood of the mouse cohorts administered 0.3 mg/kg and 1.0 mg/kg Cre mRNA at the 16 week time point.

本实例可以用于证明所提供的脂质引起至HSPC的持久LNP递送。此外，本实例可以用于证明转染的HSPC尅重建造血室。This example can be used to demonstrate that the lipids provided cause sustained LNP delivery to HSPC. In addition, this example can be used to demonstrate that transfected HSPC can reconstitute the blood compartment.

实例15：将示例性LNP制剂体外递送至人CD34+HSPCExample 15: In vitro delivery of exemplary LNP formulations to human CD34+ HSPCs

本实施例提供强效递送到如本文所述的各种细胞类型的示范性LNP组合物、制剂、纳米粒子和/或纳米材料。因此，本实例可以用于证明所提供的脂质在ApoE的存在下递送至特定细胞类型时引起报告基因表达增加。This example provides an exemplary LNP composition, formulation, nanoparticle and/or nanomaterial that is potently delivered to various cell types as described herein. Therefore, this example can be used to demonstrate that the provided lipids cause increased reporter gene expression when delivered to specific cell types in the presence of ApoE.

选择LNP制剂以确认体外人CD34+HSPC的功效结果。The LNP formulation was chosen to confirm the efficacy results in human CD34+ HSPCs in vitro.

简言之，将人CD34+细胞解冻，并且然后以在细胞培养板中40,000个细胞/孔铺在48孔组织培养板中。细胞在补充有10％青霉素-链霉素和StemSpan CD34+扩增补充剂(StemCell Technologies)的StemSpan细胞培养基(Stem Cell Technologies)中孵育48小时以达到稳态。在以包裹在LNP中的1000ng报告基因mRNA给药之前，将细胞以300xG离心并重悬于补充培养基中，如先前所述。为了测试ApoE是否是LNP摄取所必需的，将2μL重组人脱辅基蛋白E3(ApoE3，R&D Systems)添加到每个所关注的孔中。24小时之后，用人TruStain FcX(Biolegend)封闭细胞Fc受体，用PBS洗涤，并且对报告基因表达以及表型标记和活性染料进行荧光染色。In brief, human CD34+ cells were thawed and then plated in 48-well tissue culture plates at 40,000 cells/well in cell culture plates. Cells were incubated in a StemSpan cell culture medium (Stem Cell Technologies) supplemented with 10% penicillin-streptomycin and StemSpan CD34+ amplification supplement (StemCell Technologies) for 48 hours to reach steady state. Before administration with 1000ng reporter gene mRNA encapsulated in LNP, cells were centrifuged at 300xG and resuspended in supplemented culture medium, as previously described. In order to test whether ApoE is necessary for LNP uptake, 2 μL of recombinant human apoprotein E3 (ApoE3, R&D Systems) was added to each well of interest. After 24 hours, cell Fc receptors were blocked with human TruStain FcX (Biolegend), washed with PBS, and reporter gene expression and phenotypic markers and active dyes were fluorescently stained.

实例16：LNP制剂的制备和表征Example 16: Preparation and Characterization of LNP Formulations

此外，本实施例提供示范性LNP制剂。In addition, this example provides exemplary LNP formulations.

脂质纳米粒子组分以指定脂质组分摩尔比溶解于100％乙醇中。将核酸(NA)货物溶解于10mM柠檬酸盐、100mM NaCl(pH 4.0)中，使得NA货物的浓度大约为0.22mg/mL。在一些实施例中，NA货物包含以1:10到10:1的功能性NA与条形码的质量比混合的功能性NA和报告DNA条形码两者。如本文所述，NA可为siRNA、反义、表达DNA或mRNA。The lipid nanoparticle components are dissolved in 100% ethanol at a specified lipid component molar ratio. The nucleic acid (NA) cargo is dissolved in 10 mM citrate, 100 mM NaCl (pH 4.0) so that the concentration of the NA cargo is approximately 0.22 mg/mL. In some embodiments, the NA cargo comprises both functional NA and reporter DNA barcodes mixed at a mass ratio of 1:10 to 10:1 of functional NA to barcode. As described herein, the NA can be siRNA, antisense, expressed DNA, or mRNA.

LNP以47.5％可电离脂质(例如，本文提供的化合物):40％胆固醇:2.5％PEG-2000DMG:以及10％DPSC以及4-8的N/P比范围来进行制备。根据制造商方案，通过使用Precision Nanosystems NanoAssemblr Spark或Benchtop系列仪器对脂质和NA溶液进行微流体混合，形成LNP。在使用不同差分速率进行混合期间，水溶液与有机溶剂的比率保持在大约2:1或3:1。混合之后，收集LNP，在磷酸盐缓冲盐水(PBS)或Tris缓冲盐水(TBS)(大约1:1v/v)中稀释。以20kDa过滤器为背景，在4℃使用PBS或TBS透析4至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP制剂经由动态光散射(DLS)表征，以测量大小(例如，直径)和多分散性。合并特定直径和多分散范围内的LNP，并且以100kDa透析盒为背景，在4℃进一步对PBS或TBS透析1至4小时。在第二次透析之后，使用0.22μm过滤器无菌过滤LNP并存储于4℃以供进一步使用。LNP is prepared with 47.5% ionizable lipid (e.g., compound provided herein): 40% cholesterol: 2.5% PEG-2000DMG: and 10% DPSC and N/P ratio range of 4-8. According to the manufacturer's protocol, lipid and NA solution are mixed by microfluidics using Precision Nanosystems NanoAssemblr Spark or Benchtop series instruments to form LNP. During mixing using different differential rates, the ratio of aqueous solution to organic solvent is maintained at about 2: 1 or 3: 1. After mixing, LNP is collected and diluted in phosphate buffered saline (PBS) or Tris buffered saline (TBS) (about 1: 1v/v). With 20kDa filter as background, PBS or TBS is dialyzed for 4 to 24 hours at 4°C for further buffer exchange. After this initial dialysis, each individual LNP preparation is characterized via dynamic light scattering (DLS) to measure size (e.g., diameter) and polydispersity. LNPs within a specific diameter and polydispersity range were pooled and further dialyzed against PBS or TBS for 1 to 4 hours at 4° C. against a 100 kDa dialysis cassette. After the second dialysis, the LNPs were sterile filtered using a 0.22 μm filter and stored at 4° C. for further use.

LNP表征LNP characterization

使用高通量动态光散射(DLS)(Stunner,Unchained Labs)测量LNP流体动力学直径和多分散指数(PDI)。用1X TBS或PBS将LNP稀释到适当浓度并进行分析。LNP hydrodynamic diameter and polydispersity index (PDI) were measured using high throughput dynamic light scattering (DLS) (Stunner, Unchained Labs). LNPs were diluted to appropriate concentrations with IX TBS or PBS and analyzed.

RNA浓度和包裹效率通过Ribogreen测定(BioTek Synergy LX读板器)来确定。RNA concentration and packaging efficiency were determined by Ribogreen assay (BioTek Synergy LX plate reader).

表2总结了使用所提供的脂质制备的LNP的表征数据。Table 2 summarizes the characterization data of LNPs prepared using the provided lipids.

表2.Table 2.

实例17：LNP施用和生物分布Example 17: LNP Administration and Biodistribution

将大约8至12周龄的雄性和雌性小鼠用于本实施例描述的研究。每只小鼠都被暂时束缚，经由尾静脉注射每次实验向最多五只动物IV施用混合的LNP。还使用年龄匹配的小鼠通过尾静脉注射向每次实验最多三只动物施用媒剂(1X TBS)。还可以采用另外的施用途径，包含脑室内(ICV)、脑池内(ICM)、鞘内(IT)、肌肉内(IM)、雾化、鼻内(IN)、皮下(SC)、关节内和皮内(ID)。在给药后4小时，收集包含肝脏、脾脏、骨髓、睾丸、腓肠肌、四头肌、肺、心脏、肾脏和胰腺的组织进行分析。Male and female mice of about 8 to 12 weeks of age were used for the research described in this embodiment. Each mouse was temporarily restrained and the mixed LNPs were administered IV to up to five animals per experiment via tail vein injection. Age-matched mice were also used to administer vehicle (1X TBS) to up to three animals per experiment via tail vein injection. Other routes of administration can also be used, including intracerebroventricular (ICV), intracisternal (ICM), intrathecal (IT), intramuscular (IM), atomization, intranasal (IN), subcutaneous (SC), intraarticular and intradermal (ID). 4 hours after administration, tissues including liver, spleen, bone marrow, testis, gastrocnemius, quadriceps, lung, heart, kidney and pancreas were collected for analysis.

流量flow

肝脏、肾脏、肺和肌肉(例如骨骼和心脏)组织可以进行机械消化，然后使用蛋白酶混合物进行酶消化，然后通过70μm过滤器以生成单细胞悬浮液。机械消化脾脏组织以产生单细胞悬浮液。所有组织均用ACK缓冲液处理以裂解红细胞，并且然后用荧光标记抗体染色以进行流式细胞术和荧光激活细胞分选(FACS)。本实例中使用市售抗体。使用BDFACSMelody(Becton Dickinson)，经由流式细胞术采集样品以在分选之前生成门。一般来说，门控结构是大小→单峰细胞→活细胞→所关注的细胞。T细胞被定义为CD45+CD3+，单核细胞被定义为CD45+CD11b+，并且B细胞被定义为CD45+CD19+。内皮细胞被定义为CD31+，单核细胞和库普弗细胞被定义为CD45+CD11b+，并且在肝脏和肌肉中肝细胞和肌细胞分别被定义为CD31-/CD45-。来自给药媒介物的小鼠的组织用于设置用于分选的门。Liver, kidney, lung and muscle (such as bone and heart) tissue can be mechanically digested, then enzymatically digested using a protease mixture, and then passed through a 70 μm filter to generate a single cell suspension. Mechanically digest spleen tissue to produce a single cell suspension. All tissues are treated with ACK buffer to lyse red blood cells, and then stained with fluorescently labeled antibodies to perform flow cytometry and fluorescence activated cell sorting (FACS). Commercially available antibodies are used in this example. Using BDFACS Melody (Becton Dickinson), samples are collected via flow cytometry to generate gates before sorting. In general, the gating structure is size → single peak cells → live cells → cells of interest. T cells are defined as CD45+CD3+, monocytes are defined as CD45+CD11b+, and B cells are defined as CD45+CD19+. Endothelial cells are defined as CD31+, monocytes and Kupffer cells are defined as CD45+CD11b+, and hepatocytes and myocytes are defined as CD31-/CD45- in liver and muscle, respectively. Tissue from mice dosed with vehicle was used to set gates for sorting.

使用Reliaprep RNA提取试剂盒来提取来自组织的RNA，并且使用RNAseq试剂盒来制备文库并在MiSeq或NextSeq500上进行测序。RNA from tissues was extracted using the Reliaprep RNA extraction kit, and libraries were prepared using the RNAseq kit and sequenced on a MiSeq or NextSeq500.

为了筛选至靶组织的优先LNP递送，测量一个或多个靶组织中每个条形码的相对丰度并将每个条形码的相对丰度与其相应的相对输入丰度进行比较，以确定每个条形码的倍数输入(FAI)。如果靶组织中条形码的FAI高于阈值或高于同一组织中不同LNP的FAI，则可以将该LNP识别为优先递送至靶组织。To screen for preferential LNP delivery to a target tissue, the relative abundance of each barcode in one or more target tissues is measured and compared to its corresponding relative input abundance to determine the multiple input (FAI) of each barcode. If the FAI of a barcode in a target tissue is above a threshold or above the FAI of a different LNP in the same tissue, the LNP can be identified as being preferentially delivered to the target tissue.

FAI是所选样本中条形码如与其在输入中的频率相比的归一化相对丰度。条形码的FAI指示LNP的丰度相对于LNP库的其余部分如何变化。条形码的FAI值是通过将分离样品中条形码序列计数的相对丰度归一化为其在施用输入中的相对丰度来计算的。例如，FAI值为1表示LNP在分离样品中以与在施用池中相同的频率出现，从而表示相对于同一施用池中的其他LNP群体，它对测量的细胞类型显示出中性向性。然后，FAI指示LNP相对于输入LNP组合物的性能。The FAI is the normalized relative abundance of the barcode in the selected sample as compared to its frequency in the input. The FAI of a barcode indicates how the abundance of the LNP varies relative to the rest of the LNP library. The FAI value of a barcode is calculated by normalizing the relative abundance of the barcode sequence counts in the isolated sample to its relative abundance in the applied input. For example, a FAI value of 1 indicates that the LNP appears in the isolated sample at the same frequency as in the applied pool, thereby indicating that it shows a neutral tropism to the measured cell type relative to other LNP populations in the same applied pool. The FAI then indicates the performance of the LNP relative to the input LNP composition.

表3、表4、表5和表6中总结了使用所提供的脂质制备的LNP的生物分布数据。每个表描述了从一次筛选实验获得的数据。The biodistribution data for LNPs prepared using the provided lipids are summarized in Tables 3, 4, 5, and 6. Each table describes the data obtained from one screening experiment.

表3.Table 3.

可电离脂质Ionizable lipidsFAI(肺)FAI (lung)7-647-640.220.22MC3*MC3*0.500.50

*MC3是(6Z,9Z,28Z,31Z)-三十七-6,9,28,31-四烯-19-基4-(二甲氨基)丁酸酯，通常在-MC3-DMA中称为DL。*MC3 is (6Z,9Z,28Z,31Z)-triacontahedta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, commonly referred to as DL in -MC3-DMA.

表4.Table 4.

*MC3是(6Z,9Z,28Z,31Z)-三十七-6,9,28,31-四烯-19-基4-(二甲氨基)丁酸酯，通常在-MC3-DMA中称为DL。*MC3 is (6Z,9Z,28Z,31Z)-triacontahedta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, commonly referred to as DL in -MC3-DMA.

表5.Table 5.

可电离脂质Ionizable lipidsFAI(肺)FAI (lung)FAI(脾脏)FAI (Spleen)FAI(肝脏)FAI (liver)7-167-161.161.160.700.700.460.467-187-180.830.831.691.691.081.087-377-372.432.432.912.910.710.717-517-510.980.980.770.770.570.577-537-531.191.192.392.391.411.417-597-590.430.430.190.19NDND7-607-600.610.610.270.27NDND7-657-650.280.28NDNDNDND7-707-700.600.600.600.600.310.31MC3*MC3*1.511.510.610.610.300.30

*MC3是(6Z,9Z,28Z,31Z)-三十七-6,9,28,31-四烯-19-基4-(二甲氨基)丁酸酯，通常在-MC3-DMA中称为DL。*MC3 is (6Z,9Z,28Z,31Z)-triacontahedrite-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, commonly referred to as DL in -MC3-DMA.

ND＝未确定。ND = not determined.

表6.Table 6.

可电离脂质Ionizable lipidsFAI(肺)FAI (lung)FAI(脾脏)FAI (Spleen)FAI(肝脏)FAI (liver)7-587-580.440.440.290.290.550.557-647-641.731.733.373.371.801.807-667-660.820.820.510.510.860.867-687-680.520.520.390.390.680.68MC3*MC3*1.681.681.501.500.730.73

*MC3是(6Z,9Z,28Z,31Z)-三十七-6,9,28,31-四烯-19-基4-(二甲氨基)丁酸酯，通常在-MC3-DMA中称为DL。*MC3 is (6Z,9Z,28Z,31Z)-triacontahedta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, commonly referred to as DL in -MC3-DMA.

实例18：利用LNP制剂在脾脏、肝脏和骨髓中进行碱基编辑Example 18: Base Editing in Spleen, Liver, and Bone Marrow Using LNP Formulations

本实例提供了在各种细胞类型中赋予基因编辑(例如，使用碱基编辑器)的示例性LNP组合物、制剂、纳米粒子和/或纳米材料。本文所述的筛选平台可以用于识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对于体内各种细胞类型的碱基编辑最有效(例如，可以在小鼠中进行细胞的碱基编辑)。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials that confer gene editing (e.g., using base editors) in various cell types. The screening platform described herein can be used to identify several highly efficient LNP formulations to determine which type of LNP formulation is most effective for base editing of various cell types in vivo (e.g., base editing of cells can be performed in mice).

本实例可以用于证明所提供的脂质可以用于在各种细胞类型(包括骨髓、脾脏和肝脏)中执行碱基编辑和/或改善LNP制剂的至特定细胞类型的递送。本实例也可以用于证明含有示例性化合物的LNP制剂中所含的mRNA在递送期间在LNP中更加稳定。This example can be used to demonstrate that the provided lipids can be used to perform base editing in various cell types (including bone marrow, spleen, and liver) and/or improve the delivery of LNP formulations to specific cell types. This example can also be used to demonstrate that the mRNA contained in the LNP formulation containing the exemplary compound is more stable in the LNP during delivery.

方法method

选择示例性LNP制剂以确定每种制剂在Balb/C小鼠模型中执行碱基编辑的能力，如本文所述。Exemplary LNP formulations were selected to determine the ability of each formulation to perform base editing in the Balb/C mouse model, as described herein.

将脂质纳米粒子组分以指定的脂质组分摩尔比溶解在100％乙醇中。将编码腺嘌呤碱基编辑器的mRNA和经化学修饰的sgRNA以1:1的质量比溶解在50mM柠檬酸盐(pH 4.0)中，导致NA货物的浓度为大约0.1mg/mL。The lipid nanoparticle components were dissolved in 100% ethanol at the indicated lipid component molar ratios. The mRNA encoding the adenine base editor and the chemically modified sgRNA were dissolved in 50 mM citrate (pH 4.0) at a 1:1 mass ratio, resulting in a concentration of NA cargo of approximately 0.1 mg/mL.

调配LNP制剂，其中摩尔比为47.5％可电离脂质:40％胆固醇:2.5％PEG2000-DMG:10％DSPC，总脂质与NA质量比为6至12。The LNP preparation was formulated with a molar ratio of 47.5% ionizable lipid:40% cholesterol:2.5% PEG2000-DMG:10% DSPC, and a total lipid to NA mass ratio of 6 to 12.

根据制造商的方案，通过使用Precision Nanosystems NanoAssemblr Ignite系列仪器对脂质和NA溶液进行微流体混合，形成LNP制剂。LNP formulations were formed by microfluidic mixing of lipid and NA solutions using a Precision Nanosystems NanoAssemblr Ignite series instrument according to the manufacturer's protocol.

在混合期间使用不同的流速维持3:1的水与有机溶剂比。在混合之后，收集LNP制剂，稀释于TBS中(大约7:1v/v)，并且以20kDa过滤器为背景，在4℃使用TBS透析8至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP制剂经由动态光散射(DLS)表征，以测量大小和多分散性。LNP制剂亚群的pKa经由TNS测定进行测量。Different flow rates were used to maintain a 3: 1 water to organic solvent ratio during mixing. After mixing, the LNP preparations were collected, diluted in TBS (approximately 7: 1 v/v), and further buffer exchange was performed using TBS dialysis for 8 to 24 hours at 4 ° C with a 20 kDa filter as background. After this initial dialysis, each individual LNP preparation was characterized via dynamic light scattering (DLS) to measure size and polydispersity. The pKa of the LNP preparation subpopulation was measured via TNS determination.

在透析之后，使用0.22微米的无菌过滤器无菌过滤LNP制剂并存储于4℃以供进一步使用。在一些实施例中，可以根据制造商的方案使用100kDa Amicon过滤器来浓缩LNP制剂。After dialysis, the LNP formulations were sterile filtered using a 0.22 micron sterile filter and stored at 4°C for further use. In some embodiments, the LNP formulations can be concentrated using a 100 kDa Amicon filter according to the manufacturer's protocol.

LNP表征LNP characterization

使用高通量动态光散射(DLS)(DynaPro plate reader II,Wyatt)测量DLS-LNP制剂流体动力学直径和多分散指数(PDI)。用1X PBS将LNP制剂稀释到适当浓度且进行分析。The hydrodynamic diameter and polydispersity index (PDI) of DLS-LNP preparations were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). The LNP preparations were diluted to appropriate concentrations with IX PBS and analyzed.

浓度和包裹功效Concentration and packaging efficacy

按照制造商说明书，通过Qubit microRNA试剂盒(用于siRNA)或HS RNA试剂盒(用于mRNA)测定NA的浓度。通过测量未裂解和裂解的LNP来测定包裹效率。The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

LNP施用LNP administration

大约8-12周龄的雄性Balb/C小鼠用于本文所述的实验。将每只小鼠暂时束缚，并通过尾静脉注射按0.15mg/kg总RNA将LNP制剂施用至Balb/c小鼠中。Male Balb/C mice of approximately 8-12 weeks of age were used for the experiments described herein. Each mouse was temporarily restrained and the LNP formulations were administered to the Balb/c mice via tail vein injection at 0.15 mg/kg total RNA.

也使用年龄匹配的小鼠经由尾静脉注射施用媒剂(1X TBS)作为对照。给药后四到六天，收集包括肝脏、脾脏和骨髓的组织。碱基编辑是通过使用提取的基因组DNA在ALAS1靶位点处执行靶向深度测序分析来确定的。根据制造商说明书，使用Nextera DNA FlexLibrary Prep Kit(Illumina)(使用96孔板Nextera索引引物(Illumina))来对基因组DNA进行分离和片段化以及接头连接。文库大小和浓度由片段分析仪(Agilent)确认，并使用Illumina HiSeq进行全基因组测序分析。Age-matched mice were also administered vehicle (1X TBS) via tail vein injection as a control. Four to six days after administration, tissues including liver, spleen, and bone marrow were collected. Base editing was determined by performing targeted deep sequencing analysis at the ALAS1 target site using extracted genomic DNA. According to the manufacturer's instructions, genomic DNA was separated and fragmented and connected to the linker using Nextera DNA FlexLibrary Prep Kit (Illumina) (using 96-well plate Nextera index primers (Illumina)). Library size and concentration were confirmed by a fragment analyzer (Agilent), and whole genome sequencing analysis was performed using Illumina HiSeq.

实例19：利用LNP制剂在脾脏、肝脏和骨髓中进行碱基编辑Example 19: Base Editing in Spleen, Liver, and Bone Marrow Using LNP Formulations

本实例提供了在各种细胞类型中赋予基因编辑(例如，使用碱基编辑器)的示例性LNP组合物、制剂、纳米粒子和/或纳米材料。本文所述的筛选平台可以用于识别几种高效的LNP制剂，以确定哪种类型的LNP制剂对于体内各种细胞类型的碱基编辑最有效(例如，可以在小鼠中进行细胞的碱基编辑)。This example provides exemplary LNP compositions, formulations, nanoparticles and/or nanomaterials that confer gene editing (e.g., using base editors) in various cell types. The screening platform described herein can be used to identify several highly efficient LNP formulations to determine which type of LNP formulation is most effective for base editing of various cell types in vivo (e.g., base editing of cells can be performed in mice).

本实例可以用于证明所提供的脂质可以用于在各种细胞类型(包括骨髓、脾脏和肝脏)中执行碱基编辑和/或改善LNP制剂的至特定细胞类型的递送。本实例也可以用于证明含有示例性化合物的LNP制剂中所含的mRNA在递送期间在LNP中更加稳定。This example can be used to demonstrate that the provided lipids can be used to perform base editing in various cell types (including bone marrow, spleen, and liver) and/or improve the delivery of LNP formulations to specific cell types. This example can also be used to demonstrate that the mRNA contained in the LNP formulation containing the exemplary compound is more stable in the LNP during delivery.

方法method

选择示例性LNP制剂以确定每种制剂在NBSGW小鼠模型中执行碱基编辑的能力，如本文所述。Exemplary LNP formulations were selected to determine the ability of each formulation to perform base editing in the NBSGW mouse model, as described herein.

将脂质纳米粒子组分以指定的脂质组分摩尔比溶解在100％乙醇中。将编码腺嘌呤碱基编辑器的mRNA和经化学修饰的sgRNA以1:1的质量比溶解在10mM柠檬酸盐(pH 3.0)中，导致NA货物的浓度为大约0.2mg/mL。The lipid nanoparticle components were dissolved in 100% ethanol at the indicated lipid component molar ratios. The mRNA encoding the adenine base editor and the chemically modified sgRNA were dissolved in 10 mM citrate (pH 3.0) at a 1:1 mass ratio, resulting in a concentration of NA cargo of approximately 0.2 mg/mL.

调配LNP制剂，其中摩尔比为50％可电离脂质:38.5％胆固醇:1.5％PEG2000-DMG:10％DSPC，总脂质与NA质量比为19至1。The LNP preparation was formulated with a molar ratio of 50% ionizable lipid:38.5% cholesterol:1.5% PEG2000-DMG:10% DSPC, and a total lipid to NA mass ratio of 19 to 1.

根据制造商的方案，通过使用Precision Nanosystems NanoAssemblr Spark系列仪器对脂质和NA溶液进行微流体混合，形成LNP制剂。LNP formulations were formed by microfluidic mixing of lipid and NA solutions using a Precision Nanosystems NanoAssemblr Spark series instrument according to the manufacturer's protocol.

在混合期间使用不同的流速维持3:1的水与有机溶剂比。在混合之后，收集LNP制剂，稀释于PBS中(大约2:1v/v)，并且以20kDa过滤器为背景，在4℃使用PBS透析8至24小时进行进一步的缓冲液交换。在此初始透析之后，每一个别LNP制剂经由动态光散射(DLS)表征，以测量大小和多分散性。LNP制剂亚群的pKa经由TNS测定进行测量。Different flow rates were used to maintain a 3: 1 water to organic solvent ratio during mixing. After mixing, the LNP preparations were collected, diluted in PBS (approximately 2: 1 v/v), and further buffer exchange was performed using PBS dialysis for 8 to 24 hours at 4°C with a 20 kDa filter as background. After this initial dialysis, each individual LNP preparation was characterized via dynamic light scattering (DLS) to measure size and polydispersity. The pKa of the LNP preparation subpopulation was measured via TNS determination.

在透析之后，使用0.22微米的无菌过滤器无菌过滤LNP制剂并存储于4℃以供进一步使用。在一些实施例中，LNP制剂可以按照制造商的方案使用100kDa Amicon过滤器进行浓缩。After dialysis, the LNP formulations were sterile filtered using a 0.22 micron sterile filter and stored at 4°C for further use. In some embodiments, the LNP formulations can be concentrated using a 100 kDa Amicon filter according to the manufacturer's protocol.

LNP表征LNP characterization

使用高通量动态光散射(DLS)(DynaPro plate reader II,Wyatt)测量DLS-LNP制剂流体动力学直径和多分散指数(PDI)。用1X PBS将LNP制剂稀释到适当浓度且进行分析。The hydrodynamic diameter and polydispersity index (PDI) of DLS-LNP preparations were measured using high throughput dynamic light scattering (DLS) (DynaPro plate reader II, Wyatt). The LNP preparations were diluted to appropriate concentrations with IX PBS and analyzed.

浓度和包裹功效Concentration and packaging efficacy

按照制造商说明书，通过Qubit microRNA试剂盒(用于siRNA)或HS RNA试剂盒(用于mRNA)测定NA的浓度。通过测量未裂解和裂解的LNP来测定包裹效率。The concentration of NA was determined by the Qubit microRNA kit (for siRNA) or HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring uncleaved and cleaved LNPs.

LNP施用和碱基编辑LNP administration and base editing

给药前四周，将大约8-12周龄的NBSGW小鼠植入5e5人CD34+细胞并用于本文所述的实验。植入后四周，每只小鼠暂时被束缚，并经由尾静脉注射以3mg/kg IV施用LNP制剂。Four weeks before dosing, NBSGW mice of approximately 8-12 weeks of age were implanted with 5e5 human CD34+ cells and used for the experiments described herein. Four weeks after implantation, each mouse was temporarily restrained and LNP formulations were administered IV at 3 mg/kg via tail vein injection.

也使用年龄匹配的小鼠经由尾静脉注射施用媒剂(1X PBS)作为对照。给药后四到六天，收集包括肝脏、脾脏和骨髓的组织。碱基编辑是通过使用提取的基因组DNA在人ALAS1靶位点处执行靶向深度测序分析来确定的。根据制造商说明书，使用Nextera DNA FlexLibrary Prep Kit(Illumina)(使用96孔板Nextera索引引物(Illumina))来对基因组DNA进行分离和片段化以及接头连接。文库大小和浓度由片段分析仪(Agilent)确认，并使用Illumina HiSeq进行全基因组测序来进行测序。Age-matched mice were also administered vehicle (1X PBS) via tail vein injection as a control. Four to six days after administration, tissues including liver, spleen, and bone marrow were collected. Base editing was determined by performing targeted deep sequencing analysis at the human ALAS1 target site using extracted genomic DNA. According to the manufacturer's instructions, genomic DNA was separated and fragmented and connected to the linker using Nextera DNA FlexLibrary Prep Kit (Illumina) (using 96-well plate Nextera index primers (Illumina)). Library size and concentration were confirmed by a fragment analyzer (Agilent) and sequenced using Illumina HiSeq for whole genome sequencing.

等同物Equivalent

本领域中的技术人员仅使用常规实验将认识到或能够确定本文所描述的本发明具体实施例的许多等同物。本发明的范围不旨在限于上述说明，而是如随附权利要求书中所述。Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the claims that follow.

Claims (63)

Translated fromChinese

1.一种式I化合物：1. A compound of formula I:或其N-氧化物、或其药学上可接受的盐，其中：or an N-oxide thereof, or a pharmaceutically acceptable salt thereof, wherein:L¹、L^1'和L^1”中的每一者独立地为不存在的、-C(O)-或-OC(O)-；Each of L¹ , L^1′ and L^1″ is independently absent, —C(O)— or —OC(O)—;L²、L^2'和L^2”中的每一者独立地为不存在的、任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链或-(CH₂)_m-Cy^A-(CH₂)_m'-；Each of L² , L^2′ and L^2″ is independently absent, an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain or -(CH₂ )_m -Cy^A -(CH₂ )_m′ -;每个Cy^A独立地为选自亚苯基和3至12元饱和或部分不饱和的亚碳环基的任选地被取代的环；each Cy^A is independently an optionally substituted ring selected from phenylene and a 3- to 12-membered saturated or partially unsaturated carbocyclylene;m和m'中的每一者独立地为0、1或2；Each of m and m' is independently 0, 1 or 2;L³、L^3'和L^3”中的每一者独立地为不存在的、-C(O)-、-C(O)O-、-OC(O)-、-O-或-OC(O)O-；each of L³ , L^3′, and L^3″ is independently absent, —C(O)—, —C(O)O—, —OC(O)—, —O—, or —OC(O)O—;R¹、R^1'和R^1”中的每一者独立地为-(CH₂)_p-Cy^B、或任选地被取代的饱和或不饱和的直链或支链C_1-20烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each of R¹ , R^1′ and R^1″ is independently -(CH₂ )_p -Cy^B , or an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;每个Cy^B独立地为选自以下的任选地被取代的环：3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧和硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each Cy^{B is} independently an optionally substituted ring selected from: a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;每个p独立地为0、1、2或3；Each p is independently 0, 1, 2 or 3;每个L^3a独立地为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-10烃链，其中1-3个亚甲基单元任选地且独立地被-O-或-NR-置换；Each L^3a is independently absent or optionally substituted divalent saturated or unsaturated linear or branched C_1-10 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O- or -NR-;每个R^a独立地为氢或选自以下的任选地被取代的基团：C_6-20脂肪族、3至12元饱和或部分不饱和的碳环基、具有0-4个独立地选自氮、氧和硫的杂原子的7至12元饱和或部分不饱和的桥接双环基、1-金刚烷基、2-金刚烷基、甾醇基和苯基；each^{Ra is} independently hydrogen or an optionally substituted group selected from the group consisting of a_C6-20 aliphatic, a 3- to 12-membered saturated or partially unsaturated carbocyclyl, a 7- to 12-membered saturated or partially unsaturated bridged bicyclic group having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;X¹为不存在的、-O-、-S-或-NR-；^X1 is absent, -O-, -S- or -NR-;X²为不存在的或任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换；^X2 is absent or optionally substituted divalent saturated or unsaturated linear or branched_C1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or^-CyC- ;Cy^C为选自以下的任选地被取代的环：3至7元饱和或部分不饱和的亚碳环、亚苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的亚杂环或具有1-3个独立地选自氮、氧和硫的杂原子的5至6元亚杂芳基；Cy^C is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, a phenylene group, a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or a 5- to 6-membered heteroarylene group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur;X³为氢或选自以下的任选地被取代的环：3至7元饱和或部分不饱和的碳环基、苯基、具有1-3个独立地选自氮、氧和硫的杂原子的3至7元饱和或部分不饱和的杂环基以及具有1-3个独立地选自氮、氧和硫的杂原子的5至6元杂芳基；并且^X3 is hydrogen or an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a phenyl, a 3- to 7-membered saturated or partially unsaturated heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, and a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and每个R独立地为氢或任选地被取代的C_1-6脂肪族基团。Each R is independently hydrogen or an optionally substituted C_1-6 aliphatic group.2.根据权利要求1所述的化合物，其中所述化合物具有式I-A：2. The compound according to claim 1, wherein the compound has formula I-A:或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.3.根据权利要求1或2所述的化合物，其中所述化合物具有式I-B：3. The compound according to claim 1 or 2, wherein the compound has formula I-B:或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.4.根据前述权利要求中任一项所述的化合物，其中所述化合物具有式I-C：4. A compound according to any one of the preceding claims, wherein the compound has formula I-C:或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.5.根据前述权利要求中任一项所述的化合物，其中所述化合物具有式I-D：5. A compound according to any one of the preceding claims, wherein the compound has formula I-D:或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.6.根据前述权利要求中任一项所述的化合物，其中所述化合物具有式I-E：6. A compound according to any one of the preceding claims, wherein the compound has formula I-E:或其N-氧化物、或其药学上可接受的盐。or an N-oxide thereof, or a pharmaceutically acceptable salt thereof.7.根据权利要求1所述的化合物，其中L¹为-C(O)-。The compound according to claim 1 , wherein L¹ is —C(O)—.8.根据权利要求1所述的化合物，其中L^1'为-C(O)-。The compound according to claim 1 , wherein L^1′ is —C(O)—.9.根据权利要求1所述的化合物，其中L^1”为-C(O)-。9. The compound according to claim 1, wherein L^1" is -C(O)-.10.根据权利要求1-9中任一项所述的化合物，其中L²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。10. The compound according to any one of claims 1 to 9, wherein^L2 is an optionally substituted divalent saturated or unsaturated linear or branched_C1-12 hydrocarbon chain.11.根据权利要求1-10中任一项所述的化合物，其中L^2'为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。11. The compound according to any one of claims 1 to 10, wherein L^2' is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain.12.根据权利要求1-11中任一项所述的化合物，其中L^2”为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链。12. The compound according to any one of claims 1 to 11, wherein L^2" is an optionally substituted divalent saturated or unsaturated linear or branched C_1-12 hydrocarbon chain.13.根据权利要求1-9或11-12中任一项所述的化合物，其中L²为不存在的。13. The compound according to any one of claims 1-9 or 11-12, wherein^L2 is absent.14.根据权利要求1-10或12-13中任一项所述的化合物，其中L^2'为不存在的。14. The compound according to any one of claims 1-10 or 12-13, wherein L^2' is absent.15.根据权利要求1-11或13-14中任一项所述的化合物，其中L^2”为不存在的。15. The compound according to any one of claims 1-11 or 13-14, wherein L^2" is absent.16.根据权利要求1-15中任一项所述的化合物，其中L³为不存在的。16. The compound according to any one of claims 1-15, wherein^L3 is absent.17.根据权利要求1-16中任一项所述的化合物，其中L^3'为不存在的。17. The compound according to any one of claims 1-16, wherein L^3' is absent.18.根据权利要求1-17中任一项所述的化合物，其中L^3”为不存在的。18. The compound according to any one of claims 1 to 17, wherein L^3" is absent.19.根据权利要求1-15中任一项所述的化合物，其中L³为-C(O)O-或-OC(O)-。19. The compound according to any one of claims 1 to 15, wherein L³ is -C(O)O- or -OC(O)-.20.根据权利要求1-16和18-19中任一项所述的化合物，其中L^3'为-C(O)O-或-OC(O)-。20. The compound according to any one of claims 1-16 and 18-19, wherein L^3' is -C(O)O- or -OC(O)-.21.根据权利要求1-17和19-20中任一项所述的化合物，其中L^3”为-C(O)O-或-OC(O)-。21. The compound according to any one of claims 1-17 and 19-20, wherein L^3" is -C(O)O- or -OC(O)-.22.根据权利要求1-21中任一项所述的化合物，其中R¹为-(CH₂)_p-Cy^B。22. The compound according to any one of claims 1-21, wherein^R1 is -(_CH2 )_p -^CyB .23.根据权利要求1-21中任一项所述的化合物，其中R¹为23. A compound according to any one of claims 1 to 21, wherein R¹ is24.根据权利要求1-21中任一项所述的化合物，其中R¹为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。24. The compound according to any one of claims 1 to 21, wherein R¹ is an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.25.根据权利要求1-24中任一项所述的化合物，其中R^1'为-(CH₂)_p-Cy^B。25. The compound according to any one of claims 1-24, wherein R1^' is -(_CH2 )_p -^CyB .26.根据权利要求1-24中任一项所述的化合物，其中R^1'为26. A compound according to any one of claims 1 to 24, wherein R^1' is27.根据权利要求1-24中任一项所述的化合物，其中R^1'为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。27. The compound according to any one of claims 1 to 24, wherein R^1' is an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.28.根据权利要求1-27中任一项所述的化合物，其中R^1”为-(CH₂)_p-Cy^B。28. The compound according to any one of claims 1-27, wherein R^1" is -(_CH2 )_p -^CyB .29.根据权利要求1-27中任一项所述的化合物，其中R^1”为29. A compound according to any one of claims 1 to 27, wherein R^1" is30.根据权利要求1-27中任一项所述的化合物，其中R^1”为任选地被取代的饱和或不饱和的直链或支链C_1-20烃链。30. The compound according to any one of claims 1-27, wherein R^1" is an optionally substituted saturated or unsaturated linear or branched C_1-20 hydrocarbon chain.31.根据权利要求1-30中任一项所述的化合物，其中每个Cy^B为1-金刚烷基。31. The compound of any one of claims 1-30, wherein each Cy^B is 1-adamantyl.32.根据权利要求1-31中任一项所述的化合物，其中每个p独立地为0或1。32. A compound according to any one of claims 1-31, wherein each p is independently 0 or 1.33.根据权利要求1-32中任一项所述的化合物，其中X¹为-O-。33. The compound according to any one of claims 1-32, wherein X¹ is -O-.34.根据权利要求1-32中任一项所述的化合物，其中X¹为-NR-。34. A compound according to any one of claims 1-32, wherein X¹ is -NR-.35.根据权利要求1-34中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_1-12烃链，其中1-3个亚甲基单元任选地且独立地被-O-、-NR-或-Cy^C-置换。35. according to the compound described in any one of claims 1-34, wherein X² is an optionally substituted divalent saturated or unsaturated straight or branched C_1-12 hydrocarbon chain, wherein 1-3 methylene units are optionally and independently replaced by -O-, -NR- or -Cy^C -.36.根据权利要求1-35中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_4-8烃链，其中1个亚甲基单元被-NR-置换。36. The compound according to any one of claims 1 to 35, wherein^X2 is an optionally substituted divalent saturated or unsaturated linear or branched_C4-8 hydrocarbon chain in which one methylene unit is replaced by -NR-.37.根据权利要求1-35中任一项所述的化合物，其中X²为任选地被取代的二价饱和或不饱和的直链或支链C_4-8烃链，其中1个亚甲基单元被-Cy^C-置换。37. A compound according to any one of claims 1 to 35, wherein X² is an optionally substituted divalent saturated or unsaturated linear or branched C_4-8 hydrocarbon chain in which one methylene unit is replaced by -Cy^C -.38.根据权利要求1-37中任一项所述的化合物，其中X³为氢。38. A compound according to any one of claims 1 to 37, wherein X³ is hydrogen.39.根据权利要求1-36中任一项所述的化合物，其中X³为具有1-3个独立地选自氮、氧和硫的杂原子的任选地被取代的3至7元饱和或部分不饱和的杂环基。39. according to the compound described in any one of claims 1-36, wherein X³ is an optionally substituted 3 to 7 membered saturated or partially unsaturated heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur.40.根据前述权利要求中任一项所述的化合物，其中-X²-X³为40. A compound according to any one of the preceding claims, wherein -X² -X³ is41.根据权利要求1-40中任一项所述的化合物，R为氢。41. The compound of any one of claims 1-40, wherein R is hydrogen.42.根据权利要求1-40中任一项所述的化合物，R为任选地被取代的C_1-6脂肪族。42. The compound of any one of claims 1-40, R is an optionally substituted_C1-6 aliphatic.43.一种选自表1的化合物、或其药学上可接受的盐。43. A compound selected from Table 1, or a pharmaceutically acceptable salt thereof.44.一种脂质纳米粒子(LNP)制剂，其包含根据权利要求1-43中任一项所述的可电离脂质。44. A lipid nanoparticle (LNP) formulation comprising an ionizable lipid according to any one of claims 1-43.45.一种脂质纳米粒子(LNP)制剂，其包含：45. A lipid nanoparticle (LNP) formulation comprising:根据权利要求1-43中任一项所述的可电离脂质；The ionizable lipid according to any one of claims 1-43;磷脂；Phospholipids;甾醇；和Sterols; and缀合物-连接子脂质(例如，聚乙二醇脂质)。Conjugate - Linker lipid (eg, polyethylene glycol lipid).46.根据权利要求44所述的LNP制剂，其进一步包括治疗剂和/或预防剂。46. The LNP formulation of claim 44, further comprising a therapeutic and/or prophylactic agent.47.根据权利要求46所述的LNP制剂，其中所述治疗剂和/或预防剂为或包含一种或多种核酸。47. The LNP formulation of claim 46, wherein the therapeutic and/or prophylactic agent is or comprises one or more nucleic acids.48.根据权利要求47所述的LNP制剂，其中所述一种或多种核酸为或包含RNA。48. The LNP formulation of claim 47, wherein the one or more nucleic acids is or comprises RNA.49.根据权利要求47所述的LNP制剂，其中所述一种或多种核酸为或包含DNA。49. The LNP formulation of claim 47, wherein the one or more nucleic acids is or comprises DNA.50.根据权利要求46-49中任一项所述的LNP制剂，其中所述LNP制剂被调配成将所述治疗剂和/或预防剂递送至靶细胞。50. The LNP formulation of any one of claims 46-49, wherein the LNP formulation is formulated to deliver the therapeutic and/or prophylactic agent to a target cell.51.根据权利要求50所述的LNP制剂，其中所述靶细胞为或包括脾细胞(例如，脾B细胞、脾T细胞、脾单核细胞)、肝脏细胞(例如，肝细胞)、骨髓细胞(例如，骨髓单核细胞)、免疫细胞、肾细胞、肌肉细胞、心脏细胞、肺细胞或中枢神经系统中的细胞。51. The LNP formulation of claim 50, wherein the target cell is or comprises a spleen cell (e.g., a spleen B cell, a spleen T cell, a spleen monocyte), a liver cell (e.g., a hepatocyte), a bone marrow cell (e.g., a bone marrow mononuclear cell), an immune cell, a kidney cell, a muscle cell, a heart cell, a lung cell, or a cell in the central nervous system.52.根据权利要求51所述的LNP制剂，其中所述靶细胞为或包括造血干细胞(HSC)。52. The LNP formulation of claim 51, wherein the target cell is or comprises a hematopoietic stem cell (HSC).53.一种药物组合物，其包含根据权利要求44-52中任一项所述的LNP制剂以及药学上可接受的赋形剂。53. A pharmaceutical composition comprising the LNP formulation according to any one of claims 44-52 and a pharmaceutically acceptable excipient.54.一种用于向有此需要的受试者施用治疗剂和/或预防剂的方法，所述方法包括：向所述受试者施用根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物。54. A method for administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising: administering to the subject the LNP formulation of any one of claims 44-52 or the pharmaceutical composition of claim 53.55.一种用于治疗有此需要的受试者的疾病或病症的方法，所述方法包括：向所述受试者施用根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物，其中所述治疗剂和/或预防剂对治疗所述疾病有效。55. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the LNP formulation of any one of claims 44-52 or the pharmaceutical composition of claim 53, wherein the therapeutic and/or prophylactic agent is effective for treating the disease.56.一种用于延迟和/或遏制有此需要的受试者的疾病或病症的进展的方法，所述方法包括：向所述受试者施用根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物，其中所述治疗剂和/或预防剂对治疗所述疾病有效。56. A method for delaying and/or arresting the progression of a disease or condition in a subject in need thereof, the method comprising: administering to the subject the LNP formulation of any one of claims 44-52 or the pharmaceutical composition of claim 53, wherein the therapeutic and/or prophylactic agent is effective for treating the disease.57.一种将治疗剂和/或预防剂递送至来源于受试者的哺乳动物细胞的方法，所述方法包括：接触已经施用了根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物的所述受试者的所述细胞。57. A method of delivering a therapeutic and/or prophylactic agent to mammalian cells derived from a subject, the method comprising: contacting the cells of the subject to which the LNP formulation of any one of claims 44-52 or the pharmaceutical composition of claim 53 has been administered.58.一种在哺乳动物细胞中产生所关注的多肽的方法，所述方法包括：使所述细胞与根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物接触，其中所述治疗剂和/或预防剂为或包含mRNA，并且其中所述mRNA编码所述所关注的多肽，其中所述mRNA能够在所述细胞中被翻译以产生所述所关注的多肽。58. A method for producing a polypeptide of interest in a mammalian cell, the method comprising: contacting the cell with an LNP formulation according to any one of claims 44-52 or a pharmaceutical composition according to claim 53, wherein the therapeutic and/or prophylactic agent is or comprises mRNA, and wherein the mRNA encodes the polypeptide of interest, wherein the mRNA can be translated in the cell to produce the polypeptide of interest.59.一种抑制哺乳动物细胞中所关注的多肽的产生的方法，所述方法包括：使所述细胞与根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物接触，其中所述治疗剂和/或预防剂为或包含RNA，其中所述RNA能够抑制所述所关注的多肽的产生。59. A method for inhibiting the production of a polypeptide of interest in a mammalian cell, the method comprising: contacting the cell with an LNP formulation according to any one of claims 44-52 or a pharmaceutical composition according to claim 53, wherein the therapeutic and/or prophylactic agent is or comprises RNA, wherein the RNA is capable of inhibiting the production of the polypeptide of interest.60.一种将治疗剂和/或预防剂特异性地递送至哺乳动物器官、组织、细胞或细胞群体的方法，所述方法包括：使哺乳动物器官、组织、细胞或细胞群体与根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物接触，其中所述治疗剂和/或预防剂被递送至所述器官、组织、细胞或细胞群体。60. A method for specifically delivering a therapeutic and/or prophylactic agent to a mammalian organ, tissue, cell or cell population, the method comprising: contacting the mammalian organ, tissue, cell or cell population with the LNP formulation according to any one of claims 44-52 or the pharmaceutical composition according to claim 53, wherein the therapeutic and/or prophylactic agent is delivered to the organ, tissue, cell or cell population.61.根据权利要求60所述的方法，其包括：向受试者施用根据权利要求44-52中任一项所述的LNP制剂或向所述受试者施用根据权利要求53所述的药物组合物。61. The method of claim 60, comprising administering to a subject the LNP formulation of any one of claims 44-52 or administering to the subject the pharmaceutical composition of claim 53.62.一种通过施用根据权利要求44-52中任一项所述的LNP制剂或根据权利要求53所述的药物组合物来接种疫苗的方法。62. A method of vaccination by administering the LNP formulation of any one of claims 44-52 or the pharmaceutical composition of claim 53.63.一种在受试者中诱导适应性免疫应答的方法，所述方法包括：向所述受试者施用有效量的组合物，所述组合物包含至少一种RNA；其中所述组合物包含LNP制剂，所述LNP制剂包含根据权利要求1-43中任一项所述的化合物或其药学上可接受的盐。63. A method of inducing an adaptive immune response in a subject, the method comprising: administering to the subject an effective amount of a composition comprising at least one RNA; wherein the composition comprises an LNP formulation comprising a compound according to any one of claims 1-43 or a pharmaceutically acceptable salt thereof.