Disclosure of Invention
The present disclosure provides ionizable cationic lipids capable of targeting immune cells, compositions comprising the same, and uses thereof. The ionizable cationic lipid provided by the disclosure can be used for delivering therapeutic or prophylactic agents such as nucleic acid molecules, small-molecule compounds, polypeptides or proteins, has a simple preparation method, has strong targeting to immune cells, can carry pharmaceutical active ingredients to transfect cells with higher transfection efficiency, has lower cytotoxicity, and can improve delivery efficiency and safety.
The technical scheme provided by the disclosure may include, for example:
[1] a (cationic lipid) compound, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has a structure according to formula (I):
,
Wherein:
r1 is a substituted or unsubstituted N-containing heterocycle;
r2 and R3 are each independently a substituted or unsubstituted C10-16 linear or branched alkene or alkane, R2 and R3 are the same or different;
L1 and L2 are each independently unsubstituted C2-8 linear alkylene;
M1 is- (CH2)n -, -C (O) NH-or-NHC (O) -, wherein n is 0, 1, 2,3 or 4;
M2 and M3 are each independently any one of-ch=ch-, -C (O) O-and-C (O) N-, M2 and M3 are the same or different.
In the compound of formula (I) provided by the present invention, the number of carbon atoms of R2 and R3 may be 10, 11, 12, 13, 14, 15, 16, independently of each other, or may be a range formed by any two of the above values, or any intermediate value (integer) in the range.
In the compound of formula (I) provided by the present invention, R2 and R3 may each independently be a linear alkyl group, a branched alkyl group, a linear alkenyl group, or a branched alkenyl group.
In the compound of formula (I) provided by the present invention, the number of carbon atoms of L1 and L2 may be 2, 3, 4, 5, 6, 7, 8, or may be a range formed by any two of the above values, or any intermediate value (integer) in the range.
[2] The compound according to [1], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is、Or (b);
And/or R2 is、、、、Or unsubstituted C10-16 linear alkanes;
And/or R3 is、、、、Or unsubstituted C10-16 linear alkanes;
and/or L1 is- (CH2)2-、-(CH2)3-、-(CH2)5 -or- (CH2)8 -;
And/or L2 is- (CH2)2-、-(CH2)3-、-(CH2)5 -or- (CH2)8 -;
And/or M1 is-CH2 -or-NHC (O) -;
and/or M2 is-CH=CH-, -C (O) O-, or-C (O) N-;
and/or the number of the groups of groups, M3 is-ch=ch-; -C (O) O-or-C (O) N-.
[3] The compound according to [1] or [2], or an N-oxide, a solvate, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the compound has any one of the following structures YK-1501 to YK-1518:
、
、
、
、
、
、
、
、
、
、
、
、
、
、
、
、
And
。
Preferably, the compound has the structure of any one of YK-1503, YK-1504YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516.
[4] A carrier, wherein the carrier comprises a cationic lipid, wherein the cationic lipid comprises a compound of any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.
[5] The carrier according to [4], wherein the cationic lipid is 25 to 75% by mole of the carrier. That is, the content of the cationic lipid is 25 to 75mol% based on the total molar amount of the carrier.
For example, the content of the cationic lipid in the carrier may be 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol%, 75mol%, or may be a range formed by any two of the above values, or any intermediate value in the range.
[6] The vector of [4] or [5], wherein the vector further comprises a neutral lipid;
And/or, the carrier further comprises a structural lipid;
And/or the carrier further comprises a polymer conjugated lipid.
[7] The carrier according to [6], wherein the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof;
and/or the structural lipid is selected from any one or a combination of at least two of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, and corticosteroids;
And/or the polymer conjugated lipid is selected from any one or a combination of at least two of the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.
[8] The carrier according to [6] or [7], wherein the neutral lipid is selected from any one or a combination of at least two of 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphoric acid, 1-hexadecyl-sn-3-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, hexaleoyl-glycero-3-phosphorylcholine 1, 2-di-phytanoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-stearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-linolenoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-di-docosahexaenoic acid-sn-glycerol-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt, di-palmitoyl phosphatidylglycerol, palmitoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, 1-stearoyl-ethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, sphingomyelin, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylcholine.
[9] The carrier according to any one of [6] to [8], wherein the neutral lipid is selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine and/or 1, 2-distearoyl-sn-glycero-3-phosphorylcholine.
[10] The vector according to [6] or [7], wherein the structural lipid is cholesterol.
[11] The carrier according to [6] or [7], wherein the polymer conjugated lipid is selected from any one or a combination of at least two of the group consisting of distearyl phosphatidylethanolamine polyethylene glycol 2000, dimyristoyl glycerol-3-methoxypolyethylene glycol 2000 and methoxypolyethylene glycol ditetradecylamide.
[12] The carrier according to any one of [6] to [11], wherein the neutral lipid accounts for 5 to 25% by mole of the carrier;
and/or, the structural lipid accounts for 15-65% of the carrier by mole percent;
And/or the polymer conjugated lipid accounts for 0.5-10% of the carrier by mole percent.
That is, the neutral lipid content is 5 to 25mol% and/or the structural lipid content is 15 to 65mol% and/or the polymer conjugated lipid content is 0.5 to 10mol% based on the total molar amount of the carrier.
For example, the content of the neutral lipid in the carrier may be 5mol%, 10mol%, 15mol%, 20mol%, 25mol%, or may be a range formed by any two of the above values, or any intermediate value in the range.
For example, the content of the structural lipid in the carrier may be 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, or may be a range formed by any two of the above values, or any intermediate value in the range.
For example, the content of the polymer-conjugated lipid in the carrier may be 0.5mol%, 1mol%, 2mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8mol%, 9mol%, 10mol%, or may be a range formed by any two of the above values, or any intermediate value in the range.
[13] The carrier according to any one of [4] to [12], wherein a molar ratio of the cationic lipid to the neutral lipid in the carrier is 1:1 to 15:1;
and/or, the molar ratio of cationic lipid to structural lipid in the carrier is 0.6:1-3:1;
And/or, the molar ratio of the cationic lipid to the polymer conjugated lipid in the carrier is 4.5:1-32.5:1.
[14] The carrier according to any one of [4] to [13], wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid in the carrier is (25-75): 5-25): 15-65): 0.5-10.
[15] The carrier according to [14], wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid in the carrier is (35-49): 7.5-15): 35-5): 1-5.
[16] The carrier according to [14] or [15], wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid in the carrier is 49:10:39.5:1.5.
[17] The carrier of any one of [4] to [16], wherein the cationic lipid further comprises one or more other cationic lipid compounds.
"Other cationic lipid compounds" refers to cationic lipid compounds other than the compounds of formula (I) provided herein, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof. Any cationic lipid compound available in the art for use in the preparation of a carrier (particularly, a carrier for supporting a biologically active molecule such as a nucleic acid, a protein, a peptide, etc.) can be suitably used in the present invention, and the source thereof is not particularly limited, and may be obtained by a commercially available route or may be prepared by itself according to the prior art.
[18] A composition comprising an active ingredient and a carrier as described in any one of [4] to [17 ].
"Active ingredient" refers to an active molecule having a function or action, for example, an active molecule having a disease treatment/prevention function (e.g., a small molecule drug, a therapeutic/prophylactic nucleic acid, a therapeutic/prophylactic protein or peptide, etc.), an active molecule having a biomarker function (e.g., GFP or a gene encoding the same, etc.), etc. "Carrier" refers to a molecule or composition for supporting an active ingredient, for protection, transport, etc. Generally, the carrier itself does not have a function or activity, but the choice of carrier has a certain influence on the activity of the active ingredient.
[19] The composition according to [18], wherein the composition is a nanoparticle preparation having an average particle diameter of 10nm to 300nm, and a polydispersity index (PDI) of 0.5 or less. The average particle size, polydispersity, etc. of the nanoparticle formulation can be measured by conventional methods, for example, by detection using an instrument such as a laser particle sizer.
[20] The composition according to [18] or [19], wherein the nanoparticle preparation has an average particle diameter of 40nm to 240nm, and the nanoparticle preparation has a polydispersity of 0.4 or less.
For example, the nanoparticle formulation may have an average particle diameter of 40nm、50nm、60nm、70nm、72nm、74nm、76nm、78nm、80nm、82nm、84nm、86nm、88nm、90nm、92nm、94nm、96nm、98nm、100nm、110nm、120nm、130nm、140nm、150nm、180nm、200nm、220nm、240nm, or may be in a range consisting of any two of the above values, or any intermediate value in the range.
For example, the nanoparticle formulation may have a polydispersity of 0.0001、0.0005、0.001、0.005、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、0.35、0.4, or may be in the range of any two values described above, or any intermediate value in the range.
[21] The composition of any one of [18] to [20], wherein the active ingredient comprises a therapeutic or prophylactic agent.
[22] The composition according to any one of [18] to [21], wherein the mass ratio of carrier to the therapeutic or prophylactic agent is 10:1 to 30:1.
For example, the mass ratio of carrier to the therapeutic or prophylactic agent may be 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, or may be a range of any two ratios described above, or any intermediate ratio in the range.
[23] The composition according to [22], wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1.
[24] The composition according to [23], wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 13:1 to 17:1.
[25] The composition of any one of [21] to [24], wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
[26] The composition according to [25], wherein the therapeutic or prophylactic agent is selected from any one or a combination of at least two of the group consisting of a nucleic acid, a small molecule compound, a polypeptide, or a protein.
[27] The composition according to [26], wherein the therapeutic or prophylactic agent is a nucleic acid.
[28] The composition according to [27], wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).
[29] The composition of [28], wherein the ribonucleic acid is selected from any one or a combination of at least two of the group consisting of small interfering RNA, asymmetric interfering RNA, microRNA, dicer-substrate RNA, small hairpin RNA, messenger RNA.
[30] The composition of [29], wherein the ribonucleic acid is a messenger RNA.
[31] The composition of any one of [18] to [30], wherein the composition further comprises a pharmaceutically acceptable excipient and/or diluent.
[32] Use of a compound of any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier of any one of [4] to [17], or a composition of any one of [18] to [31] in the manufacture of a medicament.
[33] The use according to [32], wherein the active component of the medicament is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides or proteins.
Preferably, the drug is a nucleic acid drug (i.e., a drug whose active ingredient is a nucleic acid). The nucleic acid used in the nucleic acid drug may be a nucleic acid selected from the aforementioned therapeutic or prophylactic agents, and will not be described in detail herein.
[34] Use of a compound of any one of [1] to [3] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a vector of any one of [4] to [17], for increasing the transfection efficiency of cells and/or reducing cytotoxicity.
[35] The use according to [34], wherein the use is in increasing the efficiency of cell transfection and/or reducing cytotoxicity in vitro.
[36] Use of the compound of any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of [4] to [17], to increase targeting of a nucleic acid to a target, and/or to increase expression of a nucleic acid in a target, wherein the target is selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue and a target cell.
[37] The use of [36], wherein the target organ or target tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph and muscle;
And/or the target cell is selected from any one or a combination of at least two of the group consisting of B cells, NK cells, DC cells, T cells and macrophages.
[38] The use according to [37], wherein the target organ or target tissue is the spleen;
and/or the target cell is selected from any one or a combination of at least two of the group consisting of B cells, DC cells, T cells and macrophages.
[39] Use of a compound of any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier of any one of [4] to [17], or a composition of any one of [18] to [31], in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.
[40] The use according to [39], wherein the disease or disorder is characterized by malfunction or abnormality of a protein or polypeptide.
[41] The use according to [39] or [40], wherein the disease or disorder is selected from any one or a combination of at least two of the group consisting of infectious diseases (e.g., diseases caused by viral infections), cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.
[42] The use of [39], wherein the subject is a mammal.
[43] The use of [42], wherein the subject is a human.
[44] The use of any one of [39] to [43], wherein the medicament is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.
[45] The use according to [44], wherein the medicament is administered subcutaneously.
[46] The use of any one of [39] to [45], wherein the medicament is used in an amount such that a therapeutic or prophylactic agent in a dose of about 0.001mg/kg to about 10mg/kg is administered to the subject.
It should be understood that in the above technical solutions, the use provided by the present disclosure may include both therapeutic and diagnostic use and non-therapeutic and non-diagnostic use. For example, therapeutic uses may include packaging the (drug) active ingredient(s) in a compound provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or in a carrier provided by the present disclosure and delivering it to a target organ/tissue/cell, or using a composition of the present disclosure to deliver the active ingredient(s) contained therein to a target organ/tissue/cell, thereby effecting a disease treatment, ameliorating a condition, modulating physiological activity in vivo, etc., diagnostic uses may include packaging the active ingredient(s) for disease diagnosis in a compound provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or in a carrier thereof, causing the active ingredient(s) to be delivered to a target organ/tissue/cell, thereby effecting a disease diagnosis, or non-therapeutic/non-diagnostic uses may include packaging the active ingredient(s) provided by the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or in a carrier provided by the present disclosure, thereby detecting the active ingredient(s) to be delivered to a target organ/tissue/cell, performing a new therapeutic mechanism, research, development of a new mechanism of the drug, development of the disease, non-therapeutic mechanism, or the like.
The beneficial effects of the present disclosure include at least:
The cationic lipid compounds and lipid compositions of the present disclosure (which may also be referred to as "carriers" in the present disclosure) are useful for encapsulation of pharmaceutically active ingredients such as nucleic acids (e.g., mRNA, etc.).
The mRNA-LNP composition prepared by the cationic lipid has at least the advantages of being capable of remarkably improving the protein expression quantity in a subject (such as a mouse) and meanwhile having remarkable spleen targeting, being capable of remarkably improving the in vivo (in vivo) and in vitro (in intro) protein expression quantity, and remarkably improving the spleen and immune cell targeting of the mRNA-LNP composition prepared by the cationic lipid compared with the prior art by adopting the ionizable cationic lipid.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be described below with reference to the accompanying drawings. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. Based on the described embodiments of the present disclosure, one of ordinary skill in the art may implement other specific forms without departing from the basic attributes and gist of the present disclosure. It is to be understood that any and all embodiments of the present disclosure may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The present disclosure includes additional embodiments resulting from such combinations.
All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with the use or term used in the present disclosure, the use or term of the present disclosure controls.
The section headings used in this disclosure are for purposes of organizing articles only and should not be construed as limiting the subject matter.
Unless otherwise specified, all technical and scientific terms used in this disclosure have the ordinary meaning in the art to which claimed subject matter belongs. In case there are multiple definitions for a term, the present disclosure controls.
Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of quantitative properties such as dosages set forth in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited in this disclosure is intended to include all sub-ranges within that range and any combination of the individual endpoints of that range or sub-range. When a range of values is disclosed in this disclosure, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed in this disclosure are to be understood to include any and all subranges subsumed therein.
The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term and its equivalents, but do not exclude the presence of other elements. The terms "comprising," "including," or "comprising," as used in this disclosure, may be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of, or" consisting of.
The term "pharmaceutically acceptable" in this disclosure means that the compound or composition is chemically and/or toxicologically compatible with the other components of the formulation and/or with the human or mammal with which the disease or disorder is to be prevented or treated.
The term "subject" or "patient" in this disclosure includes mammals, such as common laboratory animals (e.g., mice, rats, guinea pigs, rabbits, pigs, monkeys, etc.), and also, for example, humans.
The term "treatment" as used in this disclosure refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, ameliorate or affect the disease or symptoms of the disease. In the context of the present disclosure, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.
The term "solvate" refers in the present disclosure to a complex of a compound of formula (I) or a pharmaceutically acceptable salt thereof, in association with a solvent (e.g., ethanol or water). It will be appreciated that any solvate of the compound of formula (I) used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will result in the compound of formula (I) once absorbed into a subject, such that the use of the compound of formula (I) encompasses the use of any solvate of the compound of formula (I), respectively.
The term "hydrate" refers to the case where the solvent in the above term "solvate" is water.
It is further understood that the compound of formula (I) or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present disclosure. For example, a compound of formula (I) or a pharmaceutically acceptable salt thereof may exist in unsolvated forms or solvated forms in which are associated with pharmaceutically acceptable solvents (such as water, ethanol, and the like).
The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure. See, for example, S.M. Bere et al, "Pharmaceutical Salts", J.Pharm. Sci.1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc., organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glyceric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid may be used to form pharmaceutically acceptable salts with the compounds of formula (I).
The nitrogen-containing compounds of formula (I) of the present disclosure may be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, the presently claimed compounds include not only nitrogen-containing compounds of the formula, but also N-oxide derivatives thereof, where valence and structure permit.
Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers diastereoisomers and enantiomers. Thus, the presently claimed compounds also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or lower side effects than the other stereoisomers. The single stereoisomers and the mixture with optical activity can be obtained by chiral source synthesis methods, chiral catalysis methods, chiral resolution methods and the like. The racemate can be chiral resolved by chromatographic resolution or chemical resolution. For example, separation can be performed by adding chiral acid resolving agents such as chiral tartaric acid, chiral malic acid, and the like to form salts with the compounds of the present disclosure, utilizing differences in the physicochemical properties of the products, such as solubility.
The present disclosure also includes all suitable isotopic variations of the compounds of the present disclosure. Isotopic variations are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found or predominantly present in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as2 H (deuterium),3 H (tritium),11C、13C、14C、15N、17 O, and18 O, respectively.
The term "alkyl" is meant in this disclosure to include both branched and straight chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene" is meant in this disclosure to include both branched and straight chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. Cn-m is meant to include groups having a number of carbon atoms from n to m. For example, C2-5 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene.
The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced with another chemical group. Useful substituents may include halogen, ester, cyano, sulfonyl, and the like.
A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or condition. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or condition. The amount of therapeutic agent constituting the "therapeutically effective amount" or the amount of prophylactic agent of the "prophylactically effective amount" varies with the therapeutic agent/prophylactic agent, the disease state and severity thereof, the age, weight, etc. of the patient/subject to be treated/prevented. One of ordinary skill in the art can routinely determine therapeutically effective and prophylactically effective amounts based on their knowledge and disclosure.
In the present disclosure, when the names of the compounds are not identical to the structural formula, the structural formula is taken into consideration.
It is to be understood that the term "compounds of the present disclosure" as used in the present disclosure may include, depending on the context, compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.
The term "cationic lipid" as used in the present disclosure refers to a lipid that is positively charged at a selected pH or range.
Cationic lipids readily bind to negatively charged nucleic acids, i.e., interact with negatively charged phosphate groups in nucleic acids by electrostatic forces, forming Lipid Nanoparticles (LNPs).
The inventors have found, upon examining and screening a large number of lipid compounds, that it is very difficult to obtain cationic lipid compounds suitable for use in nucleic acid drug carriers while satisfying the following conditions (1) having structural differences from the cationic lipids currently used in the prior art for supporting nucleic acid drugs, (2) having high transfection efficiency and low cytotoxicity, and (3) having high expression and sustained expression in organisms.
In long-term studies, the inventors have unexpectedly discovered compounds, such as YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 in the present disclosure, which are capable of delivering nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, significantly improved expression levels in animals, and spleen targeting, as compared to cationic lipids in the prior art.
The present disclosure is based at least on the following findings:
The cationic lipid compounds of the present disclosure can be used to deliver nucleic acids, small molecule compounds, polypeptides, or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds disclosed by the invention have the advantages that the transfection efficiency is higher, the cytotoxicity is smaller, the expression level in the spleen of animals is obviously improved, and the delivery efficiency is improved.
A second aspect of the present disclosure provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof as described above.
In one embodiment, the composition is a nanoparticle formulation having an average size of 10nm to 300nm, preferably 40nm to 240m, and a polydispersity of 0.5 or less, preferably 0.4 or less.
Cationic lipids
In one embodiment of the composition/carrier of the present disclosure, the cationic lipid is one or more selected from the compounds of formula (I) above or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. In one embodiment, the cationic lipid is a compound of formula (I) selected from the group consisting of those described above. For example, cationic lipids are compounds. In a preferred embodiment, the cationic lipid is a compound YK-1501 to YK-1518, and in a preferred embodiment, the cationic lipid is a compound YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 or YK-1516.
In another embodiment of the composition/carrier of the present disclosure, the cationic lipid comprises (a) one or more selected from the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof described above, and (b) one or more other ionizable lipid compounds different from (a). Wherein, (b) the cationic lipid compound may be a commercially available cationic lipid, or a cationic lipid compound reported in the literature. For example, (b) the cationic lipid compound may be SM-102 in CN201080026228.8, MC3 in CN201080026228.8, or C16 in CN 202380010167.3.
In one embodiment, the cationic lipid comprises 25% to 75%, such as 30%, 40%, 50%, 55%, 60%, 65%, 70% of the carrier by mole.
The carrier may be used for the delivery of (pharmaceutical) active ingredients such as therapeutic and/or prophylactic agents. The active ingredient may be enclosed within the carrier or combined with the carrier in any form.
For example, examples of the therapeutic/prophylactic agent may be one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Such nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNAs (sirnas), asymmetric interfering RNAs (airnas), micrornas (mirnas), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shrnas), messenger RNAs (mrnas), mixtures thereof, and the like.
Neutral lipids
The carrier may comprise neutral lipids. Neutral lipids in the present disclosure refer to lipids that exist in an uncharged form or in a neutral ionic form at a selected pH or range. The neutral lipids may modulate nanoparticle mobility into lipid bilayers (lipid bilayer) and increase efficiency by promoting lipid phase changes, while also potentially affecting target organ specificity.
In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1-15:1, such as about 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. Also for example, in a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.
For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
The carrier component of the cationic lipid-containing composition may comprise one or more neutral lipid-phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
Neutral lipids may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, the phospholipid may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cell recognition, or to couple the composition with a useful component such as a targeting or imaging moiety (e.g., dye).
Neutral lipids useful in these compositions may be selected from the non-limiting group consisting of: 1, 2-Dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C), 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1-dioleoyl-sn-3-phosphorylcholine (CPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine, 1-dioleoyl-2-glycero-3-phosphorylcholine (POPC) 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG), 1, 2-di-oleoyl-rac-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.
In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.
Structured lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more structural lipids. Structured lipids refer in the present disclosure to lipids that enhance the stability of the nanoparticle by filling the interstices between the lipids.
In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1-5:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
The structural lipid may be selected from the group consisting of, but not limited to, cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherols, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids such as prednisolone (prednisolone), dexamethasone, prednisone (prednisone), and hydrocortisone (hydrocortisone), or combinations thereof.
Polymer conjugated lipids
The carrier of the composition comprising the cationic lipid may also comprise one or more polymer conjugated lipids. The polymer conjugated lipid mainly refers to polyethylene glycol (PEG) modified lipid. Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.
In one embodiment, the polymer conjugated lipid is selected from one or more of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol, and PEG modified dialkylglycerol. The PEG modified PEG molecular weight is typically 350-5000Da.
For example, the polymer conjugated lipid is selected from one or more of distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG 2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), and methoxypolyethylene glycol ditetradecylamide (ALC-0159).
In one embodiment of the composition/carrier of the present disclosure, the polymer conjugated lipid is DMG-PEG2000.
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises neutral lipids, structural lipids, and polymer conjugated lipids, the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer conjugated lipids is (25-75): (5-25): (15-65): (0.5-10), such as (30-49): (7.5-15): (35-55): (1-5), more preferably (40-49): (8-12): (39-45): (1-3). The sum of the molar ratios of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 100.
In one embodiment of the composition/carrier of the present disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer conjugated lipid, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid being 40:10:48.5:1.5 or 49:10:39.5:1.5.
Therapeutic and/or prophylactic agent
The composition may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of carrier to the therapeutic or prophylactic agent is 10:1-30:1, e.g., 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.
In one embodiment, the mass ratio of carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1, preferably 13 to 17:1, more preferably 15:1.
The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
The vectors of the present disclosure may deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and/or prophylactic agent and/or contacting mammalian cells with the composition. Accordingly, the present disclosure provides for the use of a compound of the present disclosure or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition of the present disclosure in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.
The present disclosure also provides the use of a compound of the present disclosure or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition of the present disclosure in the preparation of a nucleic acid drug, vaccine, chemical drug, polypeptide drug or protein drug.
Therapeutic and/or prophylactic agents include bioactive substances and are alternatively referred to as "active agents". The therapeutic and/or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in the cell or organ or other body tissue or system. Such species may be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder, or condition. examples of drugs that may be used in the compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin (doxorubicin), mitoxantrone (mitoxantrone), camptothecin, cisplatin (cispratin), bleomycin (bleomycin), cyclophosphamide (cyclophosphamide), methotrexate, and streptozotocin), antineoplastic agents (e.g., actinomycin D (actinomycin D)), Vincristine, vinblastine (vinblastine), cytosine arabinoside (cytosine arabinoside), anthracyclines (anthracyclines), alkylating agents, platinoids, antimetabolites and nucleoside analogues such as methotrexate and purine and pyrimidine analogues), anti-infective agents, local anesthetics (e.g. dibucaine (dibucaine) and chlorpromazine (chlorpromazine)), beta-adrenergic blockers (e.g. propranolol (propranolol), molol (timolol) and labetalol (labetalol)), Antihypertensives (e.g., clonidine and hydralazine (hydralazine)), antidepressants (e.g., imipramine (imipramine), amitriptyline (AMITRIPTYLINE) and doxepin (doxepin)), antispasmodics (e.g., phenytoin (phenytoin)), antihistamines (e.g., diphenhydramine (DIPHENHYDRAMINE), chlorpheniramine (chlorpheniramine) and promethazine (promethazine)), antibiotics/antibacterial agents (e.g., gentamicin (gentamycin), antibiotics/antibacterial agents (e.g., gentamicin), Ciprofloxacin (ciprofloxacin) and cefoxitin (cefoxitin)), antifungal agents (e.g., miconazole (miconazole), terconazole (terconazole), econazole (econazole), econazole (isoconazole), butoconazole (butaconazole), clotrimazole (clotrimazole), itraconazole (itraconazole), nystatin (nystatin), netifen (naftifine) and amphotericin B (amphotericin B)), and pharmaceutical compositions, Antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, vitamins, sedatives and imaging agents.
In some embodiments, the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include, but are not limited to, taxol (taxol), cytochalasin B (cytochalasin B), gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), canamycin (emetine), mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine, vinblastine, colchicine (colchicine), doxorubicin, daunorubicin (daunorubicin), dihydroxyanthracene dione (dihydroxy anthracin dione), and, Mitoxantrone, mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol, puromycin, maytansinoids (maytansinoids) such as maytansinol (maytansinol), azithromycin (rachelmycin) (CC-1065), and analogs or homologs thereof. Radioions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Examples of vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human Papilloma Virus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis, for example may include mRNA encoding a pathogenic antigen (pathogenic antigen) and/or an epitope thereof, and may also include compounds and formulations that direct an immune response against cancer cells, for example may include mRNA encoding a tumor cell-derived antigen, epitope and/or neoepitope. compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and/or compound capable of eliciting an immune response is administered intramuscularly through a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg) or (III) (e.g., compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122). Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard (mechlorethamine), thiotepa (thiotepa), chlorambucil (chlorambucil), azithromycin (CC-1065), melphalan (melphalan), carmustine (carmustine, BSNU), robustin (lomustine, CCNU), and combinations thereof, Cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin (daunomycin)) and doxorubicin), antibiotics (e.g., dactinomycin (dactinomycin) (formerly dactinomycin), bleomycin, mithramycin (mithramycin) and aflatoxin (anthramycin, AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol and maytansinoids).
In other embodiments, the therapeutic and/or prophylactic agent is a protein. Therapeutic proteins useful in the nanoparticles in the present disclosure include, but are not limited to, gentamicin, amikacin (amikacin), insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing Hormone Releasing Hormone (LHRH) analogs, interferons, heparin, hepatitis b surface antigens, typhoid vaccines, and cholera vaccines.
In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" is intended to include in its broadest sense any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing factors, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, RNA that induces triple helix formation, aptamers, and the like. In some embodiments, the therapeutic and/or prophylactic agent is RNA. RNAs useful in the compositions and methods described in this disclosure may be selected from the group consisting of, but not limited to shortmer, antagomir, antisense RNA, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNA), messenger RNAs (mRNAs), and mixtures thereof. In certain embodiments, the RNA is mRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.
In other embodiments, the therapeutic and/or prophylactic agent is an siRNA. siRNA is capable of selectively reducing expression of a gene of interest or down-regulating expression of the gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced after administration of a composition comprising the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.
In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA may be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect mRNA translation of cellular genes.
In some embodiments, the therapeutic and/or prophylactic agent is an shRNA or a vector or plasmid encoding the same. shRNA may be produced inside the target cell after delivery of the appropriate construct into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant arts.
Diseases or conditions
The compositions/carriers of the present disclosure can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic or prophylactic agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Thus, the compositions of the present disclosure can be used to prepare nucleic acid drugs, genetic vaccines, small molecule drugs, polypeptides or protein drugs. Because of the wide variety of therapeutic or prophylactic agents described above, the compositions of the present disclosure are useful in the treatment or prevention of a variety of diseases or conditions.
In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.
In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, rift valley fever, yellow fever, rabies, and various herpes.
Other components
The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin a or vitamin E) or sterols.
The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule may be, for example, a molecule described in U.S. patent application publication No. 2005/0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen, and derivatives and analogs thereof).
Surface modifying agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain (bromelain), papain, dyers woad (clerodendrum), bromhexine (bromhexine), carbocisteine (carbocisteine), eplerenone (eprazinone), mesna (mesna), ambroxol (ambroxol), sibiriol (sobrerol), polymitols (domiodol), letrostat (letosteine), setronine (stepronin), tiopronin (tiopronin), gelsolin (gelsolin), thymosin (thymosin) β4, streptococcal dnase α (dornase alfa), netilmicin (neltenexine), and polysteine (erdosteine), and dnase (e.g., rhDNA enzymes). The surface modifying agent may be disposed within and/or on the nanoparticle of the composition (e.g., by coating, adsorption, covalent attachment, or other means).
The composition may further comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups effective to facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
In addition to these components, the composition may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, and the like. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, e.g., remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, MD, 2006).
Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof.
In some embodiments, compositions comprising one or more lipids described in the present disclosure may further comprise one or more adjuvants, such as Glucopyranosyl Lipid Adjuvants (GLA), cpG oligodeoxyribonucleotides (e.g., class a or class B), poly (I: C), aluminum hydroxide, and Pam3CSK4.
The compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present disclosure may be prepared by methods well known in the pharmaceutical arts. For example, sterile injectable solutions can be prepared by incorporating the therapeutic or prophylactic agent in the required amount with various of the other ingredients described above in the appropriate solvent such as sterile distilled water and then filter-sterilizing. Surfactants may also be added to promote the formation of a uniform solution or suspension.
For example, the compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the composition is administered subcutaneously.
The compositions of the present disclosure are administered in therapeutically effective amounts, which may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attendant physician or clinician. For example, a dose of about 0.001mg/kg to about 10mg/kg of the therapeutic or prophylactic agent may be administered to a subject (preferably a mammal, such as a human).
Examples
The present disclosure is further described below in connection with the following examples, but the present disclosure is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. In the specific examples of the present disclosure, all raw materials used are commercially available. All temperatures are given in degrees celsius unless otherwise indicated. Technical features of the various embodiments of the present disclosure may be combined with each other as long as they do not collide with each other.
In the examples below, the abbreviations represent the following meanings:
NaBH4 sodium borohydride, boc2 O di-tert-butyl dicarbonate, DMAP 4-dimethylaminopyridine, EDCI 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, HATU 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, DIEA N, N-diisopropylethylamine, DCM, DMF, N, N-dimethylformamide, THF, tetrahydrofuran, meOH, methanol
In the examples below, the operation was carried out at room temperature (25.+ -. 5 ℃ C.) without specific explanation.
EXAMPLE 1 Synthesis of cationic lipid Compound
1.1 Synthesis of intermediate INT-1
Step 1 Synthesis of INT-1-PM1
(S) -1-amino-3-chloro-2-propanol hydrochloride (10.0 g,68.48 mmol) was dissolved in methylene chloride (150 mL), triethylamine (27.72 g,273.95 mmol) was added, di-tert-butyl dicarbonate (55.30 g,253.40 mmol) was slowly added dropwise thereto, the temperature was raised to 40℃for 24 hours, and the TLC was monitored to complete the reaction. The heating was stopped, the reaction was quenched by adding saturated aqueous sodium bicarbonate solution to the reaction system, the liquid was separated, the aqueous phase was extracted 2 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was dried by spin-drying under reduced pressure, the obtained residue was purified by silica gel column chromatography, and the collected product was concentrated to give INT-I-PM1 (9.10 g,43.40mmol, 63.3%). C8H16ClNO3, MS(ES):m/z(M+H+) 210.1.
Step 2 Synthesis of INT-1-PM2
INT-I-PM1 (2.81 g,13.40 mmol) was dissolved in acetonitrile (40 mL), followed by addition of tetrahydropyrrole (1.14 g,16.08 mmol), potassium carbonate (5.55 g,40.20 mmol) and potassium iodide (0.44 g,2.68 mmol), followed by reaction at 70℃for 8h, and TLC monitoring was carried out until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain INT-I-PM2（2.43g,9.94mmol,74.2%）.C12H24N2O3, MS(ES):m/z(M+H+)245.2.
Step 3 Synthesis of INT-1
INT-I-PM2 (2.43 g,9.94 mmol) and a solution of hydrochloric acid in 1,4 dioxane (24 mL) were added and reacted at room temperature for 2h, and LCMS was monitored until the starting material was reacted. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in methanol and concentrated under reduced pressure, followed by repeating the above steps for 3 times to give INT-1 (2.05 g, crude product). C7H16N2O, MS(ES):m/z(M+H+) 145.2.
1.2 Synthesis of intermediate INT-2
Step 1 Synthesis of INT-2
2-Hexyldecanol (24.24 g,99.97 mmol) was dissolved in cyclohexane (250 mL), 6-bromohexanoic acid (23.40 g,119.97 mmol) and p-toluenesulfonic acid hydrate (0.28 g,1.49 mmol) were added, a water separator was installed, and the reaction was carried out at 110℃for 7h, and TLC monitoring was carried out until the starting material reaction was completed. The heating was stopped, and the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, separating the solution, extracting the aqueous phase with n-hexane for 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to give INT-2 (41.42 g,98.73mmol, 98.7%). C22H43BrO2, MS(ES):m/z(M+H+) 419.2.
1.3 Synthesis of intermediate INT-3
Step 1 Synthesis of INT-3
N-decanol (25.00 g,157.94 mmol) was dissolved in cyclohexane (250 mL), 4-bromobutyric acid (31.65 g,189.53 mmol) and p-toluenesulfonic acid hydrate (0.30 g,1.57 mmol) were added, a water separator was installed, and the reaction was carried out at 110℃for 8h, and TLC monitoring was carried out until the starting material was reacted. The heating was stopped, and the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, separating the solution, extracting the aqueous phase with n-hexane for 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to give INT-3 (47.31 g,153.96mmol, 97.4%). C14H27BrO2, MS(ES):m/z(M+H+) 307.1.
1.4 Synthesis of intermediate INT-4
Step 1 Synthesis of INT-4
Heptadecan-9-ol (5.00 g,19.49 mmol) was dissolved in dichloromethane (50 mL), 6-bromohexanoic acid (3.80 g,19.49 mmol), EDCI (5.60 g,29.24 mmol) and DMAP (0.47 g,3.889 mmol) were added, reacted at room temperature for 16h, and TLC monitoring the reaction until the starting material was complete. The reaction was quenched by adding saturated aqueous sodium hydrogencarbonate solution, separating the liquid, extracting the aqueous phase with dichloromethane 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, purifying the obtained residue by silica gel column chromatography, and concentrating the collected product to give INT-4 (8.00 g,18.45mmol, 94.6%). C23H45BrO2, MS(ES):m/z(M+H+) 433.2.
1.5 Synthesis of intermediate INT-5
Step 1 Synthesis of INT-5
2-Octyl decanol (25.00 g,92.42 mmol) was dissolved in cyclohexane (250 mL), 6-bromohexanoic acid (21.63 g,110.90 mmol) and p-toluenesulfonic acid hydrate (0.26 g,1.38 mmol) were added, a water separator was installed, and the reaction was carried out at 110℃for 8h, and TLC monitoring was carried out until the starting material reaction was completed. The heating was stopped, and the reaction was quenched by adding saturated aqueous sodium bicarbonate solution, separating the solution, extracting the aqueous phase with n-hexane for 1 time, combining the organic phases, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to give INT-5 (40.19 g,89.80mmol, 97.1%). C24H47BrO2, MS(ES):m/z(M+H+) 447.2.
1.6 Synthesis of intermediate INT-6
Step 1 Synthesis of INT-6
Hexadecylamine (10.0 g,41.41 mmol) and TEA (12.57 g,124.24 mmol) were dissolved in DCM (100 ml) in this order under nitrogen, cooled to 0℃and a solution of acryloyl chloride (4.49 g,49.69 mmol) in dichloromethane (40 ml) was added dropwise and the reaction was allowed to warm to room temperature after the dropwise addition. The reaction was monitored by TLC. After the reaction was completed, the system was cooled to 5 ℃ and quenched with aqueous NaHCO3, the aqueous phase was extracted 2 times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was dried under reduced pressure, the resulting residue was purified by silica gel column chromatography and the product was collected and concentrated to INT-6 (9.78 g,33.09mmol, 79.9%). C19H37NO, MS(ES):m/z(M+H+) 296.3.
1.7 Synthesis of intermediate INT-7
Step 1 Synthesis of INT-7-PM1
INT-7-SM (47.00 g, 0.43 mol) was dissolved in dichloromethane (350 mL), triethylamine (97.10 g, 0.96 mol) and Boc anhydride (279.40 g, 1.28 mol) were added at room temperature and the reaction was stirred at room temperature overnight. After the reaction, adding saturated sodium bicarbonate aqueous solution, extracting with dichloromethane, mixing the organic phases, washing with pure water, drying the organic phases with anhydrous sodium sulfate, spin-drying the solvent under reduced pressure, and purifying by silica gel chromatography (0% -50% ethyl acetate/n-hexane) to obtain the product INT-7-PM1（44.00 g, 0.21 mol, 48.8%）.C8H16ClNO3,MS (ES): m/z (M + H+) = 210.70.
Step 2 Synthesis of INT-7-PM2
2-Methyl-3H-imidazole (5.00 g, 60.90 mmol) and INT-7-PM1 (15.32 g, 73.08 mmol) were dissolved in DMF (50 mL), potassium carbonate (25.25 g, 182.70 mmol) and potassium iodide (2.02 g, 12.18 mmol) were added at room temperature and the reaction was stirred 75, oC and monitored by TLC. After the reaction, adding pure water for dilution, extracting with ethyl acetate, merging the organic phases, washing with saturated sodium chloride aqueous solution, drying the organic phase with anhydrous sodium sulfate, drying the solvent by spin-drying under reduced pressure, and purifying by silica gel chromatography (0% -20% methanol/dichloromethane) to obtain the product INT-7-PM2（5.05 g, 19.78 mmol, 32.5%）.C12H21N3O3,MS (ES): m/z (M + H+) = 256.05.
Step 3 Synthesis of INT-7
INT-7-PM2 (5.00 g, 19.58 mmol) was dissolved in 1, 4-dioxane solution (100 mL) of hydrochloric acid and stirred overnight at room temperature. After the reaction was completed, the solution was spin-dried to give crude INT-7 (5.00 g) with hydrochloride, which was used directly in the next step.
1.8 Synthesis of YK-1501
Step 1 Synthesis of YK-1501-PM1
Didecylamine (20.00 g,67.21 mmol) was dissolved in DMF (100 mL), boc- β -alanine (12.71 g,67.21 mmol), HATU (38.33 g,100.81 mmol) and DIEA (26.05 g,201.63 mmol) were added, the reaction was allowed to proceed at room temperature for 3h, and LCMS was monitored until the starting material was complete. Quenching with water, extracting with ethyl acetate for 2 times, mixing the organic phases, washing with saturated salt water for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, spin-drying the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain the final product YK-1501-PM1（23.40g,49.91mmol,74.2%）.C28H56N2O3, MS(ES):m/z(M+H+)469.4.
Step 2 Synthesis of YK-1501-PM2
YK-1501-PM1 (23.40 g,49.91 mol) and a1, 4 dioxane solution of hydrochloric acid (120 mL) were added and reacted at room temperature for 2h, and LCMS was monitored until the starting material was reacted. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in methanol and concentrated under reduced pressure, followed by repeating the above steps for 3 times to obtain YK-1501-PM2 (18.40 g, crude product). C23H48N2O, MS(ES):m/z(M+H+) 369.4.
Step 3 Synthesis of YK-1501-PM3
YK-1501-PM2 (7.70 g,20.89 mmol) and TEA (6.34 g,62.66 mmol) were dissolved in DCM (80 ml) in this order under nitrogen, cooled to 0deg.C, and a solution of acryloyl chloride (2.27 g,25.06 mmol) in dichloromethane (20 ml) was added dropwise, and the mixture was allowed to react at room temperature after the dropwise addition. The reaction was monitored by TLC. After the reaction, the system was cooled to 5 ℃, quenched with aqueous NaHCO3, extracted 2 times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was dried under reduced pressure, the residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1501-PM3（5.88g,13.91mmol,66.6%）.C26H50N2O2, MS(ES):m/z(M+H+)423.4.
Step 4 Synthesis of YK-1501
INT-1 (90 mg,0.62 mmol) was dissolved in xylene (3 mL), YK-1501-PM3 (659 mg,1.56 mmol) and potassium carbonate (258 mg,1.87 mmol) were added sequentially, the temperature was raised to 90℃for 3 days, and LCMS was monitored until the starting material was reacted. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1501 (35 mg,0.03mmol, 5.6%).
C59H116N6O5, MS(ES):m/z(M+H+)989.6.1H NMR (400 MHz, CDCl3) δ 3.58 – 3.42 (m, 5H), 3.34 – 3.21 (m, 5H), 3.21 – 3.14 (m, 4H), 3.01 – 2.68 (m, 5H), 2.57 – 2.47 (m, 5H), 2.33 (s, 4H), 2.13 (s, 4H), 1.50 (d, J = 7.1 Hz, 9H), 1.39 – 1.23 (m, 58H), 0.90 – 0.86 (t, 12H).
1.9 Synthesis of YK-1502
Step 1 Synthesis of YK-1502-PM1
INT-1 (90 mg,0.62 mmol) was dissolved in xylene (3 mL), YK-1501-PM3 (237 mg,0.56 mmol) and potassium carbonate (258 mg,1.87 mmol) were added sequentially, the temperature was raised to 90℃for 3h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain YK-1502-PM1（150mg,0.26mmol,42.3%）.C33H66N4O3, MS(ES):m/z(M+H+)567.5.
Step 2 Synthesis of YK-1502
YK-1502-PM1 (150 mg,0.26 mmol) was dissolved in xylene (3 mL), INT-6 (93 mg,0.31 mmol) and potassium carbonate (109 mg,0.79 mmol) were added sequentially, the temperature was raised to 90℃for 3 days, and LCMS was monitored until the starting material was reacted. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1502 (30 mg,0.03mmol, 13.1%).
C52H103N5O4, MS(ES):m/z(M+H+)862.8.1H NMR (400 MHz, CDCl3) δ 3.49 (d, J = 47.3 Hz, 2H), 3.18 (dd, J = 9.5, 4.1 Hz, 6H), 2.87 – 2.63 (m, 6H), 2.56 – 2.40 (m, 4H), 2.33 (dt, J = 9.8, 5.4 Hz, 3H), 1.51 (s, 6H), 1.31 – 1.24 (m, 55H), 0.90 – 0.86 (t, 9H).
1.10 Synthesis of YK-1503
Step 1 Synthesis of YK-1503-PM1
INT-1 (0.71 g,4.92 mmol) was dissolved in acetonitrile (25 mL), then INT-2 (1.65 g,3.93 mmol), potassium carbonate (2.04 g,14.76 mmol) and potassium iodide (0.16 g,0.98 mmol) were added sequentially, the temperature was raised to 70℃for 7h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain YK-1503-PM1（0.70g,1.44mmol,29.4%）.C29H58N2O3, MS(ES):m/z(M+H+)483.4.
Step 2 Synthesis of YK-1503
YK-1503-PM1 (650 mg,1.34 mmol) was dissolved in acetonitrile (10 mL), then INT-3 (413 mg,1.34 mmol), potassium carbonate (578 mg,4.03 mmol) and potassium iodide (44 mg,0.26 mmol) were added sequentially, the temperature was raised to 70℃for 8h, and LCMS was monitored until the reaction was completed. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1503 (390 mg,0.54mmol, 40.8%).
C43H84N2O3, MS(ES):m/z(M+H+)709.6.1H NMR (400 MHz, CDCl3) δ 4.08 – 4.02 (m, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.22 – 2.96 (m, 5H), 2.60 – 2.34 (m, 6H), 2.34 – 2.26 (t, 4H), 2.08 – 1.98 (m, 4H), 1.82 – 1.70 (m, 2H), 1.67 – 1.57 (m, 5H), 1.49 – 1.39 (m, 2H), 1.38 – 1.18 (m, 41H), 0.90 – 0.86 (t, 9H).
1.11 Synthesis of YK-1504
Step 1 Synthesis of YK-1504
INT-1 (0.71 g,4.92 mmol) was dissolved in acetonitrile (25 mL), then INT-2 (1.65 g,3.93 mmol), potassium carbonate (2.04 g,14.76 mmol) and potassium iodide (0.16 g,0.98 mmol) were added sequentially, the temperature was raised to 70℃for 7h, and LCMS was monitored until the starting material was reacted. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1504 (0.26 g,0.31mmol, 6.4%).
C51H100N2O5, MS(ES):m/z(M+H+)821.7.1H NMR (400 MHz, CDCl3) δ 4.43 – 4.38 (m, 1H), 3.96 (d, J = 5.8 Hz, 4H), 3.43 (dd, J = 17.3, 8.0 Hz, 3H), 3.34 (t, J = 13.8 Hz, 2H), 2.90 (dd, J = 12.9, 8.6 Hz, 1H), 2.70 – 2.47 (m, 4H), 2.33 – 2.29 (t, 4H), 2.19 – 2.11 (m, 4H), 1.70 – 1.47 (m, 10H), 1.37 – 1.26 (m, 52H), 0.90 – 0.86 (t, 12H).
1.12 Synthesis of YK-1505
Step 1 Synthesis of YK-1505
INT-1 (80 mg,0.55 mmol) was dissolved in acetonitrile (7 mL), INT-4 (480 mg,1.10 mmol), potassium carbonate (229 mg,1.66 mmol) and potassium iodide (18 mg,0.11 mmol) were added sequentially, the temperature was raised to 70℃for 8h, and LCMS was monitored until the starting material was reacted. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1505 (19 mg,0.02mmol, 4.0%).
C53H104N2O5, MS(ES):m/z(M+H+)849.7.1H NMR (400 MHz, CDCl3) δ 4.88 – 4.81 (m, 2H), 3.45 – 3.43 (dd, J = 27.9, 16.8 Hz, 5H), 2.94 – 2.69 (m, 6H), 2.32 – 2.27 (dd, J = 14.3, 6.9 Hz, 4H), 2.16 – 2.11 (m, 4H), 1.69 – 1.46 (m, 16H), 1.40 – 1.25 (m, 52H), 0.89 – 0.86 (t, 12H).
1.13 Synthesis of YK-1506
Step 1 Synthesis of YK-1506-PM1
INT-1 (0.96 g,6.65 mmol) was dissolved in DMF (20 mL), INT-5 (1.48 g,3.32 mmol) and potassium carbonate (1.84 g,13.31 mmol) were added sequentially, the temperature was raised to 70℃for 7h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain YK-1506-PM1（0.24g,0.46mmol,7.0%）.C31H62N2O3, MS(ES):m/z(M+H+)511.4.
Step 2 Synthesis of YK-1506
YK-1506-PM1 (200 mg,0.39 mmol) was dissolved in DMF (5 mL), then INT-3 (180 mg,0.58 mmol), potassium carbonate (162 mg,1.17 mmol) and potassium iodide (64 mg,0.39 mmol) were added sequentially, the temperature was raised to 70℃for 8h, and LCMS was monitored until the starting material was reacted. Heating was stopped, filtration was performed, the filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to give YK-1506 (40 mg,0.05mmol, 13.8%).
C45H88N2O5, MS(ES):m/z(M+H+)737.6.1H NMR (400 MHz, CDCl3) δ 4.14 – 4.01 (m, 3H), 3.96 (d, J = 5.8 Hz, 2H), 3.52 – 3.23 (m, 2H), 3.17 (dd, J = 14.5, 6.6 Hz, 1H), 2.97 – 2.52 (m, 4H), 2.55 – 2.35 (m, 3H), 2.34 – 2.26 (m, 2H), 2.03 – 1.91 (m, 1H), 1.87 (dd, J = 22.5, 5.4 Hz, 3H), 1.61 (ddd, J = 18.0, 13.4, 5.4 Hz, 6H), 1.47 – 1.37 (m, 2H), 1.32 – 1.25 (m, 39H), 0.89 – 0.86 (s, 9H).
1.14 Synthesis of YK-1507
Step 1 Synthesis of YK-1507
INT-I (0.96 g,6.65 mmol) was dissolved in DMF (20 mL), then INT-5 (1.48 g,3.32 mmol) and potassium carbonate (1.84 g,13.31 mmol) were added sequentially, the temperature was raised to 70℃for 7h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain YK-1507（0.12g,0.13mmol,2.0%）.C55H108N2O5, MS(ES):m/z(M+H+)877.8.1H NMR (400 MHz, CDCl3) δ 3.96 (d, J = 5.8 Hz, 4H), 2.61 – 2.41 (m, 4H), 2.34 – 2.26 (m, 4H), 2.06 – 1.99 (m, 3H), 1.70 – 1.56 (m, 7H), 1.51 – 1.40 (m, 4H), 1.34 – 1.26 (m, 62H), 0.89 – 0.86 (t, 12H).
1.15 Synthesis of YK-1508
Step 1 Synthesis of YK-1508
INT-I (200 mg,1.38 mmol) was dissolved in DMF (20 mL), then bromolinolene (348 mg,1.38 mmol), potassium carbonate (383 mg,2.77 mmol) and potassium iodide (46 mg,0.27 mmol) were added sequentially, the temperature was raised to 70℃for 8h, and LCMS was monitored until the starting material was reacted. Stopping heating, filtering, concentrating the filtrate under reduced pressure, purifying the residue by silica gel column chromatography, collecting the product, concentrating to obtain YK-1508（80mg,0.12mmol,8.9%）.C43H80N2O, MS(ES):m/z(M+H+)641.6.1H NMR (400 MHz, CDCl3) δ 5.42 – 5.29 (m, 8H), 3.30 (s, 2H), 3.00 (d, J = 8.3 Hz, 1H), 2.85 – 2.75 (m, 7H), 2.10 – 2.02 (m, 11H), 1.65 – 1.61 (m, 3H), 1.42 – 1.22 (m, 34H), 0.90 – 0.85 (s, 6H).
1.16 Synthesis of YK-1509
Step 1 Synthesis of YK-1509-PM1
INT-7 (1.00 g, crude) and INT-2 (1.60 g, 3.90 mmol) were dissolved in acetonitrile (10 mL), potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature and the reaction stirred at 75 oC for LCMS monitoring. After the reaction is finished, the mixture is filtered, the filter cake was washed with a small amount of acetonitrile. After the filtrate was spin-dried, the product YK-1509-PM1 (200 mg, 0.41 mmol, two step yield 10.5%), C29H55N3O3,MS (ES): m/z (M + H+) = 494.80 was purified by silica gel chromatography (0% -50% methanol/dichloromethane with 10% aqueous ammonia).
Step 2 Synthesis of YK-1509
YK-1509-PM1 (100 mg, 0.20 mmol) and INT-3 (75 mg, 0.24 mmol) were dissolved in acetonitrile (2 mL), potassium carbonate (84 mg, 0.60 mmol) and potassium iodide (7 mg, 0.040 mmol) were added at room temperature and the reaction was stirred at 75, oC, and LCMS monitored. After the reaction is finished, the mixture is filtered, the filter cake was washed with a small amount of acetonitrile. The filtrate is dried by spin, and purified by silica gel chromatography (0% -50% methanol/dichloromethane containing 10% ammonia water) to obtain the product YK-1509（35 mg, 0.049 mmol, 24.3%）,C43H81N3O5,MS (ES): m/z (M + H+) = 721.15.
1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.41 (s, 1H), 4.27 - 4.18 (m, 2H), 4.11 - 4.04 (m, 2H), 3.99 - 3.86 (m, 3H), 3.62 - 3.51 (m, 2H), 3.49 - 3.37 (m, 2H), 3.18 - 3.02 (m, 5H), 2.90 - 2.82 (m, 3H), 2.51 - 2.43 (m, 2H), 2.37 - 2.27 (m, 2H), 2.22 - 2.12 (m, 1H), 2.01 - 1.90 (m, 1H), 1.71 - 1.56 (m, 6H), 1.34 - 1.22 (m, 38H), 0.92 - 0.84 (m, 9H).
1.17 Synthesis of YK-1510
Step 1 Synthesis of YK-1509-PM1
INT-7 (1.28 g, crude) and INT-2 (4.20 g, 10.02 mmol) were dissolved in acetonitrile (10 mL), potassium carbonate (3.60 g, 26.05 mmol) and potassium iodide (172 mg, 1.04 mmol) were added at room temperature, the reaction was stirred at 75 oC, and LCMS monitored. After the reaction is finished, the mixture is filtered, the filter cake was washed with a small amount of acetonitrile. After the filtrate was spin-dried, the product YK-1510 (550 mg, 0.66 mmol, 13.2% two step yield), C51H97N3O5,MS (ES): m/z (M + H+) = 833.35 was purified by silica gel chromatography (0% -50% methanol/dichloromethane with 10% aqueous ammonia).
1H NMR (400 MHz, CDCl3) δ 7.56 (d,J= 17.6 Hz, 2H), 4.28 (t,J= 7.5 Hz, 4H), 4.01 - 3.90 (m, 5H), 2.82 (s, 3H), 2.38 - 2.26 (m, 5H), 1.94 - 1.83 (m, 4H), 1.73 - 1.55 (m, 7H), 1.50 - 1.38 (m, 5H), 1.35 - 1.17 (m, 49H), 0.94 - 0.83 (m, 12H).
1.18 Synthesis of YK-1511
Step 1 Synthesis of YK-1511
INT-7 (150 mg, crude) and INT-4 (512 mg, 1.18 mmol) were dissolved in acetonitrile (2 mL), potassium carbonate (361 mg, 2.61 mmol) and potassium iodide (17.3 mg, 0.10 mmol) were added at room temperature, the reaction was stirred at 75 oC and monitored by LCMS. After the reaction is finished, the mixture is filtered, the filter cake was washed with a small amount of acetonitrile. After the filtrate was spin-dried, the product YK-1511 (8 mg, 0.044 mmol, two step yield 7.5%) was purified by silica gel chromatography (0% -50% methanol/dichloromethane with 10% aqueous ammonia), C53H101N3O5,MS (ES): m/z (M + H+) = 861.40.
1H NMR (400 MHz, CDCl3) δ 7.54 (d,J= 17.6 Hz, 2H), 4.95 - 4.75 (m, 2H), 4.19 - 4.00 (m, 3H), 3.56 - 2.93 (m, 3H), 2.86 - 2.76 (m, 3H), 2.38 - 2.20 (m, 4H), 2.07 - 1.82 (m, 3H), 1.73 - 1.58 (m, 6H), 1.56 - 1.38 (m, 12H), 1.34 - 1.17 (m, 50H), 0.95 - 0.76 (m, 12H).
1.19 Synthesis of YK-1512
Step 1 Synthesis of YK-1512-PM1
INT-7 (1.00 g, crude) and INT-5 (1.75 g, 3.92 mmol) were dissolved in DMF (10 mL), potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature and the reaction stirred at 75 oC for LCMS monitoring. After the reaction, pure water was added to dilute, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated aqueous sodium chloride solution, the organic phases were dried over anhydrous sodium sulfate, the solvent was spin-dried under reduced pressure, and the product YK-1512-PM1 (300 mg, 0.57 mmol, two-step yield 14.5%) was purified by silica gel chromatography (0% -50% methanol/dichloromethane with 10% ammonia) to give C31H59N3O3,MS (ES): m/z (M + H+) = 523.03.
Step 2 Synthesis of YK-1512
YK-1512-PM1 (150 mg, 0.29 mmol) and INT-3 (89 mg, 0.29 mmol) were dissolved in DMF (2 mL), potassium carbonate (120 mg, 0.87 mmol) and potassium iodide (7 mg, 0.040 mmol) were added at room temperature and the reaction was stirred 75, oC and monitored by LCMS. After the reaction, adding pure water for dilution, extracting with ethyl acetate, merging the organic phases, washing with saturated sodium chloride aqueous solution, drying the organic phase with anhydrous sodium sulfate, drying the solvent by spin-drying under reduced pressure, and purifying by silica gel chromatography (0% -50% methanol/dichloromethane containing 10% ammonia water) to obtain the product YK-1512（60 mg, 0.080 mmol, 27.6%）,C45H85N3O5,MS (ES): m/z (M + H+) = 749.46.
1H NMR (400 MHz, CDCl3) δ 6.99 - 6.87 (m, 2H), 4.10 - 4.03 (m, 4H), 3.99 - 3.91 (m, 4H), 2.45 (s, 3H), 2.35 - 2.26 (m, 4H), 1.67 - 1.57 (m, 7H), 1.48 - 1.38 (m, 2H), 1.37 - 1.21 (m, 49H), 0.93 - 0.84 (m, 9H).
1.20 Synthesis of YK-1513
Step 1 Synthesis of YK-1513
INT-7 (500 mg, crude) and INT-5 (1.75 g, 3.92 mmol) were dissolved in DMF (10 mL), potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature, the reaction was stirred at 75 oC and monitored by LCMS. After the reaction, pure water was added to dilute, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated aqueous sodium chloride solution, the organic phases were dried over anhydrous sodium sulfate, the solvent was spin-dried under reduced pressure, and the product YK-1513 (100 mg, 0.11: 0.11 mmol, two-step yield 5.6%) was purified by silica gel chromatography (0% -50% methanol/dichloromethane with 10% ammonia) to give C55H105N3O5,MS (ES): m/z (M + H+) = 889.41.
1H NMR (400 MHz, CDCl3) δ 6.96 (d,J= 15.0 Hz, 2H), 3.99 (d,J= 5.8 Hz, 4H), 3.95 - 3.82 (m, 2H), 2.61 - 2.49 (m, 2H), 2.49 - 2.43 (m, 4H), 2.38 - 2.27 (m, 4H), 1.70 - 1.60 (m, 6H), 1.55 - 1.39 (m, 5H), 1.37 - 1.21 (m, 63H), 0.96 - 0.84 (m, 12H).
1.21 Synthesis of YK-1514
Step 1 Synthesis of YK-1514
INT-7 (200 mg, crude) and Compound A (516 mg, 1.57 mmol) were dissolved in DMF (2 mL), potassium carbonate (434 mg, 3.14 mmol) and potassium iodide (33 mg, 0.20 mmol) were added at room temperature, the reaction was stirred at 75 oC and monitored by LCMS. After the reaction, pure water was added to dilute, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated aqueous sodium chloride solution, the organic phases were dried over anhydrous sodium sulfate, the solvent was dried by spin-drying under reduced pressure, and the product YK-1514 (20 mg, 0.14: 0.14 mmol, two-step yield 3.1%) was obtained by purification through silica gel chromatography (0% -50% methanol/dichloromethane with 10% ammonia) and C43H77N3O,MS (ES): m/z (M + H+) = 653.03.
1H NMR (400 MHz, CDCl3) δ 6.94 (d,J= 15.0 Hz, 2H),5.48 - 5.32 (m, 8H), 3.34 (s, 4H), 3.03 (d,J= 6.4 Hz, 2H), 2.86 (d,J= 6.8 Hz, 3H), 2.42 (s, 3H), 2.17 – 2.03 (m, 11H), 1.67 (s, 3H), 1.44 - 1.24 (m, 34H), 0.92 (t,J= 6.8 Hz, 6H).
1.22 Synthesis of YK-1515
Step 1 Synthesis of YK-1515-PM1
Starting from (S) -1-amino-3-chloro-2-propanol hydrochloride (10.00 g, 68.49 mmol), (Boc)2 O (55.30 g,253.40 mmol), TEA (27.72 g, 273.90 mmol) in dichloromethane (50.0, mL) at 40 ℃ for 24 hours, the spotting reaction was complete. The reaction solution was slowly added to a saturated aqueous sodium bicarbonate solution, stirred for 10 minutes, separated, extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by spin-drying. Wet loading, n-hexane/ethyl acetate=0-18%, spin drying to obtain YK-1515-PM1 (9.1 g, 43.40 mmol, 63.3%).
Step 2 Synthesis of YK-1515-PM2
YK-1515-PM1 (3.10 g, 14.78 mmol), N-methylpiperazine (1.77 g, 17.74 mmol), K2CO3 (6.13 g, 44.35 mmol) and KI (0.49 g,2.95 mmol) were used as raw materials, dissolved in acetonitrile (40.0 mL), heated to 70 ℃, reacted for 12 hours and the spot-plating reaction was complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, wherein dichloromethane/methanol=0-15%, and spin-drying to obtain YK-1515-PM2(3.28 g, 12.00 mmol, 81.1%).C13H27N3O3, MS(ES): m/z(M+H+)274.2.
Step 3 Synthesis of YK-1515-PM3
YK-1515-PM2 (3.28 g, 12.00 mmol) was weighed into a 1,4 dioxane solution (30 ml) of hydrochloric acid slowly at room temperature and reacted at room temperature for 12 hours with complete spot plate reaction. Spin-drying to obtain crude YK-1515-PM3 (3.05 g, crude). C8H19N3O, MS(ES): m/z(M+H+) 174.2.
Step 4 Synthesis of YK-1515-PM4 with YK-1515
YK-1515-PM3(0.83 g, 3.95 mmol)、INT-2(1.32 g, 3.16 mmol)、K2CO3(1.64 g, 11.87 mmol)、KI(131 mg,0.79 mmol) Is taken as a raw material, dissolved in acetonitrile (20.0 mL), heated to 70 ℃, reacted for 12 hours, and the spot-plate reaction is complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, wherein dichloromethane/methanol=0-16%, spin-drying to obtain YK-1515-PM4 (582 mg, 1.14 mmol, 28.7%).C30H61N3O3, MS(ES): m/z(M+H+)512.2. dichloromethane/methanol=0-8%, spin-drying to obtain YK-1515 (370 mg, 0.44 mmol, 10.9%).C52H103N3O5, MS(ES): m/z(M+H+)850.4.
1H NMR (400 MHz, CDCl3) δ 4.00 (d,J= 5.8 Hz, 4H), 3.13 (s, 11H), 2.74 (s, 3H), 2.62 (d,J= 7.6 Hz, 2H), 2.37 (t,J= 7.3 Hz, 4H), 1.83 (s, 5H), 1.70 (m,J= 24.2, 8.2 Hz, 7H), 1.44 (m,J= 14.6, 7.1 Hz, 5H), 1.30 (s, 49H), 0.92 (t,J= 6.7 Hz, 12H).
1.23 Synthesis of YK-1516
Step 1 Synthesis of YK-1516
YK-1515-PM3(100 mg, 0.47 mmol)、INT-2 (413 mg, 0.95 mmol)、K2CO3(197 mg, 1.43 mmol)、KI(15.8 mg,0.09 mmol) Is taken as a raw material, dissolved in acetonitrile (5.0 mL), heated to 70 ℃, reacted for 12 hours, and the spot-plate reaction is complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, wherein dichloromethane/methanol=0-39%, and spin-drying to obtain YK-1516 (55 mg, 0.06 mmol, 13.1%).C54H107N3O5, MS(ES): m/z(M+H+)879.2.
1H NMR (400 MHz, CDCl3) δ 4.93 - 4.81 (m, 2H), 2.71 (d,J= 73.1 Hz, 11H), 2.57 - 2.43 (m, 5H), 2.33 (t,J= 7.4 Hz, 4H), 1.82 - 1.58 (m, 9H), 1.54 (d,J= 5.2 Hz, 9H), 1.47 - 1.19 (m, 54H), 0.91 (t,J= 6.6 Hz, 12H).
1.24 Synthesis of YK-1517
Step 1 Synthesis of YK-1517
YK-1515-PM3(512 mg, 1.00 mmol)、INT-3(307 mg, 1.00 mmol)、K2CO3(414 mg, 3.00 mmol)、KI(33 mg,0.20 mmol) Is taken as a raw material, dissolved in acetonitrile (10.0 mL), heated to 70 ℃, reacted for 8 hours, and the spot-plate reaction is complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, wherein dichloromethane/methanol=0-16%, and spin-drying to obtain YK-1517 (133 mg, 0.18 mmol, 18.0%).C44H87N3O5, MS(ES): m/z(M+H+)738.2.
1H NMR (400 MHz, CDCl3) δ 4.08 (t,J= 6.8 Hz, 2H), 3.99 (d,J= 5.8 Hz, 2H), 2.81 - 2.38 (m, 18H), 2.38 - 2.28 (m, 4H), 1.80 (m,J= 12.5, 5.4 Hz, 2H), 1.72 - 1.58 (m, 5H), 1.51 (m,J= 13.4, 6.5 Hz, 3H), 1.31 (d,J= 14.3 Hz, 41H), 0.91 (t,J= 6.6 Hz, 9H).
1.25 Synthesis of YK-1518
Step 1 Synthesis of YK-1518-PM1
Starting from 2-hexyl N-decanol (5.00 g, 20.62 mmol), N-Boc-6-aminocaproic acid (5.70 g, 24.64 mmol), EDCI (5.00 g, 26.60 mmol), DMAP (756 mg, 6.22 mmol) and dichloromethane (50.0 mL) were dissolved and reacted at room temperature for 12 hours to complete the spot-plating reaction. Wet loading, n-hexane/ethyl acetate=0-50%, spin drying to obtain YK-1518-PM1 (8.6 g, 18.87 mmol, 91.5%).C27H53NO4, MS(ES): m/z(M+H+) 456.2.
Step 2 Synthesis of YK-1518-PM2
YK-1518-PM1 (8.60 g, 18.87 mmol) was weighed into HCl/dioxane (50 ml) slowly at room temperature and reacted at room temperature for 12 hours with complete spot plate reaction. Spin drying to obtain crude product YK-1518-PM2 (6.50 g, 18.28 mmol, 96.8%).C22H45NO2, MS(ES): m/z(M+H+) 356.2.
Step 3 Synthesis of YK-1518-PM3
YK-1518-PM2 (2.00 g, 5.62 mmol), INT-2 (2.12 g, 5.06 mmol) and K2CO3 (2.33 g, 26.60 mmol) are taken as raw materials, dissolved in acetonitrile (20.0 mL), heated to 70 ℃, reacted for 12 hours, and the spot-plating reaction is complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, wherein dichloromethane/methanol=0-10%, and spin-drying to obtain YK-1518-PM3(1.6 g, 2.31 mmol, 40.9%).C45H89NO4, MS(ES): m/z(M+H+) 694.3.
Step 4 Synthesis of YK-1518-PM4
CDI (555 mg, 3.44 mmol) was weighed and dissolved in methylene chloride (10.0 mL), YK-1518-PM3 (600 mg, 0.98 mmol) was slowly added at 0℃and the temperature was returned to room temperature after the addition, stirred for 5 hours, and the spot-plating reaction was completed. Stopping the reaction, adding water for three times, extracting with dichloromethane, drying, filtering, and spin-drying. Obtaining the product YK-1518-PM4(350 mg, 0.50 mmol, 51.4%).C48H89N3O5, MS(ES): m/z(M+H+) 788.6.
Step 5 Synthesis of YK-1518
YK-1518-PM4(350 mg, 0.50 mmol)、YK-1515-PM3(182 mg, 1.24 mmol)、K2CO3(207 mg, 1.50 mmol) Is taken as a raw material, dissolved in acetonitrile (5.0 mL), heated to 70 ℃, reacted for 12 hours, and the spot-plate reaction is complete. The reaction was stopped, filtered, washed with dichloromethane and dried by spin. Dissolving a small amount of dichloromethane, loading by wet method, performing spin drying on dichloromethane/methanol (10% ammonia water) =0-10% to obtain YK-1518(15 mg, 0.02 mmol, 3.8%).C53H104N4O6, MS(ES): m/z(M+H+) 893.7.
1H NMR (400 MHz, CDCl3) δ 6.53 (s, 1H), 4.7 (d,J= 5.8 Hz, 4H), 3.23 (s, 11H), 2.75 (s, 3H), 2.71 (d,J= 7.6 Hz, 2H), 2.43 (t,J= 7.3 Hz, 4H), 1.83 (s, 5H), 1.75 (m,J= 24.2, 8.2 Hz, 8H), 1.42 (m,J= 14.6, 7.1 Hz, 4H), 1.25-1.34 (m, 49H), 0.92 (t,J= 6.7 Hz, 12H).
1.26 Synthesis of C16
Synthesized according to the procedure for the synthesis of C16 in CN 202380010167.3, 44mg of C16 were obtained.
1.27 Synthesis of 9322-O17S
According to the synthetic route 9322-O17S in Imidazole-Based Synthetic Lipidoids for In Vivo mRNA Delivery into Primary T Lymphocytes, Xuewei Zhao et al, ANGEW CHEM INT ED Engl, 59 (45): 20083-20089, 55mg of 9322-O17S were synthesized.
1.28.76-017 Se synthesis
38Mg of 76-O17Se were synthesized according to the method for synthesizing 76-O17Se in In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery, Zhongfei Ye et al, ACS Biomater Sci Eng, 2022 Feb 14;8 (2): 722-733.
Example 2 mRNA-LNP formulation optimization
Specific procedures for the cell transfection experiments employed in this example include:
And 1, cell resuscitating and passaging, namely resurrecting Jurkat cells, and culturing and passaging the Jurkat cells in a culture dish until the number of the cells is required.
Step2, plating, namely digesting and counting cells in a culture dish, plating 15 ten thousand cells per hole in a 12-hole plate, and culturing overnight until the cells adhere to the wall.
Step 3, cell transfection, namely adding mRNA-LNP preparations encapsulated with eGFP-mRNA by different carriers into a cell culture solution of a 12-well plate (1.5 mug of mRNA-LNP preparation is added to each well), continuously culturing for 24 hours, observing by a fluorescence microscope, and observing the transfection efficiency according to the fluorescence intensity.
2.1 Vector (liposome) and mRNA ratio optimization
Step 1 the cationic lipids YK-1503, YK-1504, YK-1505 and YK-1507 synthesized in example 1 were dissolved in ethanol with DSPC (Ai Weita (Shanghai) medical science Co., ltd.), cholesterol (Ai Weita (Shanghai) medical science Co., ltd.) and DMG-PEG2000, respectively, according to the molar ratio of DSPC to cholesterol to DMG-PEG2000 of 49:10:39.5:1.5, to obtain a solution A. Solution a was added rapidly to citrate buffer (ph=4.5±0.5) by ethanol injection and vortexed for 30s to obtain an ethanol lipid solution.
Step 2. EGFP-mRNA (Shanghai laboratory Start. Reagent Co., ltd.) was diluted in citrate buffer (pH=4.5.+ -. 0.5) to give an aqueous eGFP-mRNA solution.
And 3, mixing the ethanol lipid solution prepared in the step 1 with the mRNA aqueous solution prepared in the step 2 at a flow rate of 10 mL/min by using a microfluidic device according to the mass ratio of the carrier to the mRNA of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1 to prepare a corresponding liposome solution. After dilution of the liposome solution to 10 volumes using PBS, ultrafiltration was performed using a 300 KDa ultrafiltration tube to remove ethanol. The mixture was then subjected to PBS to a proper volume and filtered through a 0.2 μm sterile filter to obtain a cationic lipid (YK-1503, YK-1504, YK-1505, or YK-1507)/DSPC/cholesterol/DMG-PEG 2000 in a molar ratio of 49:10:39.5:1.5, respectively, as an eGFP-mRNA-encapsulating mRNA-LNP preparation.
The results of cell transfection experiments show that when the mass ratio of the vector to the mRNA is in the range of 10:1-30:1, the corresponding mRNA-LNP composition has a good transfection effect, wherein the transfection effect is preferably 15:1, and when the mass ratio is 5:1 and 35:1, the proper transfection effect cannot be obtained.
2.2 Cationic lipid and neutral lipid ratio optimization
MRNA-LNP compositions encapsulating eGFP-mRNA were prepared in analogy to 2.1, with the molar ratios of cationic lipids (YK-1503, YK-1504, YK-1505 or YK-1507) to neutral lipid DSPC adjusted to 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.
Cell transfection experiments show that when the molar ratio of cationic lipid to neutral lipid is in the range of 1:1-15:1, the corresponding mRNA-LNP composition is capable of transfecting cells, wherein the transfection efficiency is 4.9:1 at the highest.
2.3 Optimization of carrier proportion of polymer conjugated lipid
MRNA-LNP compositions encapsulating eGFP-mRNA were prepared in analogy to the procedure in 2.1, wherein the cationic lipids were YK-1503, YK-1504, YK-1505 or YK-1507 and the polymer conjugated lipid DMG-PEG2000 was present in the carrier in 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15%, respectively.
Cell transfection experiment results show that when the mole percentage of the polymer conjugated lipid in the carrier is in the range of 0.5% -10%, the corresponding mRNA-LNP composition can be used for transfecting cells, and the transfection efficiency is highest at 1.5% and lowest at 10%.
2.4 Optimization of the ratio of the components in the carrier
MRNA-LNP preparations encapsulating eGFP-mRNA were prepared in analogy to the procedure in 2.1, with cationic lipids (YK-1503, YK-1504, YK-1505 or YK-1507), neutral lipids DSPC, structured lipid cholesterol and polymer conjugated lipid DMG-PEG2000 in molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5, respectively, in step 1.
As can be seen from cell transfection experiments, the cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid have good transfection effect in the range of the ratio of (35-49): (7.5-15): (35-55): (1-5) in the molar ratio of (75:5:15:5), 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5, and the transfection effect is the best when the molar ratio is 49:10:39.5:1.5.
Example 3 cell transfection of mRNA-LNP preparation encapsulating eGFP-mRNA
Specific procedures for cell transfection employed in this example include:
And 1, cell resuscitating and passaging, namely resurrecting Jurkat cells, and culturing and passaging the Jurkat cells in a culture dish until the number of the cells is required.
Step2, plating, namely digesting and counting cells in a culture dish, plating 15 ten thousand cells per hole in a 12-hole plate, and culturing overnight until the cells adhere to the wall.
Step 3 cell transfection the transfection efficiency was examined by fluorescence intensity by observation under a fluorescence microscope after adding 1.5. Mu.g of the mRNA-LNP preparation prepared in example 2 and encapsulating eGFP-mRNA, wherein the cationic lipid was YK-1503, YK-1504, YK-1505 or YK-1507, respectively, to the cell culture solution of the 12-well plate, followed by further culturing for 24 hours.
Based on transfection efficiency results, an mRNA-LNP preparation with a carrier to mRNA mass ratio of 15:1, a cationic lipid to neutral lipid molar ratio of 4.9:1, a polymer conjugated lipid molar ratio of 1.5% to liposomes, and a cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid molar ratio of 49:10:39.5:1.5 was selected for the examples below.
EXAMPLE 4 preparation of mRNA-LNP preparation
TABLE 1 cationic lipid Compounds
4.1
The corresponding ethanolic lipid solutions of the cationic lipids in table 1 were prepared according to the method in step 1 of example 2.1.
4.2
EGFP-mRNA (Shanghai Start-Up reagent Co., ltd.) or Fluc-mRNA (Shanghai Start-Up reagent Co., ltd.) was diluted in citrate buffer (pH=4.5.+ -. 0.5) to give a corresponding aqueous mRNA solution.
4.3
The ethanol lipid solution obtained in 4.1 was mixed with the eGFP-mRNA aqueous solution or Fluc mRNA aqueous solution obtained in 4.2 at a volume ratio of 1:3 using a microfluidic device at a flow rate of 10 mL/min, and the corresponding liposome solution was prepared at a mass ratio of carrier (liposome) to mRNA of about 15:1. After dilution of the liposome solution to 10 volumes using PBS, ultrafiltration was performed using a 300 KDa ultrafiltration tube to remove ethanol. And then the mixture is subjected to PBS volume fixation to a proper volume, and filtered by a 0.2 mu m sterile filter to obtain the mRNA-LNP preparation encapsulated with eGFP-mRNA or Fluc-mRNA, wherein the molar ratio of the cationic lipid to DSPC to cholesterol to DMG-PEG2000 is 49:10:39.5:1.5.
Example 5 determination of mRNA-LNP particle size, polydispersity index (PDI) and encapsulation efficiency
Particle size and Polydispersity (PDI) were determined using dynamic light scattering using a malvern laser particle sizer.
10 Μl of the mRNA-LNP solution prepared in example 4 was diluted to 1mL with RNase-free deionized water, and added to the sample well, and the measurement was repeated 3 times for each sample. The measurement conditions are 90 ℃ scattering angle and 25 ℃;
the encapsulation efficiency of LNP was determined using the Quant-iTTM RiboGreen RNA quantification kit (Thermo FISHER SCIENTIFIC, UK) according to the manufacturer's instructions and the results of the assay are shown in Table 2:
TABLE 2 mRNA particle size, polydispersity (PDI), encapsulation efficiency of LNP
As can be seen from Table 2, the nanolipid particles prepared in example 4, having a particle size between 70-90 nm, can be used to deliver mRNA. The polydispersity numbers were all less than 0.15, indicating good particle size uniformity. And has high encapsulation efficiency, and the encapsulation efficiency is more than 90 percent.
Example 6 in vitro (in vitro) delivery Performance and toxicity of LNP
Methods of cell resuscitating and passaging and plating refer to step 1 and step 2 of example 3.
The 96-well plate obtained in step 2, in which Jurkat cells were cultured, was supplemented with an appropriate volume of Jurkat cell culture solution, and after adding mRNA-LNP preparation (prepared in example 4) containing 0.3. Mu.g of Fluc-mRNA to the 96-well plate and continuing to culture for 24h, a corresponding reagent was added according to Gaussia Luciferase Assay Kit (Thermo Fisher) instructions, the relative fluorescence intensity of each well was detected by an IVIS fluorescence detection system, and finally 10. Mu.L of CCK-8 solution was added to each well of the above-mentioned 24-hour-cultured well plate, and after incubating the culture plate in an incubator for 1 hour, the absorbance at 450 nm was measured by a microplate reader, thereby detecting the cell viability. The results of relative fluorescence intensity and cell viability are shown in Table 3.
TABLE 3 fluorescent detection results of Fluc-mRNA
The relative fluorescence intensities of the above mRNA-LNP compositions (which may be indicative of translation efficiency of the mRNA) were significantly different, and the relative fluorescence intensities of the mRNA-LNP compositions prepared from YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 were significantly higher than those of the mRNA-LNP compositions prepared from SM-102, MC3, 9322-O17S, 76-017Se and C16, specifically:
1. The cell transfection efficiency of the mRNA-LNP composition prepared by YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 is remarkably improved compared with that of the representative cationic lipid in the prior art. For example, YK-1507 can achieve 2.8 times of SM-102 and 6.0 times of MC 3.
The cell transfection efficiency of the mRNA-LNP composition prepared by YK-1503, YK-1504, YK-1505 and YK-1507 is obviously improved compared with that of 76-017Se cationic lipid with the same tetrahydropyrrole head structure. For example, YK-1507 can be transfected with 4.9 times as high as 76-017 Se.
Cell transfection efficiency of mRNA-LNP compositions prepared by YK-1510, YK-1511, YK-1513 was significantly improved compared to 9322-O17S cationic lipids also having a 2-methylimidazole head structure. For example, YK-1513 can be transfected with 4.6 times as much as 9322-O17S.
The cell transfection efficiency of the mRNA-LNP composition prepared by YK-1515 and YK-1516 is remarkably improved compared with that of the C16 cationic lipid with piperazine head structure. For example, YK-1516 can be transfected with 1.8 times as much as C16.
Example 7 in vivo (in vivo) delivery Performance of LNP
The Fluc-mRNA-LNP composition prepared in example 4 was injected into female BALB/c albino mice of age 4-6 weeks and weight 17-19 g (amount of Fluc-mRNA/mouse is about 5 μg) by tail vein, and a fluorogenic substrate was injected into the mice by intraperitoneal injection after 6 hours of administration, and the mice were free to move for 5 minutes, and then the total radiation intensity (corresponding to the fluorescent protein expression intensity, i.e., protein expression amount) of the protein expressed in the mice by mRNA carried by the mRNA-LNP composition was detected by IVIS Spectrum small animal in vivo imager. After the sampling is completed, the mice are sacrificed and dissected, and the internal organs liver and spleen of the mice are precisely separated. The total radiation intensity (corresponding to the fluorescent protein expression intensity, i.e., protein expression level) of the protein expressed by the Fluc-mRNA in each organ of the mice was examined by an IVIS Spectrum small animal in vivo imager. The results of in vivo imaging of mice and detection of protein expression in the liver and spleen are shown in Table 4 and FIGS. 1-3.
TABLE 4 Experimental data on imaging of mice living and organs
MRNA-LNP compositions prepared from YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 can deliver mRNA to the spleen with high efficiency, with significantly enhanced delivery compared to SM-102, MC3, 9322-O17S, 76-017Se and C16. Specifically, the mRNA-LNP compositions prepared from YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 of the present disclosure, respectively, were significantly enhanced in spleen total radiation intensity and in vivo total radiation intensity, such as the mRNA-LNP compositions prepared from YK-1507, 1.8 times, 2.2 times, 2.4 times, and 1.9 times, respectively, compared to the mRNA-LNP compositions prepared from the prior art ionizable cationic lipids (SM-102, MC3, 9322-O17S, 76-017Se, C16 and YK-301), respectively, the mRNA-LNP compositions prepared from SM-102, MC3, 9322-O17S, 76-017Se and C16 and prepared from the present disclosure, respectively, were 1.8 times, 2.2 times, 2.4 times, and 1.9 times, respectively, the spleen total radiation intensity was 1.2 times, 2.4 times, 2.7 times, 2 times, and 2.7 times, and 7 times, respectively, as compared to the SM-102, MC-17S, 76-017Se and C16 and YK-301.
Example 8 targeting of mRNA-LNP compositions to mouse spleen cells
1. The eGFP-mRNA compositions of different cationic lipids prepared in example 4 were injected into female C57BL/6 mice of age 4-6 weeks and weight 17-19 g by tail vein (about 20. Mu.g of eGFP-mRNA per mouse) and the mice were sacrificed at 24 hours after administration and dissected to precisely isolate the spleens of the mice.
2. Preparation of single cells
1) The isolated spleen tissue was ground to single-cell spleen tissue and sieved.
2) 10 Times volume (about 4 mL) of erythrocyte lysate is added to lyse and remove the erythrocyte in the tissue.
3) Cell counts, 5×106 cells were taken into the flow tube (ensuring consistent cell count for each sample).
3. Spleen tissue immune cell detection
1) To each single cell suspension 100. Mu.L of surface antibody MIX (surface antibody MIX composition detailed in Table 5, incubated for 15min at room temperature in the dark (one negative control) was added.
TABLE 5 reagents and sources for surface antibody MIX in mouse spleen cell flow experiments
2) 2 ML of PBS was added, and the supernatant was discarded after centrifugation at 500 g for 5 minutes.
3) Cells were resuspended in 200 μl PBS, (after 200 mesh nylon mesh filtration) and detected on-machine by flow cytometer Cytoflex S. The cells were analyzed for percent GFP content. The sequence of each cell line was as follows:
T cell GFP proportion CD45+→CD3+→GFP+
Proportion of B-cell GFP CD45+→CD3-CD19+→GFP+
DC GFP proportion CD45+→CD11c+→GFP+
Macrophage GFP proportion CD45+→F4/80+→GFP+
Mice injected with an equal volume of 0.9% sodium chloride solution were set as a blank.
The percentage of eGFP positive cells in mouse spleen cells is shown in table 6 and fig. 4.
TABLE 6 percent eGFP positive cells for mouse spleen cells (%)
As can be seen from the data in table 6 and fig. 4, the mRNA-LNP composition prepared from the cationic lipid containing the present disclosure can significantly increase the ratio of cells expressing antigen in immune cells (T cells, B cells, DC cells, and macrophages) in spleen, and can significantly increase the ratio of cells expressing antigen in immune cells (T cells, B cells, DC cells, and macrophages) in spleen as compared to the mRNA-LNP composition prepared from SM-102 and MC 3. For example, YK-1507 produced mRNA-LNP increased the ratio of T cells, B cells, DC cells and macrophages in spleen by 1.8-fold, 1.5-fold, 2.4-fold and 5.0-fold compared to MC3 produced mRNA-LNP.
The preferred embodiments of the present disclosure are described in detail above, but the present disclosure is not limited thereto. Within the scope of the technical idea of the present disclosure, various simple modifications may be made to the technical solution of the present disclosure, including that each technical feature is combined in any other suitable manner, and these simple modifications and combinations should also be regarded as the content of the disclosure, which falls within the protection scope of the present disclosure.