CN114668852A - A combination of polyethylene glycol-polypeptide and protein drugs - Google Patents
A combination of polyethylene glycol-polypeptide and protein drugsDownload PDFInfo
- Publication number
- CN114668852A CN114668852ACN202210270700.7ACN202210270700ACN114668852ACN 114668852 ACN114668852 ACN 114668852ACN 202210270700 ACN202210270700 ACN 202210270700ACN 114668852 ACN114668852 ACN 114668852A
- Authority
- CN
- China
- Prior art keywords
- polyethylene glycol
- linker
- integer
- present
- combination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
- C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
- C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
- C07C215/08—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic with only one hydroxy group and one amino group bound to the carbon skeleton
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
- C08G65/48—Polymers modified by chemical after-treatment
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Polymers & Plastics (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
Abstract
Translated fromChinese
Description
Translated fromChinese技术领域technical field
本发明涉及医药技术领域，具体涉及一种聚乙二醇-多肽和蛋白类药物的结合物，特别是一种聚乙二醇-白细胞介素(如白介素2)的结合物。The invention relates to the technical field of medicine, in particular to a combination of polyethylene glycol-polypeptide and protein drugs, in particular to a combination of polyethylene glycol and interleukin (eg, interleukin-2).
背景技术Background technique
目前，在临床上，多肽和蛋白类药物口服给药时，由于进入消化道后容易受各种蛋白酶、肽酶等水解环境破坏，药效降低甚至丧失，如某些药物对胃部有刺激性或不耐酸，易被胃酸破坏，因此不适宜口服，其主要给药途径为注射给药，直接注入人体组织或血管，不经过消化系统和肝脏，不会受到消化液的破坏和食物的影响，药物吸收快，血药浓度升高迅速，给药剂量精确，但在临床应用中，由于注射给药后药物常常会迅速分布全身，对病灶部位如肿瘤组织的靶向性较差，生物利用度不高，药效相对不高，且不良反应出现迅速，应对处理相对困难，另外，常常需要多次给药，且给药时需严格遵守无菌操作原则，需专业人士如医生和护士来操作，不利于病人的依从性，因此药物的临床应用常常遭遇瓶颈。At present, in clinical practice, when polypeptide and protein drugs are administered orally, they are easily damaged by various proteases, peptidases and other hydrolysis environments after entering the digestive tract, and the efficacy of the drugs is reduced or even lost. For example, some drugs are irritating to the stomach. Or not acid-resistant, easily destroyed by gastric acid, so it is not suitable for oral administration. The main route of administration is injection, which is directly injected into human tissues or blood vessels, without going through the digestive system and liver, and will not be damaged by digestive juices and food. The drug is absorbed quickly, the blood drug concentration rises rapidly, and the dosage is accurate. However, in clinical applications, because the drug is often rapidly distributed throughout the body after injection, the targeting of the lesion site such as tumor tissue is poor, and the bioavailability is poor. Not high, the efficacy of the drug is relatively low, and the adverse reactions appear quickly, and it is relatively difficult to deal with it. In addition, multiple doses are often required, and the aseptic operation principle must be strictly adhered to when administering. Professionals such as doctors and nurses are required to operate , is not conducive to patient compliance, so the clinical application of drugs often encounter bottlenecks.
现有技术中科研人员常利用水溶性聚合物如聚乙二醇修饰连接药物，来延长药物的生理半衰期，降低药物的免疫原性和毒性，但药物在体内的释放和药效有时并不理想。实验中发现通过连接子(linker)将水溶性聚合物和药物连接形成聚合物-药物结合物，药物从结合物链上的降解，可以达到缓释和控释的目的，药物在病灶(例如患癌)部位停留更长时间，可降低给药频率，减少患者用药不便。如专利文献CN200680029849.5公开了一种轭合物，其中包括含有可电离氢原子的芳香族部分如芴、间隔部分和水溶性聚合物。In the prior art, researchers often use water-soluble polymers such as polyethylene glycol to modify the linking drugs to prolong the physiological half-life of the drug and reduce the immunogenicity and toxicity of the drug, but the release and efficacy of the drug in the body are sometimes unsatisfactory. . In the experiment, it was found that the water-soluble polymer and the drug are connected to form a polymer-drug conjugate through a linker, and the degradation of the drug from the conjugate chain can achieve the purpose of sustained and controlled release. Cancer) sites stay longer, which can reduce the frequency of administration and reduce the inconvenience of medication for patients. For example, the patent document CN200680029849.5 discloses a conjugate including an aromatic moiety containing ionizable hydrogen atoms such as fluorene, a spacer moiety and a water-soluble polymer.
本发明的发明人经过大量的试验和研究，得到一种多肽和蛋白类药物通过非肽连接子与聚乙二醇的结合物，特别是一种白介素(如白介素2)通过非肽连接子与聚乙二醇的结合物，所述结合物中，药物可以从结合物结构中降解分离，可实现缓释和控释，降低给药频率，大大提高药物的生物利用度和病人的依从性。特别是，发明人对结合物的偶合度进行了更深入的研究，得到偶合度明确的结合物或其混合物，有利于后续药效的优化和药理的研究。The inventor of the present invention has obtained a combination of polypeptide and protein drugs and polyethylene glycol through a non-peptide linker, especially an interleukin (such as interleukin 2) through a non-peptide linker and The conjugate of polyethylene glycol, in which the drug can be degraded and separated from the structure of the conjugate, can achieve sustained and controlled release, reduce the frequency of administration, and greatly improve the bioavailability of the drug and the compliance of patients. In particular, the inventors conducted more in-depth research on the coupling degree of the conjugate, and obtained a conjugate or a mixture thereof with a clear coupling degree, which is beneficial to the subsequent optimization of drug efficacy and pharmacological research.
发明内容SUMMARY OF THE INVENTION
本发明的一个目的是提供一种连接子化合物。An object of the present invention is to provide a linker compound.
本发明的另一个目的是提供一种聚乙二醇与上述连接子的结合物及其衍生物。Another object of the present invention is to provide a combination of polyethylene glycol and the above-mentioned linker and derivatives thereof.
本发明的另一个目的是提供一种聚乙二醇-连接子-药物的结合物。Another object of the present invention is to provide a polyethylene glycol-linker-drug conjugate.
本发明的另一个目的是提供包含上述结合物及其与药学上可接受的载体或添加剂的药物组合物。Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned combination and a pharmaceutically acceptable carrier or additive.
本发明的还一个目的是提供一种上述结合物、药物组合物在制备用于预防和/或治疗疾病的药物中的应用。Still another object of the present invention is to provide an application of the above-mentioned combination and pharmaceutical composition in the preparation of a medicament for preventing and/or treating a disease.
具体地，本发明第一方面提供一种化合物，其具有如下结构：Specifically, a first aspect of the present invention provides a compound having the following structure:
其中，l为1-5的整数，where l is an integer from 1 to 5,
Z选自：-H和羟基保护基团，Z is selected from: -H and a hydroxyl protecting group,
A选自：氨基酸残基、多肽残基、
所述氨基酸选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、脯氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、赖氨酸、精氨酸、半胱氨酸、蛋氨酸、组氨酸、色氨酸、苯丙氨酸和酪氨酸，The amino acid is selected from: glycine, alanine, valine, leucine, isoleucine, serine, threonine, proline, aspartic acid, asparagine, glutamic acid, glutamine amide, lysine, arginine, cysteine, methionine, histidine, tryptophan, phenylalanine and tyrosine,
R1-7、R9-11独立地选自：-H、-F、-Cl、-Br、-I、C1-6的烷基、C1-6的烷氧基、C3-6的环烷基、C1-6的烯基、C6-12的芳基、C6-12芳烷基、C3-12芳族或非芳族的杂环基、C3-12的杂环烷基和-(CH2)l-O-Z，R1-7 and R9-11 are independently selected from: -H, -F, -Cl, -Br, -I, alkyl of C1-6, alkoxy of C1-6, cycloalkane of C3-6 C1-6 alkenyl, C6-12 aryl, C6-12 aralkyl, C3-12 aromatic or non-aromatic heterocyclyl, C3-12 heterocycloalkyl and -(CH2 )l -OZ,
R8和R12独立地选自C1-6的烷基，R8 and R12 are independently selected from C1-6 alkyl,
R13-16独立地选自：-H和C1-6的烷基，R13-16 are independently selected from: -H and C1-6 alkyl,
B为连接基团-B1-B2-，B is a linking group -B1 -B2 -,
其中，B1选自：-(CH2)j-、-NHCO(CH2)j-和-CONH(CH2)j-，j为0-6的整数，wherein, B1 is selected from: -(CH2 )j -, -NHCO(CH2 )j - and -CONH(CH2 )j -, and j is an integer of 0-6,
B2选自：-C＝O、-C＝S、-O-、-S-、-C(O)O-、-C(O)S-、-C(S)O-和-S-S-。B2 is selected from: -C=O, -C=S, -O-, -S-, -C(O)O-, -C(O)S-, -C(S)O- and -SS- .
所述的连接子化合物中，所述的l为1-5的整数，如1、2、3、4、5，优选为1、2或3；更优选为1。In the linker compound, the l is an integer of 1-5, such as 1, 2, 3, 4, 5, preferably 1, 2 or 3; more preferably 1.
所述的连接子化合物中，本领域技术人员可以根据实际需要选择合适的羟基保护基团，如：-CH3、-C(CH3)3、-CH2OCH3、-COCH3、-COC(CH3)3、-CH2CH＝CH2、-Si(CH3)3、
在本发明的一个实施例中，所述的Z为-H。In an embodiment of the present invention, the Z is -H.
本发明的一个实施例中，所述的连接子化合物中，所述A为氨基酸残基，所述氨基酸选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、脯氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、赖氨酸、精氨酸、半胱氨酸、蛋氨酸、组氨酸、色氨酸、苯丙氨酸和酪氨酸，优选自：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺和赖氨酸，更优选自：甘氨酸、丙氨酸和缬氨酸。In an embodiment of the present invention, in the linker compound, the A is an amino acid residue, and the amino acid is selected from: glycine, alanine, valine, leucine, isoleucine, serine , threonine, proline, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, cysteine, methionine, histidine, tryptophan, benzene Alanine and tyrosine, preferably selected from: glycine, alanine, valine, leucine, isoleucine, aspartic acid, asparagine, glutamic acid, glutamine and lysine , more preferably selected from: glycine, alanine and valine.
本发明的另一个实施例中，所述的连接子化合物中，所述A为
在本发明的另一个实施例中，所述的连接子化合物中，所述的A为-(CH2)i-、-(CH2)iNH-和-CO(CR15R16)iNH-中的一种或多种的组合。In another embodiment of the present invention, in the linker compound, the A is -(CH2 )i -, -(CH2 )i NH- and -CO(CR15 R16 )i NH - A combination of one or more of.
优选地，所述的连接子化合物中，所述的R1-7、R9-11独立地选自：-H、-F、-Cl、-Br、-I、C1-6的烷基、C1-6的烷氧基和-(CH2)l-O-Z；更优选自：-H、-F、-Cl、-CH3、-CH2CH3、-CH2CH2CH3、-CH(CH3)2、-C(CH3)3、-OCH3和-(CH2)l-O-Z；进一步优选自：-H、-F、-Cl、-CH3、-OCH3和-(CH2)l-O-Z。Preferably, in the linker compound, the R1-7 and R9-11 are independently selected from: -H, -F, -Cl, -Br, -I, C1-6 alkyl, C1-6 alkoxy and- (CH2 )1- OZ; more preferably selected from: -H, -F,-Cl ,-CH3 ,-CH2CH3 ,-CH2CH2CH3 , -CH (CH3 )2 , -C(CH3 )3 , -OCH3 and -(CH2 )1 -OZ; further preferred from: -H, -F, -Cl, -CH3 , -OCH3 and -( CH2 )l -OZ.
在本发明的一个实施例中，所述的R1-4均为-H。In an embodiment of the present invention, the R1-4 are all -H.
在本发明的一个实施例中，所述的R5-7均为-H。In an embodiment of the present invention, the R5-7 are all -H.
在本发明的一个实施例中，所述的R9-11均为-H。In an embodiment of the present invention, the R9-11 are all -H.
优选地，所述的连接子化合物中，所述R8和R12独立地选自C1-4的烷基，如甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基或叔丁基；更优选地，所述R8和/或R12为甲基。Preferably, in the linker compound, the R8 and R12 are independently selected from C1-4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl group, isobutyl group or tert-butyl group; more preferably, the R8 and/or R12 are methyl groups.
优选地，所述的连接子化合物中，R13-16独立地选自：-H和C1-3的烷基(如甲基、乙基、正丙基或异丙基)。Preferably, in the linker compound, R13-16 is independently selected from: -H and C1-3 alkyl (such as methyl, ethyl, n-propyl or isopropyl).
优选地，所述的连接子化合物中，R15为-H，R16选自：-H、甲基、乙基、正丙基和异丙基。Preferably, in the linker compound, R15 is -H, and R16 is selected from: -H, methyl, ethyl, n-propyl and isopropyl.
在本发明的一个实施例中，所述的R13和/或R14为-H。In an embodiment of the present invention, the R13 and/or R14 is -H.
优选地，所述的连接子化合物中，i为0-3的整数，如0、1、2或3。Preferably, in the linker compound, i is an integer of 0-3, such as 0, 1, 2 or 3.
优选地，所述的连接子化合物中，j为0-3的整数，如0、1、2或3。Preferably, in the linker compound, j is an integer of 0-3, such as 0, 1, 2 or 3.
优选地，在本发明的一个实施例中，式Ⅰ-1中，所述A为-COCH2NH-、-COCH(CH3)NH-、-COCH(CH(CH3)2)NH-。Preferably, in an embodiment of the present invention, in formula I-1, the A is -COCH2 NH-, -COCH(CH3 )NH-, -COCH(CH(CH3 )2 )NH-.
优选地，所述B2选自：-C＝O、-O-、-S-、-C(O)O-、-C(O)S-和-S-S-。Preferably, the B2 is selected from: -C=O, -O-, -S-, -C(O)O-, -C(O)S- and -SS-.
在本发明的一个实施例中，所述的B2为-C(O)O-或-O-。In an embodiment of the present invention, the B2 is -C(O)O- or -O-.
在本发明另一个实施例中，所述的B为-(CH2)jO-，j为0-3的整数，如0、1、2或3。In another embodiment of the present invention, the B is -(CH2 )j O-, and j is an integer of 0-3, such as 0, 1, 2 or 3.
在本发明的一个实施例中，式Ⅰ-2、Ⅰ-3中，所述-B-A-为-OCH2CH2NH-。In one embodiment of the present invention, in formulas I-2 and I-3, the -BA- is -OCH2 CH2 NH-.
在本发明的一个实施例中，所述连接子化合物选自如下结构：In one embodiment of the present invention, the linker compound is selected from the following structures:
本发明另一方面还提供一种聚乙二醇-连接子结合物，其具有如下结构：Another aspect of the present invention also provides a polyethylene glycol-linker combination, which has the following structure:
PEG-X-LPEG-X-L
(Ⅱ)(II)
其中，L为本发明上述连接子，Wherein, L is the above-mentioned linker of the present invention,
PEG为聚乙二醇残基，PEG is a polyethylene glycol residue,
X为连接基团，选自：-(CH2)a-、-(CH2)aCO-、-(CH2)aOCO-、-(CH2)aNHCO-、-NH(CH2)aCO-、-(CH2)aSO2-、-O(CH2)a-、-O(CH2)aCO-、-O(CH2)aOCO-、-O(CH2)aNHCO-和-O(CH2)aSO2-中的一种或多种的组合，a为0-10的整数。X is a linking group selected from: -(CH2 )a -, -(CH2 )a CO-, -(CH2 )a OCO-, -(CH2 )a NHCO-, -NH(CH2 )a CO-, -(CH2 )a SO2 -, -O(CH2 )a -, -O(CH2 )a CO-, -O(CH2 )a OCO-, -O(CH2 )a A combination of one or more of NHCO- and -O(CH2 )a SO2 -, a is an integer of 0-10.
在本发明一个实施例中，所述聚乙二醇-连接子结合物具有如下结构：In one embodiment of the present invention, the polyethylene glycol-linker conjugate has the following structure:
上式Ⅱ-1～Ⅱ-3中，所述R1-12、l、A、B、Z等具有本发明上述定义。In the above formulas II-1 to II-3, the R1-12 , l, A, B, Z, etc. have the above definitions in the present invention.
在本发明的一个实施方式中，所述聚乙二醇-连接子结合物中，所述X选自：-(CH2)a-、-(CH2)aCO-、-(CH2)aNHCO-、-NH(CH2)aCO-、-O(CH2)a-、-O(CH2)aCO-和-O(CH2)aNHCO-中的一种或多种的组合，优选为-(CH2)a-、-(CH2)aCO-或-(CH2)aNHCO-。In one embodiment of the present invention, in the polyethylene glycol-linker conjugate, the X is selected from: -(CH2 )a -, -(CH2 )a CO-, -(CH2 ) of one or more ofa NHCO-, -NH(CH2 )a CO-, -O(CH2 )a -, -O(CH2 )a CO- and -O(CH2 )a NHCO- Combination, preferably -(CH2 )a -, -(CH2 )a CO- or -(CH2 )a NHCO-.
优选地，所述聚乙二醇-连接子结合物中，a为0-5的整数，如0、1、2、3、4或5。Preferably, in the polyethylene glycol-linker conjugate, a is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个实施例中，所述聚乙二醇-连接子结合物中，所述X为单键、-CH2-、-CO-、-CH2CO-或-NHCO-。In an embodiment of the present invention, in the polyethylene glycol-linker conjugate, the X is a single bond, -CH2 -, -CO-, -CH2 CO- or -NHCO-.
优选地，所述聚乙二醇-连接子结合物中，所述PEG为直链、Y型、多分支的聚乙二醇残基，例如包括单甲氧基聚乙二醇(mPEG)、直链双端PEG、Y型PEG、4臂支链PEG、6臂支链PEG或8臂支链PEG等。Preferably, in the polyethylene glycol-linker combination, the PEG is a linear, Y-shaped, multi-branched polyethylene glycol residue, such as monomethoxy polyethylene glycol (mPEG), Straight-chain double-ended PEG, Y-type PEG, 4-arm branched PEG, 6-arm branched PEG or 8-arm branched PEG, etc.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为直链聚乙二醇残基，具有通式Ⅲ或Ⅳ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker combination, the PEG is a linear polyethylene glycol residue, and has the structure shown in the general formula III or IV:
其中，p和q独立地选自1-960的整数，优选为1-480的整数。wherein p and q are independently selected from integers of 1-960, preferably integers of 1-480.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为Y型聚乙二醇残基，具有通式Ⅴ或Ⅵ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker combination, the PEG is a Y-shaped polyethylene glycol residue, and has the structure shown by the general formula V or VI:
其中，i和h独立地选自1-480的整数，优选为1-240的整数。wherein i and h are independently selected from integers of 1-480, preferably integers of 1-240.
在本发明一具体实施方式中，所述聚乙二醇-连接子结合物中，所述PEG为多分支聚乙二醇残基，具有通式Ⅶ所示的结构：In a specific embodiment of the present invention, in the polyethylene glycol-linker combination, the PEG is a multi-branched polyethylene glycol residue and has the structure shown in the general formula VII:
其中，k是1-320的整数，优选为1-240的整数，wherein k is an integer of 1-320, preferably an integer of 1-240,
j是3-8的整数，j is an integer from 3-8,
R是多分支聚乙二醇的核心分子，R选自：季戊四醇、寡聚季戊四醇、甲基葡萄糖苷、蔗糖、二甘醇、丙二醇、甘油和聚甘油的残基；优选地，R选自：甘油、六聚甘油、季戊四醇、二聚季戊四醇和三聚季戊四醇残基。R is the core molecule of multi-branched polyethylene glycol, and R is selected from the residues of pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol, propylene glycol, glycerol and polyglycerol; preferably, R is selected from: Glycerol, hexaglycerol, pentaerythritol, dimerpentaerythritol and trimerpentaerythritol residues.
优选地，所述多分支聚乙二醇残基仅含有一个可连接位点，其具有如下结构：Preferably, the multibranched polyethylene glycol residue contains only one linkable site, which has the following structure:
在本发明一个实施例中，所述多分支聚乙二醇残基具有如下结构：In one embodiment of the present invention, the multi-branched polyethylene glycol residue has the following structure:
其中，k是1-320的整数，优选为1-240的整数，wherein k is an integer of 1-320, preferably an integer of 1-240,
x为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数。x is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-6.
在本发明的另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the present invention, the multi-branched polyethylene glycol residue has the following structure:
其中，k是1-320的整数，优选为1-240的整数，wherein k is an integer of 1-320, preferably an integer of 1-240,
x为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-6的整数。x is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-6.
在本发明另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the present invention, the multi-branched polyethylene glycol residue has the following structure:
其中，k是1-320的整数，优选为1-240的整数，更优选为1-120的整数，wherein, k is an integer of 1-320, preferably an integer of 1-240, more preferably an integer of 1-120,
y为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-5的整数，更优选为1-3的整数。y is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-5, more preferably an integer of 1-3.
在本发明的另一个实施例中，所述多分支聚乙二醇残基具有如下结构：In another embodiment of the present invention, the multi-branched polyethylene glycol residue has the following structure:
其中，k是1-320的整数，优选为1-240的整数，更优选为1-120的整数，wherein, k is an integer of 1-320, preferably an integer of 1-240, more preferably an integer of 1-120,
y为1-10的整数(具体如1、2、3、4、5、6、7、8、9或10)，优选为1-5的整数，更优选为1-3的整数。y is an integer of 1-10 (specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-5, more preferably an integer of 1-3.
本发明中，所述的PEG的分子量可为1-100KDa，例如1-10KDa(具体可为1、2、3、4、5、6、7、8、9、10KDa)、10-50KDa(具体可为10、15、20、25、30、35、40、45、50KDa)、50-100KDa(具体可为50、55、60、65、70、75、80、85、90、95、100KDa)等；进一步优选为10-50KDa。In the present invention, the molecular weight of the PEG can be 1-100KDa, such as 1-10KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10KDa), 10-50KDa (specifically Can be 10, 15, 20, 25, 30, 35, 40, 45, 50KDa), 50-100KDa (specifically can be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100KDa) etc.; further preferably 10-50KDa.
在本发明的一个实施方式中，所述聚乙二醇-连接子结合物选自如下结构：In one embodiment of the present invention, the polyethylene glycol-linker conjugate is selected from the following structures:
上式Ⅱ-a、Ⅱ-b、Ⅱ-c和Ⅱ-d中，PEG和X具有本发明上述相应定义。In the above formulas II-a, II-b, II-c and II-d, PEG and X have the corresponding definitions above in the present invention.
在本发明的一个实施例中，上式Ⅱ-a、Ⅱ-b、Ⅱ-c和Ⅱ-d中，所述PEG具有本发明上述通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构；In one embodiment of the present invention, in the above formula II-a, II-b, II-c and II-d, the PEG has the above general formula III, V, VI, VII-3 or VII-5 of the present invention Structure;
在本发明的一个实施例中，上式Ⅱ-a、Ⅱ-b、Ⅱ-c和Ⅱ-d中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In an embodiment of the present invention, in the above formulas II-a, II-b, II-c and II-d, the molecular weight of the PEG is 10-50KDa (specifically 10, 15, 20, 25, 30 KDa) , 35, 40, 45 or 50KDa).
在本发明的一个实施例中，上式Ⅱ-a、Ⅱ-b、Ⅱ-c和Ⅱ-d中，所述X为-CH2CO-或-CO-。In one embodiment of the present invention, in the above formulas II-a, II-b, II-c and II-d, the X is -CH2 CO- or -CO-.
此处所述PEG可以为通式Ⅲ、Ⅴ或Ⅵ所示的具有一个可连接位点的PEG结构，其连接一个连接子，如PEG具有通式Ⅲ所示结构，X为-CO-时，Ⅱ-d的结构为
优选地，上式Ⅱ-a、Ⅱ-b、Ⅱ-c和Ⅱ-d中，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构，其中，x优选为1-6的整数，y优选为1-3的整数。Preferably, in the above formulas II-a, II-b, II-c and II-d, the PEG has the structure of the above-mentioned III, V, VI, VII-3 or VII-5 of the present invention, wherein, x is preferably An integer of 1-6, and y is preferably an integer of 1-3.
本发明另一方面还提供一种聚乙二醇-连接子结合物衍生物，其具有如下结构：Another aspect of the present invention also provides a polyethylene glycol-linker conjugate derivative, which has the following structure:
PEG-X-L-P-QPEG-X-L-P-Q
(Ⅷ)(VIII)
其中，L为本发明上述连接子，Wherein, L is the above-mentioned linker of the present invention,
PEG为聚乙二醇残基，PEG is a polyethylene glycol residue,
X为PEG与L的连接基团，选自：-(CH2)a-、-(CH2)aCO-、-(CH2)aOCO-、-(CH2)aNHCO-、-NH(CH2)aCO-、-(CH2)aSO2-、-O(CH2)a-、-O(CH2)aCO-、-O(CH2)aOCO-、-O(CH2)aNHCO-和-O(CH2)aSO2-中的一种或多种的组合，a为0-10的整数，X is a linking group between PEG and L, selected from: -(CH2 )a -, -(CH2 )a CO-, -(CH2 )a OCO-, -(CH2 )a NHCO-, -NH (CH2 )a CO-, -(CH2 )a SO2 -, -O(CH2 )a -, -O(CH2 )a CO-, -O(CH2 )a OCO-, -O( A combination of one or more of CH2 )a NHCO- and -O(CH2 )a SO2 -, a is an integer of 0-10,
P为L与封端基Q的连接基团，选自：-(CH2)r-、
Q为封端基，选自：C1-C6的烷氧基、羟基、氨基、羧基、巯基、酯基、酮基、醛基、邻二硫吡啶基、叠氮基、酰肼基、炔基、硅烷基、马来酰亚胺基和琥珀酰亚胺基，Q is a capping group, selected from: C1-C6 alkoxy group, hydroxyl group, amino group, carboxyl group, mercapto group, ester group, ketone group, aldehyde group, o-dithiopyridyl group, azide group, hydrazide group, alkynyl group , silyl, maleimide and succinimide groups,
R17和R18独立地选自：-H、C1-6的烷基、C1-6的烷氧基、C3-6环烷基和C4-10亚烷基环烷基。R17 and R18 are independently selected from: -H, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, and C4-10 alkylenecycloalkyl.
在本发明一个实施方式中，所述衍生物具有如下结构：In one embodiment of the present invention, the derivative has the following structure:
上式Ⅷ-1～Ⅷ-3中，所述R1-12、l、A、B、X、PEG等具有本发明上述相应定义。In the above formulae VIII-1 to VIII-3, the R1-12 , l, A, B, X, PEG, etc. have the corresponding definitions above in the present invention.
在本发明的一个实施例中，所述X为单键、-CH2-、-CO-、-CH2CO-或-NHCO-。In one embodiment of the present invention, the X is a single bond, -CH2 -, -CO-, -CH2 CO- or -NHCO-.
优选地，R17和R18独立地选自：-H、-CH3、-CH2CH3、-CH2CH2CH3、-OCH3、-OCH2CH3和-OCH2CH2CH3，更优选自：-H、-CH3、-OCH3和-OCH2CH3，在本发明一个实施例中，R17为H，R18为-CH3、-OCH3或-OCH2CH3，在本发明一个优选实施例中，R17为H，R18为-CH3。Preferably, R17 and R18 are independently selected from: -H, -CH3 , -CH2 CH3 , -CH2 CH2 CH3 , -OCH3 , -OCH2 CH3 and -OCH2 CH2 CH3 , more preferably selected from: -H, -CH3 , -OCH3 and -OCH2 CH3 , in one embodiment of the present invention, R17 is H, and R18 is -CH3 , -OCH3 or -OCH2 CH3 , in a preferred embodiment of the present invention, R17 is H, and R18 is -CH3 .
在本发明的一个实施例中，所述P选自：-(CH2)r-、-(CH2)rCH(CH3)-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rSH-、
优选地，r为0-5的整数，如0、1、2、3、4或5。Preferably, r is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个具体实施例中，所述P选自：单键、-CH2-、-CH2CH2-、-CH2CH2CH2-、-CH2CH2CH2CH2-、-CH2CH2CH2CH2CH2-、-CH(CH3)-、-CH2CH(CH3)-、-CH2CH2CH(CH3)-、-CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH2CH(CH3)-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rSH-、
在本发明中，所述P具有两个以上连接位点时，可连接一个以上PEG-X-L-和一个以上Q，如所述P为
在本发明的一个实施例中，所述Q为酯基或酮基。In one embodiment of the present invention, the Q is an ester group or a ketone group.
在本发明的一个具体实施例中，所述的Q中，所述酯基选自：
在本发明的另一个具体实施例中，所述的Q中，所述酮基选自：
在本发明的一个优选实施例中，所述Q为
在本发明一个实施方式中中，所述衍生物具有如下结构：In one embodiment of the present invention, the derivative has the following structure:
上式Ⅷ-1～Ⅷ-3中，所述R1-12、l、A、B、X、PEG等具有本发明上述相应定义。In the above formulae VIII-1 to VIII-3, the R1-12 , l, A, B, X, PEG, etc. have the corresponding definitions above in the present invention.
在本发明的一个实施方式中，所述衍生物选自如下结构：In one embodiment of the present invention, the derivative is selected from the following structures:
上式Ⅷ-a、Ⅷ-b、Ⅷ-c和Ⅷ-d中，所述PEG和X具有本发明上述相应定义。In the above formulas VIII-a, VIII-b, VIII-c and VIII-d, the PEG and X have the corresponding definitions above in the present invention.
在本发明的一个实施例中，上式Ⅷ-a、Ⅷ-b、Ⅷ-c和Ⅷ-d中，所述PEG具有本发明上述通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构。In one embodiment of the present invention, in the above formulas VIII-a, VIII-b, VIII-c and VIII-d, the PEG has the above general formula III, V, VI, VII-3 or VII-5 of the present invention Structure.
在本发明的一个实施例中，上式Ⅷ-a、Ⅷ-b、Ⅷ-c和Ⅷ-d中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In an embodiment of the present invention, in the above formulas VIII-a, VIII-b, VIII-c and VIII-d, the molecular weight of the PEG is 10-50KDa (specifically, it can be 10, 15, 20, 25, 30 KDa) , 35, 40, 45 or 50KDa).
在本发明的一个实施例中，上式Ⅷ-a、Ⅷ-b、Ⅷ-c和Ⅷ-d中，所述X为-CH2CO-或-CO-。In one embodiment of the present invention, in the above formulas VIII-a, VIII-b, VIII-c and VIII-d, the X is -CH2 CO- or -CO-.
优选地，上式Ⅷ-a、Ⅷ-b、Ⅷ-c和Ⅷ-d中，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构，其中，x为1-6的整数，y为1-3的整数。Preferably, in the above formulas VIII-a, VIII-b, VIII-c and VIII-d, the PEG has the structure of the above-mentioned III, V, VI, VII-3 or VII-5 of the present invention, wherein, x is 1 An integer of -6, and y an integer of 1-3.
本发明另一方面还提供一种聚乙二醇-连接子-药物结合物，其具有如下结构：Another aspect of the present invention also provides a polyethylene glycol-linker-drug conjugate, which has the following structure:
(PEG-X-L-Y)n-D(PEG-XLY)n -D
(Ⅸ)(IX)
其中，PEG为聚乙二醇残基，Wherein, PEG is polyethylene glycol residue,
X为PEG与L的连接基团，选自：-(CH2)a-、-(CH2)aCO-、-(CH2)aOCO-、-(CH2)aNHCO-、-NH(CH2)aCO-、-(CH2)aSO2-、-O(CH2)a-、-O(CH2)aCO-、-O(CH2)aOCO-、-O(CH2)aNHCO-和-O(CH2)aSO2-中的一种或多种的组合，a为0-10的整数，X is a linking group between PEG and L, selected from: -(CH2 )a -, -(CH2 )a CO-, -(CH2 )a OCO-, -(CH2 )a NHCO-, -NH (CH2 )a CO-, -(CH2 )a SO2 -, -O(CH2 )a -, -O(CH2 )a CO-, -O(CH2 )a OCO-, -O( A combination of one or more of CH2 )a NHCO- and -O(CH2 )a SO2 -, a is an integer of 0-10,
Y为L与D的连接基团，选自：-(CH2)r-、
R17和R18独立地选自：-H、C1-6的烷基、C1-6的烷氧基、C3-6环烷基和C4-10亚烷基环烷基，R17 and R18 are independently selected from: -H, C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl and C4-10 alkylenecycloalkyl,
L为本发明上述连接子，L is the above-mentioned linker of the present invention,
D为多肽和蛋白质类药物，D is for polypeptide and protein drugs,
n为整数，且1≤n≤500。n is an integer, and 1≤n≤500.
在本发明一个实施方式中，所述聚乙二醇-连接子-药物结合物具有如下结构：In one embodiment of the present invention, the polyethylene glycol-linker-drug conjugate has the following structure:
上式Ⅸ-1～Ⅸ-3中，所述R1-12、l、A、B、X、PEG等具有本发明上述相应定义。In the above formulas IX-1 to IX-3, the R1-12 , I, A, B, X, PEG, etc. have the corresponding definitions above in the present invention.
在本发明的一个实施例中，上式Ⅸ-1中，所述的R1-4均为-H。In an embodiment of the present invention, in the above formula IX-1, the R1-4 are all -H.
在本发明的一个实施例中，上式Ⅸ-2中，所述的R5-7均为-H。In an embodiment of the present invention, in the above formula IX-2, the R5-7 are all -H.
在本发明的一个实施例中，上式Ⅸ-3中，所述的R9-11均为-H。In an embodiment of the present invention, in the above formula IX-3, the R9-11 are all -H.
在本发明的一个实施例中，上式Ⅸ-2、Ⅸ-3中，所述R8和/或R12为甲基。In an embodiment of the present invention, in the above formulas IX-2 and IX-3, the R8 and/or R12 are methyl groups.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述l为1。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the l is 1.
在本发明的一个实施例中，上式Ⅸ-1中，所述A为-COCH2NH-、-COCH(CH3)NH-或-COCH(CH(CH3)2)NH-。In one embodiment of the present invention, in the above formula IX-1, the A is -COCH2 NH-, -COCH(CH3 )NH- or -COCH(CH(CH3 )2 )NH-.
在本发明的一个实施例中，上式Ⅸ-2、Ⅸ-3中，所述-B-A-为-OCH2CH2NH-。In an embodiment of the present invention, in the above formulas IX-2 and IX-3, the -BA- is -OCH2 CH2 NH-.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述X为单键、-CH2-、-CO-、-CH2CO-或-NHCO-。In an embodiment of the present invention, in the above formulas IX-1 to IX-3, the X is a single bond, -CH2 -, -CO-, -CH2 CO- or -NHCO-.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构，其中，x为1-6的整数，y为1-3的整数。In an embodiment of the present invention, in the above formulas IX-1 to IX-3, the PEG has the structure of the above-mentioned III, V, VI, VII-3 or VII-5 of the present invention, wherein, x is 1-6 , and y is an integer from 1 to 3.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述的PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In an embodiment of the present invention, in the above formulas IX-1 to IX-3, the molecular weight of the PEG is 10-50KDa (specifically, it can be 10, 15, 20, 25, 30, 35, 40, 45 or 50KDa).
在本发明中所述的多肽和蛋白类药物如细胞因子(如白细胞介素、集落刺激因子、干扰素、生长因子、肿瘤坏死因子、转化生长因子-β家族或趋化因子家族等)、人血红蛋白、凝血因子、血管内皮生长因子抗体拮抗剂、蛋白类激素(如胰岛素、胰高血糖素、降钙素、下丘脑激素、垂体激素或胃肠激素等)、抗体(如单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体或抗体片断)、酶及辅酶类药物(苯丙氨酸裂解酶、精氨酸酶、精氨酸脱酰酶、胰核糖核酸酶、超氧化物歧化酶、天冬酰胺酶、葡糖脑苷脂酶或透明质酸酶)。在所述药物结合物中，药物分子通过胺基与聚乙二醇-连接子连接，所述胺基可为肽链N-末端氨基和/或肽链内氨基酸(如赖氨酸等)残基的侧链氨基；更优选地，在所述药物结合物中，聚乙二醇-连接子通过活性碳酸酯基与药物分子的伯胺基反应形成
本申请中，术语“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如：双特异性抗体)和抗体片段，只要它们表现出所需的生物活性(Miller等(2003)Jour.of Immunology,170:4854-4861)。抗体可以为鼠、人、人源化、嵌合抗体或来源于其它物种。抗体可具有任何类型(例如IgG、IgE、IgM、IgD及IgA)及类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)，其中IgM含游离氨基约450个。In this application, the term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, so long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology, 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, and IgA) and class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), where IgM contains about 450 free amino groups.
在本发明的一个实施方式中，所述的D为细胞因子；在本发明的一个实施例中，所述的细胞因子为白介素，优选为白介素2(IL-2)，更优选为重组人白介素2(rhIL-2)。In an embodiment of the present invention, the D is a cytokine; in an embodiment of the present invention, the cytokine is an interleukin, preferably interleukin 2 (IL-2), more preferably recombinant human interleukin 2 (rhIL-2).
在本发明另一个实施方式中，所述的细胞因子为集落刺激因子，优选为粒细胞集落刺激因子，更优选为重组人粒细胞集落刺激因子。In another embodiment of the present invention, the cytokine is a colony stimulating factor, preferably a granulocyte colony stimulating factor, more preferably a recombinant human granulocyte colony stimulating factor.
在本发明的另一个实施方式中，所述的D为抗体。In another embodiment of the present invention, the D is an antibody.
在本发明的一个实施方式中，所述的抗体为单克隆抗体，如抗HER2单克隆抗体，优选为重组抗HER2人源化单克隆抗体。In one embodiment of the present invention, the antibody is a monoclonal antibody, such as an anti-HER2 monoclonal antibody, preferably a recombinant anti-HER2 humanized monoclonal antibody.
在本发明的另一个实施方式中，所述D为人血红蛋白。In another embodiment of the present invention, the D is human hemoglobin.
在本发明的另一个实施方式中，所述D为酶及辅酶类药物，优选自：胰核糖核酸酶、超氧化物歧化酶和天冬酰胺酶。In another embodiment of the present invention, the D is an enzyme and a coenzyme drug, preferably selected from: pancreatic ribonuclease, superoxide dismutase and asparaginase.
在本发明的另一个实施方式中，所述D为蛋白类激素，优选为胰岛素。In another embodiment of the present invention, the D is a protein hormone, preferably insulin.
所述聚乙二醇-连接子-药物结合物中，n为1-500的整数(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500)，如所述药物为白介素2时，n可为1、2、3、4、5、6、7、8、9、10、11或12；所述药物为重组抗HER2人源化单克隆抗体时，n可为1-90的整数；所述药物为IgM时，n可为1-450的整数。In the polyethylene glycol-linker-drug conjugate, n is an integer from 1 to 500 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500), if the drug is
在本发明的一个实施方式中，所述Y选自：-(CH2)r-、-(CH2)rCH(CH3)-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rNH-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rSH-、
优选的，r为0-5的整数，如0、1、2、3、4或5。Preferably, r is an integer from 0 to 5, such as 0, 1, 2, 3, 4 or 5.
在本发明的一个具体实施例中，所述Y选自：单键、-CH2-、-CH2CH2-、-CH2CH2CH2-、-CH2CH2CH2CH2-、-CH2CH2CH2CH2CH2-、-CH(CH3)-、-CH2CH(CH3)-、-CH2CH2CH(CH3)-、-CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH2CH(CH3)-、-(CH2)rO-、-(CH2)rCO-、-(CH2)rCONH-、-(CH2)rNH-、-(CH2)rSH-、
在本发明的另一个具体的实施例中，所述Y为-(CH2)rCO-与单键、-CH2-、-CH2CH2-、-CH2CH2CH2-、-CH2CH2CH2CH2-、-CH2CH2CH2CH2CH2-、-CH(CH3)-、-CH2CH(CH3)-、-CH2CH2CH(CH3)-、-CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH(CH3)-、-CH2CH2CH2CH2CH2CH(CH3)-、-(CH2)rO-、-(CH2)rCONH-、-(CH2)rNHCO-、-(CH2)rNH-、-(CH2)rSH-、
在本发明的一个实施例中，所述聚乙二醇-连接子-药物结合物中，所述Y为-(CH2)rCO-。In an embodiment of the present invention, in the polyethylene glycol-linker-drug conjugate, the Y is -(CH2 )r CO-.
在本发明的一个另一个实施例中，所述聚乙二醇-连接子-药物结合物中，所述Y为-CO-。In another embodiment of the present invention, in the polyethylene glycol-linker-drug conjugate, the Y is -CO-.
在本发明中，所述Y具有两个以上连接位点时，可连接一个以上PEG-X-L-和D，如所述Y为
在本发明的一个实施方式中，所述聚乙二醇-连接子-药物结合物中，所述D为IL-2，n为1-12的整数，所述聚乙二醇-连接子-IL-2结合物具有如下结构：In one embodiment of the present invention, in the polyethylene glycol-linker-drug conjugate, the D is IL-2, n is an integer of 1-12, and the polyethylene glycol-linker- IL-2 conjugates have the following structure:
上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述R1-12、l、A、B、X、PEG、Y等具有本发明上述相应定义。In the above formulas IX-1-1, IX-2-1, IX-3-1, the R1-12 , l, A, B, X, PEG, Y, etc. have the corresponding definitions above in the present invention.
在本发明的一个实施例中，上式Ⅸ-1-1中，所述的R1-4均为-H。In an embodiment of the present invention, in the above formula IX-1-1, the R1-4 are all -H.
在本发明的一个实施例中，上式Ⅸ-2-1中，所述的R5-7均为-H。In an embodiment of the present invention, in the above formula IX-2-1, the R5-7 are all -H.
在本发明的一个实施例中，上式Ⅸ-3-1中，所述的R9-11均为-H。In an embodiment of the present invention, in the above formula IX-3-1, the R9-11 are all -H.
在本发明的一个实施例中，上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述R8和/或R12为甲基。In an embodiment of the present invention, in the above formulas IX-1-1, IX-2-1 and IX-3-1, the R8 and/or R12 are methyl groups.
在本发明的一个实施例中，上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述l为1。In an embodiment of the present invention, in the above formulas IX-1-1, IX-2-1 and IX-3-1, the l is 1.
在本发明的一个实施例中，上式Ⅸ-1-1中，所述A为-COCH2NH-、-COCH(CH3)NH-或-COCH(CH(CH3)2)NH-。In an embodiment of the present invention, in the above formula IX-1-1, the A is -COCH2 NH-, -COCH(CH3 )NH- or -COCH(CH(CH3 )2 )NH-.
在本发明的一个实施例中，上式Ⅸ-2-1、Ⅸ-3-1中，所述-B-A-为-OCH2CH2NH-。In an embodiment of the present invention, in the above formulas IX-2-1 and IX-3-1, the -BA- is -OCH2 CH2 NH-.
在本发明的一个实施例中，上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述X为单键、-CH2-、-CO-、-CH2CO-或-NHCO-。In an embodiment of the present invention, in the above formulas IX-1-1, IX-2-1 and IX-3-1, the X is a single bond, -CH2 -, -CO-, -CH2 CO -or -NHCO-.
在本发明的一个实施例中，上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构，其中，x为1-6的整数，y为1-3的整数。In an embodiment of the present invention, in the above formula IX-1-1, IX-2-1, IX-3-1, the PEG has the above-mentioned III, V, VI, VII-3 or VII-5 of the present invention , where x is an integer from 1 to 6 and y is an integer from 1 to 3.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述的PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In an embodiment of the present invention, in the above formulas IX-1 to IX-3, the molecular weight of the PEG is 10-50KDa (specifically, it can be 10, 15, 20, 25, 30, 35, 40, 45 or 50KDa).
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，所述Y为-CO-。In one embodiment of the present invention, in the above formulas IX-1 to IX-3, the Y is -CO-.
在本发明的一个实施例中，上式Ⅸ-1～Ⅸ-3中，n为1-12的整数，优选为1-7的整数，具体如1、2、3、4、5、6或7。In an embodiment of the present invention, in the above formulas IX-1 to IX-3, n is an integer of 1-12, preferably an integer of 1-7, specifically 1, 2, 3, 4, 5, 6 or 7.
在本发明的一个具体实施方式中，所述聚乙二醇-连接子-IL-2结合物具有如下结构：In a specific embodiment of the present invention, the polyethylene glycol-linker-IL-2 conjugate has the following structure:
上式Ⅸ-a、Ⅸ-b、Ⅸ-c和Ⅸ-d中，所述PEG、X和n具有本发明上述定义。In the above formulas IX-a, IX-b, IX-c and IX-d, the PEG, X and n have the above definitions in the present invention.
在本发明的一个实施例中，上式Ⅸ-1-1、Ⅸ-2-1、Ⅸ-3-1中，所述PEG具有本发明上述Ⅲ、Ⅴ、Ⅵ、Ⅶ-3或Ⅶ-5的结构，其中，x为1-6的整数，y为1-3的整数。当PEG为通式Ⅴ、Ⅵ或Ⅶ-1的结构时，可增加空间位阻效应，调控药物释放速度。In an embodiment of the present invention, in the above formula IX-1-1, IX-2-1, IX-3-1, the PEG has the above-mentioned III, V, VI, VII-3 or VII-5 of the present invention , where x is an integer from 1 to 6 and y is an integer from 1 to 3. When PEG is the structure of general formula V, VI or VII-1, it can increase the steric hindrance effect and control the drug release rate.
在本发明的一个实施例中，上式Ⅸ-a、Ⅸ-b、Ⅸ-c和Ⅸ-d中，所述PEG的分子量为10-50KDa(具体可为10、15、20、25、30、35、40、45或50KDa)。In an embodiment of the present invention, in the above formulas IX-a, IX-b, IX-c and IX-d, the molecular weight of the PEG is 10-50KDa (specifically, it can be 10, 15, 20, 25, 30 KDa) , 35, 40, 45 or 50KDa).
在本发明的一个实施例中，上式Ⅸ-a、Ⅸ-b、Ⅸ-c和Ⅸ-d中，所述X为-CH2CO-或-CO-。In one embodiment of the present invention, in the above formulas IX-a, IX-b, IX-c and IX-d, the X is -CH2 CO- or -CO-.
在本发明的一个实施例中，上式Ⅸ-a、Ⅸ-b、Ⅸ-c和Ⅸ-d中，所述n为1-12的整数，优选为1-7的整数，具体如1、2、3、4、5、6或7。In an embodiment of the present invention, in the above formulas IX-a, IX-b, IX-c and IX-d, the n is an integer of 1-12, preferably an integer of 1-7, specifically 1, 2, 3, 4, 5, 6 or 7.
在本发明的一个实施例中，上式Ⅸ-a、Ⅸ-b、Ⅸ-c和Ⅸ-d中，所述IL-2为rhIL-2。In one embodiment of the present invention, in the above formulas IX-a, IX-b, IX-c and IX-d, the IL-2 is rhIL-2.
在本发明的一个更具体的实施方式中，所述聚乙二醇-连接子-IL-2结合物具有如下结构：In a more specific embodiment of the present invention, the polyethylene glycol-linker-IL-2 conjugate has the following structure:
上式中，p、q、i、h、k和x分别具有本发明通式Ⅲ、Ⅴ、Ⅵ、Ⅶ-3中的定义。In the above formula, p, q, i, h, k and x have the definitions in the general formulae III, V, VI and VII-3 of the present invention, respectively.
在本发明的一个实施例中，x为1-6的整数，优选地，x为6。In an embodiment of the present invention, x is an integer of 1-6, preferably, x is 6.
在本发明的一个实施例中，上式中，所述n为1-12的整数，优选为1-7的整数，具体如1、2、3、4、5、6或7。In an embodiment of the present invention, in the above formula, the n is an integer of 1-12, preferably an integer of 1-7, specifically 1, 2, 3, 4, 5, 6 or 7.
在本发明的一个实施例中，上式中，所述IL-2为rhIL-2。In one embodiment of the present invention, in the above formula, the IL-2 is rhIL-2.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的制备方法，包括本发明上述聚乙二醇-连接子结合物衍生物与多肽和蛋白类药物反应连接的步骤。Another aspect of the present invention also provides a method for preparing the above-mentioned polyethylene glycol-linker-drug conjugate, comprising the step of reacting and linking the above-mentioned polyethylene glycol-linker conjugate derivative of the present invention with a polypeptide and a protein drug .
优选地，所述制备方法中，所述的衍生物为活性酯，更优选地，所述衍生物的反应基团为活性碳酸酯基。Preferably, in the preparation method, the derivative is an active ester, and more preferably, the reactive group of the derivative is an active carbonate group.
在本发明的一个实施例中，所述衍生物具有上述式Ⅷ-a、Ⅷ-b、Ⅷ-c或Ⅷ-d的结构。In one embodiment of the present invention, the derivative has the structure of formula VIII-a, VIII-b, VIII-c or VIII-d above.
优选地，所述制备方法中，所述多肽和蛋白类药物的反应基团为胺基，更优选为伯胺基。Preferably, in the preparation method, the reactive group of the polypeptide and the protein drug is an amine group, more preferably a primary amine group.
在本发明的一个实施例中，所述多肽和蛋白类药物为IL-2，更优选为rhIL-2。In one embodiment of the present invention, the polypeptide and protein drugs are IL-2, more preferably rhIL-2.
在本发明的一个优选实施例中，所述的聚乙二醇-连接子-药物结合物为聚乙二醇-连接子-IL-2结合物，其制备方法包括本发明上述聚乙二醇-连接子结合物衍生物与IL-2反应连接的步骤。In a preferred embodiment of the present invention, the polyethylene glycol-linker-drug conjugate is a polyethylene glycol-linker-IL-2 conjugate, and the preparation method thereof comprises the above-mentioned polyethylene glycol of the present invention - a step of reactively linking the linker conjugate derivative with IL-2.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述聚乙二醇-连接子结合物衍生物与IL-2的摩尔比为1-50:1(具体如1:1、5:1、10:1、15:1、20:1、25:1、30:1、35:1、40:1、45:1或50:1)，更优选为10-30:1。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the molar ratio of the polyethylene glycol-linker conjugate derivative to IL-2 is 1-50:1 ( Specifically, such as 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1), more preferably 10-30:1.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应pH值为6.0-10.0(具体如6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5或10.0)。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the pH value of the reaction is 6.0-10.0 (specifically, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0).
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应温度为20-30℃，更优选为室温。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the reaction temperature is 20-30°C, more preferably room temperature.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法中，所述反应在缓冲液中进行，本领域技术人员可以根据所采用的反应pH值选择合适的缓冲液种类，如磷酸盐缓冲液、硼酸盐缓冲液或碳酸氢钠缓冲液等，本发明对此不作具体限定。Preferably, in the preparation method of the polyethylene glycol-linker-IL-2 conjugate, the reaction is carried out in a buffer, and those skilled in the art can select a suitable buffer type according to the pH of the reaction used. , such as phosphate buffer, borate buffer or sodium bicarbonate buffer, etc., which are not specifically limited in the present invention.
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法还包括终止反应的步骤，更优选地，所述终止反应步骤包括加入甘氨酸溶液，所述甘氨酸溶液浓度为0.5-2M(具体如0.5、1.0、1.5或2.0M)。Preferably, the preparation method of the polyethylene glycol-linker-IL-2 conjugate further includes a step of terminating the reaction, more preferably, the step of terminating the reaction includes adding a glycine solution, and the concentration of the glycine solution is 0.5- 2M (specifically such as 0.5, 1.0, 1.5 or 2.0M).
优选地，所述聚乙二醇-连接子-IL-2结合物的制备方法还包括分离纯化反应产物的步骤。所述分离纯化的方法可采用本领域中常用的方法，如离子交换柱层析、反相高效液相色谱分离和凝胶渗透色谱分离等中的一种或多种的组合，本发明对此不作具体限定。Preferably, the preparation method of the polyethylene glycol-linker-IL-2 conjugate further comprises the step of separating and purifying the reaction product. The method for separation and purification can be a combination of one or more of the methods commonly used in the art, such as ion exchange column chromatography, reversed-phase high performance liquid chromatography separation, and gel permeation chromatography separation. There is no specific limitation.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的组合物，所述组合物中包括至少两种本发明上述具有不同n值的聚乙二醇-连接子-药物结合物。Another aspect of the present invention also provides a composition of the above polyethylene glycol-linker-drug conjugate, the composition comprising at least two of the above-mentioned polyethylene glycol-linker- drug conjugates.
在本发明的一个实施方式中，上述组合物中包括至少三种本发明上述具有不同n值的聚乙二醇-连接子-药物结合物。In one embodiment of the present invention, the above-mentioned composition includes at least three kinds of the above-mentioned polyethylene glycol-linker-drug conjugates with different n values of the present invention.
优选地，所述聚乙二醇-连接子-药物结合物聚乙二醇-连接子-IL-2结合物。Preferably, the polyethylene glycol-linker-drug conjugate polyethylene glycol-linker-IL-2 conjugate.
在本发明的一个实施例中，所述组合物包括n值为1-7的聚乙二醇-连接子-IL-2结合物。In one embodiment of the present invention, the composition includes a polyethylene glycol-linker-IL-2 conjugate with an n value of 1-7.
在本发明的一个实施例中，所述组合物包括n值为1-3的聚乙二醇-连接子-IL-2结合物。In one embodiment of the present invention, the composition includes a polyethylene glycol-linker-IL-2 conjugate with an n value of 1-3.
在本发明的一个实施例中，所述组合物包括n值为3-5的聚乙二醇-连接子-IL-2结合物。In one embodiment of the present invention, the composition includes a polyethylene glycol-linker-IL-2 conjugate with an n value of 3-5.
在本发明的一个实施例中，所述组合物包括n值为4-7的聚乙二醇-连接子-IL-2结合物。In one embodiment of the present invention, the composition includes a polyethylene glycol-linker-IL-2 conjugate with an n value of 4-7.
本发明另一方面还提供一种上述聚乙二醇-连接子-药物结合物的药学上可接受的盐、异构体、前药或溶剂化物。Another aspect of the present invention also provides a pharmaceutically acceptable salt, isomer, prodrug or solvate of the above polyethylene glycol-linker-drug conjugate.
本发明另一方面还提供一种包含上述聚乙二醇-连接子-药物结合物及其药学上可接受的盐、异构体、前药或溶剂化物和药学上可接受的载体或添加剂的药物组合物。Another aspect of the present invention also provides a polyethylene glycol-linker-drug conjugate and a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof and a pharmaceutically acceptable carrier or additive. pharmaceutical composition.
如本文所用，“药学上可接受”一词是指在对人施用后具有生理相容性并且不会引起肠胃失调、诸如头晕的过敏反应或类似反应。添加剂可为赋形剂、崩解剂、粘结剂、润滑剂、悬浮剂、稳定剂等等中的任一种。赋形剂的例子包括乳糖、甘露醇、益寿糖、微晶纤维素、硅化微晶纤维素、粉状纤维素等等。崩解剂的例子包括低取代羟丙基纤维素、交聚维酮、羧基乙酸淀粉钠、交联羧甲基纤维素钠、淀粉等等。粘结剂的例子包括羟丙基纤维素、羟丙甲纤维素、聚维酮、共聚维酮、预胶凝淀粉等等。润滑剂的例子包括硬脂酸、硬脂酸镁、富马酰硬脂酸钠等等；润湿剂的例子包括聚氧乙烯山梨糖醇酐脂肪酸酯、泊洛沙姆、聚氧乙烯蓖麻油衍生物等等。悬浮剂的例子包括羟丙甲纤维素、羟丙基纤维素、聚维酮、共聚维酮、羧甲基纤维素钠、甲基纤维素等等。稳定剂的例子包括柠檬酸、富马酸、琥珀酸等等。另外，本发明的药物组合物还可包括阻凝剂、增味剂、乳化剂、防腐剂等等中的任一种。As used herein, the term "pharmaceutically acceptable" means physiologically compatible and does not cause gastrointestinal disturbances, allergic reactions such as dizziness, or the like upon administration to humans. The additives can be any of excipients, disintegrants, binders, lubricants, suspending agents, stabilizers, and the like. Examples of excipients include lactose, mannitol, isomalt, microcrystalline cellulose, silicified microcrystalline cellulose, powdered cellulose, and the like. Examples of disintegrants include low-substituted hydroxypropylcellulose, crospovidone, sodium starch carboxyacetate, croscarmellose sodium, starch, and the like. Examples of binders include hydroxypropylcellulose, hypromellose, povidone, copovidone, pregelatinized starch, and the like. Examples of lubricants include stearic acid, magnesium stearate, sodium fumarate stearate, and the like; examples of wetting agents include polyoxyethylene sorbitan fatty acid esters, poloxamers, polyoxyethylene castor Sesame Oil Derivatives, etc. Examples of suspending agents include hypromellose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethyl cellulose, methyl cellulose and the like. Examples of stabilizers include citric acid, fumaric acid, succinic acid, and the like. In addition, the pharmaceutical composition of the present invention may further include any one of anticoagulants, flavor enhancers, emulsifiers, preservatives and the like.
本发明所述的药物组合物可以为片剂(包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等)、丸剂、粉剂、颗粒剂、胶囊剂(包括软胶囊、微胶囊)、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等、口服或胃肠外制剂(例如，静脉内、肌内、皮下、器官内、鼻内、皮内、滴注、脑内、直肠内等给药形式、给药至肿瘤的附近和直接给药至病变处)。优选地，所述的药物组合物为注射剂。The pharmaceutical composition of the present invention can be in the form of tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations (eg, immediate release formulations, sustained release formulations, sustained release microcapsules), aerosols, films ( For example, oral disintegrating film, oral mucosa-adhesive film), injection (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip, transdermal absorption preparation, ointment, lotion oral or parenteral formulations (eg, intravenous, intramuscular) , subcutaneous, intra-organ, intranasal, intradermal, instillation, intracerebral, intrarectal and other forms of administration, administration to the vicinity of the tumor and direct administration to the lesion). Preferably, the pharmaceutical composition is an injection.
本发明所述的药学上可接受的辅料优选为药学上可接受的注射剂辅料，例如等渗的无菌盐溶液(磷酸二氢钠、磷酸氢二钠、氯化钠、氯化钾、氯化钙、氯化镁等，或上述盐的混合物)，或所述药物组合物是干燥的例如是冷冻干燥的组合物，其适当地通过加入无菌水或生理盐水形成可注射溶质。The pharmaceutically acceptable adjuvant of the present invention is preferably a pharmaceutically acceptable adjuvant for injection, such as isotonic sterile saline solution (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, chloride calcium, magnesium chloride, etc., or mixtures of the foregoing salts), or the pharmaceutical composition is a dry, eg, lyophilized, composition, which is suitably formed into an injectable solute by the addition of sterile water or physiological saline.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物在制备聚乙二醇-连接子结合物衍生物中的应用。Another aspect of the present invention also provides the use of the above-mentioned linker compound and polyethylene glycol-linker conjugate in the preparation of a polyethylene glycol-linker conjugate derivative.
优选地，所述的衍生物为活性酯，更优选为活性碳酸酯。Preferably, the derivatives are activated esters, more preferably activated carbonates.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物及其衍生物在修饰药物中的应用。Another aspect of the present invention also provides the use of the above-mentioned linker compound, polyethylene glycol-linker conjugate and derivatives thereof in modifying medicines.
优选的，所述的应用为制备聚乙二醇-连接子-药物结合物；所述药物为多肽和蛋白质类药物，其具有本发明上述定义。Preferably, the application is the preparation of polyethylene glycol-linker-drug conjugates; the drugs are polypeptide and protein drugs, which have the above definitions of the present invention.
在本发明的另一个具体实施例中，所述的药物为细胞因子，所述细胞因子优选为白介素，更优选为IL-2，最优选为rhIL-2；所述抗体优选为单克隆抗体，更优选为重组抗HER2人源化单克隆抗体。In another specific embodiment of the present invention, the drug is a cytokine, and the cytokine is preferably an interleukin, more preferably IL-2, and most preferably rhIL-2; the antibody is preferably a monoclonal antibody, More preferred are recombinant anti-HER2 humanized monoclonal antibodies.
本发明另一方面还提供一种上述连接子化合物、聚乙二醇-连接子结合物及其衍生物、聚乙二醇-连接子-药物结合物及其药学上可接受的盐、异构体、前药或溶剂化物、药物组合物在制备预防和/或治疗疾病的药物中的应用。Another aspect of the present invention also provides the above-mentioned linker compound, polyethylene glycol-linker combination and derivatives thereof, polyethylene glycol-linker-drug combination and pharmaceutically acceptable salts, isomers thereof Application of body, prodrug or solvate, and pharmaceutical composition in the preparation of medicaments for preventing and/or treating diseases.
优选地，所述聚乙二醇-连接子-药物结合物为本发明所述的聚乙二醇-连接子-IL-2结合物。Preferably, the polyethylene glycol-linker-drug conjugate is the polyethylene glycol-linker-IL-2 conjugate of the present invention.
优选地，所述的疾病为肿瘤、自身免疫疾病、病毒性疾病或细菌性疾病。Preferably, the disease is tumor, autoimmune disease, viral disease or bacterial disease.
优选地，所述肿瘤疾病包括肾细胞癌、黑色素瘤、恶性血管内皮细胞瘤、皮肤T细胞瘤、卵巢癌、乳腺癌、膀胱癌、肺癌、神经胶质瘤、神经母细胞瘤、肝癌、毛细胞白血病、髓样母细胞白血病、结肠癌、癌性胸腹腔积液或非霍奇金淋巴瘤等。Preferably, the tumor diseases include renal cell carcinoma, melanoma, malignant hemangioendothelioma, cutaneous T cell tumor, ovarian cancer, breast cancer, bladder cancer, lung cancer, glioma, neuroblastoma, liver cancer, hair Cell leukemia, myeloid leukemia, colon cancer, cancerous pleural effusion or non-Hodgkin lymphoma, etc.
优选地，所述自身免疫疾病包括类风湿关节炎、系统性红斑狼疮和干燥综合征。Preferably, the autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome.
优选地，所述病毒包括肝炎病毒、乳头状病毒、HSV、HIV、EBv、冠状病毒和流感病毒等，更优选为肝炎病毒，如HBV或HCV。Preferably, the virus includes hepatitis virus, papilloma virus, HSV, HIV, EBv, coronavirus and influenza virus, etc., more preferably hepatitis virus, such as HBV or HCV.
优选地，所述细菌性疾病如麻风病、肺结核等。Preferably, the bacterial diseases such as leprosy, tuberculosis and the like.
优选地，所述应用为本发明所述的聚乙二醇-连接子-IL-2结合物在制备增强手术、放疗或化疗后的肿瘤患者的机体免疫功能的药物中的应用。Preferably, the application is the application of the polyethylene glycol-linker-IL-2 conjugate of the present invention in the preparation of a drug for enhancing the immune function of tumor patients after surgery, radiotherapy or chemotherapy.
本发明另一方面还提供一种向一名个体施用上述药物组合物的方法。Another aspect of the present invention also provides a method of administering the above-described pharmaceutical composition to an individual.
本发明提供的连接子化合物及其与聚乙二醇的结合物及其衍生物，可用于修饰药物，且修饰反应简单，容易进行，反应产率较高，修饰药物的适用范围较宽。本发明的提供的多肽和蛋白类药物通过非肽连接子与聚乙二醇的结合物，特别是一种白介素(如白介素2)通过非肽连接子与聚乙二醇的结合物，药物可以从结合物结构中降解分离，可实现缓释和控释，降低给药频率，大大提高药物的生物利用度和病人的依从性。特别是，本发明的发明人对结合物的偶合度进行了更深入的研究，得到偶联度明确的结合物或其混合物，有利于后续药效的优化和药理的研究。The linker compound and its conjugate with polyethylene glycol and its derivatives provided by the invention can be used for modifying drugs, and the modification reaction is simple and easy to carry out, the reaction yield is high, and the application range of modified drugs is wide. The polypeptide and protein drugs provided by the present invention are conjugated with polyethylene glycol through a non-peptide linker, especially a conjugate of an interleukin (such as interleukin 2) and polyethylene glycol through a non-peptide linker, and the drug can Degradation and separation from the conjugate structure can achieve sustained and controlled release, reduce the frequency of administration, and greatly improve the bioavailability of the drug and the compliance of patients. In particular, the inventors of the present invention conducted more in-depth research on the coupling degree of the conjugate, and obtained a conjugate or a mixture thereof with a clear coupling degree, which is beneficial to the subsequent optimization of drug efficacy and pharmacological research.
附图说明Description of drawings
图1所示为实施例8中各反应组制备的mPEG-L5-rhIL-2(20K)的RP-HPLC色谱图，检测波长：214nm。从上到下分别为：1-反应1组，2-反应2组，3-反应3组，4-反应4组，5-反应5组，6-反应6组。Figure 1 shows the RP-HPLC chromatogram of mPEG-L5-rhIL-2 (20K) prepared by each reaction group in Example 8, detection wavelength: 214 nm. From top to bottom: 1-
图2所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)的SEC-MALS谱图。Figure 2 shows the SEC-MALS spectrum of mPEG-L5-rhIL-2 (20K) prepared by
图3所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)经阳离子交换层析后的色谱图。Figure 3 shows the chromatogram of mPEG-L5-rhIL-2 (20K) prepared by
图4所示为实施例8中反应3组制备的mPEG-L5-rhIL-2(20K)纯化后穿透峰及各分段收集偶联物的RP-HPLC色谱图。从上到下分别为：1-穿透，2-A8，3-A7，4-A6，5-A5，6-A4，7-A3。Figure 4 shows the RP-HPLC chromatogram of the breakthrough peak and the conjugates collected in each segment after purification of mPEG-L5-rhIL-2 (20K) prepared by
图5所示为实施例14得到的药效学研究实验结果。FIG. 5 shows the experimental results of the pharmacodynamics study obtained in Example 14.
具体实施方式Detailed ways
除非另有定义，本发明中所使用的所有的技术和科学术语具有与本发明涉及领域的技术人员通常理解的相同的含义，如，“多肽和蛋白类药物”是指用于预防、治疗和诊断的多肽和蛋白质类物质，其中，多肽是α-氨基酸以肽键连接在一起形成的化合物，也可以是蛋白质水解的中间产物；N条多肽链按一定的空间结构缠绕纠结构成蛋白质。多肽和蛋白类药物按药物结构分类可分为：氨基酸及其衍生物类药物、多肽和蛋白质类药物、酶和辅酶类药物、核酸及其降解物和衍生物类药物、糖类药物、脂类药物、细胞生长因子和其他生物制品类药物。Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention relates, for example, "polypeptide and protein drugs" refer to prophylactic, therapeutic and Diagnosed polypeptides and protein substances, among which, polypeptides are compounds formed by α-amino acids linked together by peptide bonds, and can also be intermediate products of proteolysis; N polypeptide chains are entangled and tangled according to a certain spatial structure to form proteins. Polypeptide and protein drugs can be classified according to drug structure: amino acid and its derivatives drugs, polypeptide and protein drugs, enzymes and coenzyme drugs, nucleic acid and its degradation products and derivatives drugs, carbohydrate drugs, lipid drugs Drugs, cell growth factors and other biologics.
本发明中所述的IL-2可以是天然的、重组蛋白(如重组人白介素2)或同样具有天然IL-2功能的突变体(如“重组人白介素-2(IL-2)突变体克隆及在巴斯德毕赤酵母系统中的表达与纯化”，刘堰，博士学位论文，中所述的“IL-2-C125A/L18M/L19S”)，也包括通过组织培养、蛋白质合成、细胞培养(天然、重组细胞或突变体)方法得到的产品。天然、重组IL-2或突变体的提取和分离方法是本领域技术人员所熟知的。The IL-2 described in the present invention can be a natural, recombinant protein (such as recombinant human interleukin-2) or a mutant with the same function of natural IL-2 (such as "recombinant human interleukin-2 (IL-2) mutant clone" and expression and purification in the Pichia pastoris system", Liu Yan, Ph.D. dissertation, "IL-2-C125A/L18M/L19S"), also including by tissue culture, protein synthesis, cell A product obtained by a culture (native, recombinant cell or mutant) method. Methods of extraction and isolation of native, recombinant IL-2 or mutants are well known to those skilled in the art.
本发明中所述的英文缩写及其代表含义如下：The English abbreviations described in the present invention and their representative meanings are as follows:
IL-2：白介素2；rhIL-2：重组人白介素2；HSV：单纯孢疹病毒；HIV：人类免疫缺陷病毒；HBV：乙型肝炎病毒；HCV：丙型肝炎病毒；EBv：人类孢疹病毒4型。IL-2: interleukin-2; rhIL-2: recombinant human interleukin-2; HSV: herpes simplex virus; HIV: human immunodeficiency virus; HBV: hepatitis B virus; HCV: hepatitis C virus; EBv: human
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
本发明所用的化合物原料可以商购，也可以根据公开的制备方法进行制备，其并不限制本发明的范围。The raw materials of the compounds used in the present invention are commercially available, and can also be prepared according to the disclosed preparation methods, which do not limit the scope of the present invention.
实施例中所用的聚乙二醇及其衍生物由北京键凯科技股份有限公司提供，除非指明，分子量均为20K。其他为市售试剂。The polyethylene glycol and its derivatives used in the examples were provided by Beijing Jiankai Technology Co., Ltd., and the molecular weights were all 20K unless otherwise specified. Others are commercially available reagents.
实施例1：连接链(L)的合成Example 1: Synthesis of Linker (L)
将BOC-氨基酸(92.2mmol)和N,N-二环己基碳二亚胺(DCC，23.8g，115.3mmol)加到二氯甲烷(500mL)中，冰水浴冷却，再加入对羟基苯甲醇(11.4g，92.2mmol)，加完后撤去冰浴，室温反应过夜。过滤，滤饼用乙酸乙酯洗涤，滤液蒸干得粗品，柱层析纯化后得到产物1。BOC-amino acid (92.2 mmol) and N,N-dicyclohexylcarbodiimide (DCC, 23.8 g, 115.3 mmol) were added to dichloromethane (500 mL), cooled in an ice-water bath, and p-hydroxybenzyl alcohol ( 11.4 g, 92.2 mmol), remove the ice bath after the addition, and react at room temperature overnight. Filtration, the filter cake was washed with ethyl acetate, and the filtrate was evaporated to dryness to obtain the crude product, which was purified by column chromatography to obtain
1a:19.7g，收率76.0％。1H NMR:(CDCl3)：8.75(s,1H),7.22(d,2H),7.05(d,2H),4.87(s,2H),3.74(s,2H),1.52(s,9H)。1a: 19.7 g, yield 76.0%.1 H NMR: (CDCl3 ): 8.75 (s, 1H), 7.22 (d, 2H), 7.05 (d, 2H), 4.87 (s, 2H), 3.74 (s, 2H), 1.52 (s, 9H) .
1b:20.3g，收率74.8％。1H NMR:(CDCl3)：8.74(s,1H),7.21(d,2H),7.05(d,2H),4.88(s,2H),3.77(m,1H),1.51(s,9H),1.27(d,3H)。1b: 20.3 g, yield 74.8%.1 H NMR: (CDCl3 ): 8.74 (s, 1H), 7.21 (d, 2H), 7.05 (d, 2H), 4.88 (s, 2H), 3.77 (m, 1H), 1.51 (s, 9H) , 1.27(d,3H).
1c:21.6g，收率72.5％。1H NMR:(CDCl3)：8.75(s,1H),7.22(d,2H),7.05(d,2H),4.87(s,2H),3.61(d,1H),2,82(m,1H),1.52(s,9H),1.06(d,6H)。1c: 21.6 g, yield 72.5%.1 H NMR: (CDCl3 ): 8.75(s,1H), 7.22(d,2H), 7.05(d,2H), 4.87(s,2H), 3.61(d,1H), 2,82(m, 1H), 1.52 (s, 9H), 1.06 (d, 6H).
将化合物1(39.1mmol)溶于二氯甲烷(250mL)中，加入三氟乙酸(50mL)，加完后室温搅拌过夜，浓缩，残分中加入二氯甲烷，再蒸干，反复三次，最后加乙醚沉淀，过滤，得产物L。Compound 1 (39.1 mmol) was dissolved in dichloromethane (250 mL), trifluoroacetic acid (50 mL) was added, and after the addition, the mixture was stirred at room temperature overnight, concentrated, dichloromethane was added to the residue, evaporated to dryness, repeated three times, and finally Add ether for precipitation and filter to obtain product L.
L1:11.1g，收率96.7％。L1: 11.1 g, yield 96.7%.
L2:11.6g，收率97.1％。L2: 11.6 g, yield 97.1%.
L3:12.7g，收率96.3％。L3: 12.7 g, yield 96.3%.
实施例2：单甲氧基聚乙二醇乙酸与连接链的结合物(mPEG-L-40K)的合成Example 2: Synthesis of the conjugate of monomethoxy polyethylene glycol acetic acid and linking chain (mPEG-L-40K)
将单甲氧基聚乙二醇-乙酸(mPEG-CM，40K，5g，0.125mmol)、化合物L(0.25mmol，实施例1制备)和1-羟基苯并三氮唑(HOBt，16.9mg，0.125mmol)加到反应瓶中，用二氯甲烷溶解，再加入二异丙基乙基胺(45.2mg，0.35mmol)，搅拌均匀，冰浴冷却后分批加入(EDCI，47.9mg，0.25mmol)，加完后体系自然升到室温，反应过夜。次日浓缩后残分用异丙醇结晶，抽滤，干燥后得产品mPEG-L。Monomethoxy polyethylene glycol-acetic acid (mPEG-CM, 40K, 5 g, 0.125 mmol), compound L (0.25 mmol, prepared in Example 1) and 1-hydroxybenzotriazole (HOBt, 16.9 mg, 0.125 mmol) was added to the reaction flask, dissolved in dichloromethane, then diisopropylethylamine (45.2 mg, 0.35 mmol) was added, stirred well, and after cooling in an ice bath, (EDCI, 47.9 mg, 0.25 mmol) was added in batches ), after the addition, the system naturally rose to room temperature and reacted overnight. The next day after concentration, the residue was crystallized with isopropanol, filtered with suction, and dried to obtain the product mPEG-L.
mPEG-L1(40K):4.6g，收率92.4％。mPEG-L1 (40K): 4.6 g, yield 92.4%.
mPEG-L2(40K):4.5g，收率90.8％。mPEG-L2 (40K): 4.5 g, yield 90.8%.
mPEG-L3(40K):4.7g，收率93.7％。mPEG-L3 (40K): 4.7 g, yield 93.7%.
实施例3：连接链L5的制备Example 3: Preparation of Linker Chain L5
连接链L5的合成路线如下所示：The synthetic route of linking chain L5 is shown below:
化合物(2)的合成：Synthesis of compound (2):
3,4-二羟基苯甲醛(10g，72.5mmol)溶于乙腈(150mL)中，加入碳酸氢钠(8g，94.3mmol)，升温至60℃，加入溴苄(12.4g，72.5mmol)，然后升温至80℃搅拌过夜。浓缩除去乙腈，残分中加入10％盐酸水溶液(200mL)，用乙酸乙酯萃取(150mL*3)，合并后干燥，过滤，浓缩，残分用柱层析纯化得灰白色固体10g(收率60％)。1H NMR:(CDCl3)：δ9.82(s,1H),7.48-7.40(m,7H),7.05(m,1H),6.02(s,1H),5.21(s,2H)。3,4-Dihydroxybenzaldehyde (10g, 72.5mmol) was dissolved in acetonitrile (150mL), sodium bicarbonate (8g, 94.3mmol) was added, the temperature was raised to 60°C, benzyl bromide (12.4g, 72.5mmol) was added, then The temperature was raised to 80°C and stirred overnight. Concentrate to remove acetonitrile, add 10% aqueous hydrochloric acid (200 mL) to the residue, extract with ethyl acetate (150 mL*3), combine, dry, filter, concentrate, and the residue is purified by column chromatography to obtain 10 g of off-white solid (yield: 60 g) %).1 H NMR: (CDCl3 ): δ 9.82 (s, 1H), 7.48-7.40 (m, 7H), 7.05 (m, 1H), 6.02 (s, 1H), 5.21 (s, 2H).
化合物(3)的合成：Synthesis of compound (3):
化合物(2)(5g，21.9mmol)溶于DMF(80mL)中，加入碳酸钾(7.6g，54.75mmol)，碘化钾(0.73g，4.38mmol)，搅拌10分钟，加入ATN-1(9g，28.47mmol)，升至70℃搅拌过夜。后处理，往反应液中加入氯化铵饱和溶液(400mL)，用乙酸乙酯萃取(150mL*3)，合并后干燥，过滤，浓缩，残分用柱层析纯化得白色固体5g(收率61％)。1H NMR:(CDCl3)：δ9.85(s,1H),7.47-7.36(m,7H),7.03(m,1H),5.24(s,2H),5.07(s,1H),4.17(m,2H),3.59(m,2H),1.46(s,9H)。Compound (2) (5 g, 21.9 mmol) was dissolved in DMF (80 mL), potassium carbonate (7.6 g, 54.75 mmol), potassium iodide (0.73 g, 4.38 mmol) were added, stirred for 10 minutes, and ATN-1 (9 g, 28.47 mmol) was added. mmol), warmed to 70 °C and stirred overnight. After-treatment, a saturated solution of ammonium chloride (400 mL) was added to the reaction solution, extracted with ethyl acetate (150 mL*3), combined, dried, filtered, concentrated, and the residue was purified by column chromatography to obtain 5 g of white solid (yield 61%).1 H NMR: (CDCl3 ): δ 9.85 (s, 1H), 7.47-7.36 (m, 7H), 7.03 (m, 1H), 5.24 (s, 2H), 5.07 (s, 1H), 4.17 ( m, 2H), 3.59 (m, 2H), 1.46 (s, 9H).
化合物(4)的合成：Synthesis of compound (4):
将化合物(3)(5g，13.5mmol)溶于四氢呋喃(100mL)中，室温加入硼氢化钠(0.77g，20.25mmol)，室温搅拌2小时后冷却到0℃，加入醋酸(2mL)淬灭反应，减压浓缩除去溶剂，残分用柱层析纯化，得无色油状物4g(收率80％)。1H NMR:(DMSO-d6)：δ7.38-7.05(m,8H),6.80(m,1H),5.09(s,2H),5.06(m,1H),4.40(m,2H),3.98(m,2H),3.31(m,2H),1.38(s,9H)。Compound (3) (5 g, 13.5 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (0.77 g, 20.25 mmol) was added at room temperature, stirred at room temperature for 2 hours, cooled to 0 °C, and acetic acid (2 mL) was added to quench the reaction , the solvent was removed by concentration under reduced pressure, and the residue was purified by column chromatography to obtain 4 g of a colorless oil (yield 80%).1 H NMR: (DMSO-d6): δ 7.38-7.05 (m, 8H), 6.80 (m, 1H), 5.09 (s, 2H), 5.06 (m, 1H), 4.40 (m, 2H), 3.98 (m, 2H), 3.31 (m, 2H), 1.38 (s, 9H).
化合物(5)的合成：Synthesis of compound (5):
化合物(4)(1g，2.7mmol)溶于甲醇(8mL)中，加入Pd/C(10％，0.3g)，再通入氢气反应过夜。过滤，滤液浓缩，残分用柱层析纯化，得产品0.6g(收率78％).1H NMR:(DMSO-d6)：δ8.37(s,1H),7.09(s,1H),6.71(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.89(m,2H),3.31(m,2H),1.38(s,9H)。Compound (4) (1 g, 2.7 mmol) was dissolved in methanol (8 mL), Pd/C (10%, 0.3 g) was added, and hydrogen was passed through to react overnight. Filter, concentrate the filtrate, and purify the residue by column chromatography to obtain 0.6 g of product (yield 78%).1 H NMR: (DMSO-d6): δ8.37(s, 1H), 7.09(s, 1H), 6.71(s, 1H), 6.65(d, 1H), 6.56(d, 1H), 4.45(s, 2H), 3.89(m, 2H), 3.31(m, 2H), 1.38(s, 9H).
化合物(6)的合成：Synthesis of compound (6):
将化合物(5)(0.6g，2.1mmol)溶于丙酮(7mL)，加入碳酸钾(0.58g，4.2mmol)，体系冷却到0℃，加入醋酸酐(235mg，2.3mmol)，缓慢升至室温反应3小时。后处理，加入氯化铵水溶液(30mL)，乙酸乙酯萃取，合并干燥浓缩，过柱得产物0.5g(收率73％)。1H NMR:(DMSO-d6)：δ7.12(s,1H),6.89(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.89(m,2H),3.31(m,2H),2.14(s,3H),1.38(s,9H)。Compound (5) (0.6 g, 2.1 mmol) was dissolved in acetone (7 mL), potassium carbonate (0.58 g, 4.2 mmol) was added, the system was cooled to 0 °C, acetic anhydride (235 mg, 2.3 mmol) was added, and the temperature was slowly raised to room temperature The reaction was carried out for 3 hours. After post-treatment, aqueous ammonium chloride solution (30 mL) was added, extracted with ethyl acetate, combined, dried and concentrated, and passed through a column to obtain 0.5 g of the product (yield 73%).1 H NMR: (DMSO-d6): δ 7.12(s, 1H), 6.89(s, 1H), 6.65(d, 1H), 6.56(d, 1H), 4.45(s, 2H), 3.89(m , 2H), 3.31 (m, 2H), 2.14 (s, 3H), 1.38 (s, 9H).
化合物(L5)的合成：Synthesis of compound (L5):
将化合物(6)(0.5g，1.5mmol)溶于二氯甲烷(7mL)中，加入三氟乙酸(4mL),室温搅拌1小时。浓缩除去溶剂，残分中加入乙醚，有固体析出，过滤，干燥得固体产物0.3g，收率86％。1H NMR:(DMSO-d6)：δ8.30(s,1H),6.89(s,1H),6.65(d,1H),6.56(d,1H),4.45(s,2H),3.85(m,2H),3.29(m,2H),2.12(s,3H)。Compound (6) (0.5 g, 1.5 mmol) was dissolved in dichloromethane (7 mL), trifluoroacetic acid (4 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was removed by concentration, ether was added to the residue, and a solid was precipitated, which was filtered and dried to obtain 0.3 g of a solid product with a yield of 86%.1 H NMR: (DMSO-d6): δ8.30(s, 1H), 6.89(s, 1H), 6.65(d, 1H), 6.56(d, 1H), 4.45(s, 2H), 3.85(m , 2H), 3.29 (m, 2H), 2.12 (s, 3H).
实施例4：单甲氧基聚乙二醇乙酸与连接链的结合物(mPEG-L5-NHS-20K)的合成Example 4: Synthesis of the conjugate of monomethoxy polyethylene glycol acetic acid and linking chain (mPEG-L5-NHS-20K)
mPEG-L5的合成：Synthesis of mPEG-L5:
将mPEG(20K)-CM-NHS(2g，0.1mmol)溶于无水二氯甲烷中，低温冷却至0℃，加入DIPEA(78mg，0.6mmol)，然后加入化合物L5，缓慢升至室温搅拌过夜。反应液浓缩后残分用异丙醇结晶，抽滤，干燥后得产品mPEG-L5(1.8g，90％)。1H NMR:(DMSO-d6)：δ8.10(s,1H),6.85(s,1H),6.72(d,1H),6.65(d,1H),5.34(s,2H),4.20(m,2H),3.65(m,1800H),3.51(m,2H),3.24(s,3H),2.78(m,4H),2.12(s,3H)。mPEG(20K)-CM-NHS (2g, 0.1mmol) was dissolved in anhydrous dichloromethane, cooled to 0°C at low temperature, DIPEA (78mg, 0.6mmol) was added, then compound L5 was added, and the mixture was slowly raised to room temperature and stirred overnight . After the reaction solution was concentrated, the residue was crystallized with isopropanol, filtered with suction, and dried to obtain the product mPEG-L5 (1.8 g, 90%).1 H NMR: (DMSO-d6): δ8.10(s, 1H), 6.85(s, 1H), 6.72(d, 1H), 6.65(d, 1H), 5.34(s, 2H), 4.20(m , 2H), 3.65(m, 1800H), 3.51(m, 2H), 3.24(s, 3H), 2.78(m, 4H), 2.12(s, 3H).
mPEG-L5-NHS的合成：Synthesis of mPEG-L5-NHS:
将化合物mPEG-L5(1g，0.05mmol)加到反应瓶中，用二氯甲烷(6mL)溶解，N2保护下冷却，加入琥珀酰亚胺碳酸酯(19.0mg，0.075mmol)，搅拌溶解后再加入DIPEA(12.9mg，0.1mmol)，加完后撤去冷浴，室温反应过夜。反应液浓缩，残分用异丙醇结晶，得产品mPEG-L5-NHS。1H NMR:(DMSO-d6)：δ8.10(s,1H),6.85(s,1H),6.72(d,1H),6.65(d,1H),4.65(s,2H),4.15(m,2H),3.65(m,1800H),3.29(m,2H),3.24(s,3H),2.12(s,3H)。The compound mPEG-L5 (1 g, 0.05 mmol) was added to the reaction flask, dissolved in dichloromethane (6 mL), cooled under N2 protection, added with succinimidyl carbonate (19.0 mg, 0.075 mmol), stirred to dissolve Then DIPEA (12.9 mg, 0.1 mmol) was added, the cooling bath was removed after the addition, and the reaction was carried out at room temperature overnight. The reaction solution was concentrated, and the residue was crystallized with isopropanol to obtain the product mPEG-L5-NHS.1 H NMR: (DMSO-d6): δ8.10(s, 1H), 6.85(s, 1H), 6.72(d, 1H), 6.65(d, 1H), 4.65(s, 2H), 4.15(m , 2H), 3.65 (m, 1800H), 3.29 (m, 2H), 3.24 (s, 3H), 2.12 (s, 3H).
实施例5：Y-PEG-L5-NHS-20K的合成Example 5: Synthesis of Y-PEG-L5-NHS-20K
其制备方法参考实施例4。Refer to Example 4 for its preparation method.
实施例6：U-PEG-L5-NHS-20K的合成Example 6: Synthesis of U-PEG-L5-NHS-20K
其制备方法参考实施例4。Refer to Example 4 for its preparation method.
实施例7：8arm-PEG-L5-NHS-20K的合成Example 7: Synthesis of 8arm-PEG-L5-NHS-20K
其制备方法参考实施例4。Refer to Example 4 for its preparation method.
实施例8：mPEG-L5-rhIL-2(20K)的合成Example 8: Synthesis of mPEG-L5-rhIL-2 (20K)
在氮气吹扫下，将在-20℃下储存的mPEG-L5-NHS(20K，实施例4制备)升温至室温。在DMSO中制备mPEG-L5-NHS(20K)储备溶液(200mg/mL)，加入rhIL-2溶液中(反应分组、反应条件如反应物mPEG-L5-NHS：rhIL-2摩尔比(以下简称为PEG：rhIL-2摩尔比)及具体反应pH如表1所示)。混合物中rhIL-2的终浓度是0.5mg/mL。将碳酸氢钠缓冲液(1M，pH9.0)分别添加至混合物以达到20mM的终浓度，室温下反应2h以提供缀合物。2h后，分别添加1M甘氨酸(pH6.0)至终浓度100mM来终止反应。Under a nitrogen purge, mPEG-L5-NHS (20K, prepared in Example 4) stored at -20°C was warmed to room temperature. A stock solution of mPEG-L5-NHS (20K) (200 mg/mL) was prepared in DMSO and added to the rhIL-2 solution (reaction grouping, reaction conditions such as reactant mPEG-L5-NHS:rhIL-2 molar ratio (hereinafter referred to as PEG: rhIL-2 molar ratio) and specific reaction pH are shown in Table 1). The final concentration of rhIL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer (1 M, pH 9.0) was separately added to the mixture to reach a final concentration of 20 mM and reacted at room temperature for 2 h to provide the conjugate. After 2 h, the reactions were terminated by adding 1 M glycine (pH 6.0) to a final concentration of 100 mM, respectively.
对终止后的反应体系开展RP-HPLC测定不同反应组的偶联情况，根据偶联物的迁移情况(左移代表偶联度高)分析评价偶联效率和偶联度，RP-HPLC谱图如图1所示，由图1可知，偶联度及偶联效率均为反应3组〉反应4组〉反应5组〉反应2组〉反应6组〉反应1组。进一步开展SEC+MALS分析反应3组，评价偶联度，结果如图2所示。RP-HPLC was performed on the terminated reaction system to determine the coupling situation of different reaction groups, and the coupling efficiency and degree of coupling were analyzed and evaluated according to the migration of the conjugates (the left shift represents a high degree of coupling), and the RP-HPLC spectrum As shown in Figure 1, it can be seen from Figure 1 that the degree of coupling and the coupling efficiency are both
本实施例制备的偶联物mPEG-L5-rhIL-2(20K)的偶联度结果如表1所示。Table 1 shows the results of the coupling degree of the conjugate mPEG-L5-rhIL-2 (20K) prepared in this example.
表1：实施例8制备的偶联物的SEC-MALS偶联度结果表Table 1: SEC-MALS coupling degree result table of the conjugate prepared in Example 8
上表所获产品可以分为三类：偶联度为4-7的mPEG-L5-rhIL-2、偶联度为3-5的mPEG-L5-rhIL-2和偶联度为1-3的mPEG-L5-rhIL-2，最优含量可控制在大于90％。The products obtained in the above table can be divided into three categories: mPEG-L5-rhIL-2 with a coupling degree of 4-7, mPEG-L5-rhIL-2 with a coupling degree of 3-5, and mPEG-L5-rhIL-2 with a coupling degree of 1-3 The optimal content of mPEG-L5-rhIL-2 can be controlled at more than 90%.
接着分别将终止反应的混合物用纯化水稀释以提供低于0.5mS/cm(25℃)的电导率。使用冰醋酸将pH值调节至4.0，然后进行柱层析纯化，如图3所示。结合RP-HPLC分析(图4)，PEG在穿透峰中富集，mPEG-L5-rhIL-2在0～50％B(0～0.5M NaCl梯度)中洗脱，分段收集各洗脱峰(A3～A8)，观察到偶联度由高到低分布。The quenched mixtures were then individually diluted with purified water to provide conductivity below 0.5 mS/cm (25°C). The pH was adjusted to 4.0 using glacial acetic acid and then purified by column chromatography as shown in Figure 3. Combined with RP-HPLC analysis (Figure 4), PEG was enriched in the breakthrough peak, mPEG-L5-rhIL-2 was eluted in 0-50% B (0-0.5M NaCl gradient), and each elution was collected in sections Peaks (A3-A8), the degree of coupling was observed to be distributed from high to low.
实施例9：mPEG-L5-rhIL-2(40K)的合成Example 9: Synthesis of mPEG-L5-rhIL-2 (40K)
其制备方法参照实施例8。Refer to Example 8 for its preparation method.
实施例10：Y-PEG-L5-rhIL-2(20k)的合成Example 10: Synthesis of Y-PEG-L5-rhIL-2 (20k)
其制备方法参照实施例8。Refer to Example 8 for its preparation method.
实施例11：U-PEG-L5-rhIL-2(20k)的合成Example 11: Synthesis of U-PEG-L5-rhIL-2 (20k)
其制备方法参照实施例8。Refer to Example 8 for its preparation method.
实施例12：8arm-PEG-L5-rhIL-2(20K)的合成Example 12: Synthesis of 8arm-PEG-L5-rhIL-2 (20K)
其制备方法参照实施例8。Refer to Example 8 for its preparation method.
实施例13：mPEG-L1-rhIL-2(20K)的合成Example 13: Synthesis of mPEG-L1-rhIL-2 (20K)
在氮气吹扫下，将在-20℃下储存的mPEG-L1-NHS(20K，其制备方法参考实施例2)升温至室温。在DMSO中制备mPEG-L1-NHS(20K)储备溶液(200mg/mL)，加入rhIL-2溶液中(PEG：rIL-2摩尔比分别为1:1、3:1、10：1、30:1)。混合物中rhIL-2的终浓度是0.5mg/mL。将碳酸氢钠缓冲液(1M，pH9.0)分别添加至混合物以达到20mM的终浓度，室温下反应2h以提供缀合物。2h后，分别添加1M甘氨酸(pH6.0)至终浓度100mM来终止反应。接着分别将终止反应的混合物用纯化水稀释以提供低于0.5mS/cm(25℃)的电导率。使用冰醋酸将pH值调节至4.0，然后进行柱层析纯化。Under nitrogen purging, mPEG-L1-NHS (20K, whose preparation method refers to Example 2) stored at -20°C was warmed to room temperature. A stock solution of mPEG-L1-NHS (20K) (200 mg/mL) was prepared in DMSO and added to the rhIL-2 solution (PEG:rIL-2 molar ratios of 1:1, 3:1, 10:1, 30:1, respectively). 1). The final concentration of rhIL-2 in the mixture was 0.5 mg/mL. Sodium bicarbonate buffer (1 M, pH 9.0) was separately added to the mixture to reach a final concentration of 20 mM and reacted at room temperature for 2 h to provide the conjugate. After 2 h, the reactions were terminated by adding 1 M glycine (pH 6.0) to a final concentration of 100 mM, respectively. The quenched mixtures were then individually diluted with purified water to provide conductivity below 0.5 mS/cm (25°C). The pH was adjusted to 4.0 using glacial acetic acid, followed by column chromatography purification.
实施例14：经本发明修饰的IL-2的药效学研究Example 14: Pharmacodynamic studies of IL-2 modified by the present invention
采用实施例8的反应3组条件进行PEG与rIL-2偶联，偶联混合物经离子交换色谱纯化，分离得到PEG与rIL-2偶联物进行药效学实验。采用C57BL/6小鼠的皮下黑色素瘤B16模型评估mPEG-L5-rhIL-2偶联物(以下简称为PEG-rhIL-2)的抑制肿瘤效果。实验过程如下：每只5-6周龄的C57BL/6小鼠在背部中部植入106个B16细胞。肿瘤生长至可测量尺寸后，实验动物随机分组，每组6只，以不同剂量浓度和剂量方案对小鼠施用测试化合物：rhIL-2、PEG-rhIL-2及溶剂对照。每隔一天测量体重和肿瘤体积。药效实验分组如表2所示，实验结果如表3和图5所示。PEG and rIL-2 were conjugated under the reaction conditions of Example 8, and the conjugated mixture was purified by ion exchange chromatography, and the PEG and rIL-2 conjugates were separated and subjected to pharmacodynamics experiments. The tumor-inhibitory effect of mPEG-L5-rhIL-2 conjugate (hereinafter referred to as PEG-rhIL-2) was evaluated by subcutaneous melanoma B16 model of C57BL/6 mice. The experimental procedure was as follows: Each 5-6 week old C57BL/6 mouse was implanted with 106 B16 cells in the middle of the back. After the tumor grew to a measurable size, the experimental animals were randomly divided into groups of 6, and the mice were administered the test compounds: rhIL-2, PEG-rhIL-2 and solvent control at different dose concentrations and dose schedules. Body weight and tumor volume were measured every other day. The groupings of the efficacy experiments are shown in Table 2, and the experimental results are shown in Table 3 and Figure 5.
表2：药效实验分组Table 2: Pharmacodynamic test groupings
表3：药效实验结果Table 3: Pharmacodynamic test results
由数据可知，给药第9天时，与溶剂对照组肿瘤体积(1260mm3)相比，经本发明修饰的rhIL-2组肿瘤体积(310mm3)大大减小，与未修饰的rhIL-2组肿瘤体积(650mm3)相比，经本发明修饰后的mPEG-L5-rhIL-2组的抑瘤效果提高了27％，且其给药频次大大降低，作为抗肿瘤药物具有降低给药频率、大大提高药物的生物利用度和病人的依从性的优势。It can be seen from the data that on the 9th day of administration, compared with the tumor volume of the solvent control group (1260 mm3 ), the tumor volume (310 mm 3) of the rhIL-2 group modified by the present invention was greatly reduced, which was significantly lower than that of the unmodified rhIL-2 group. Compared with the tumor volume (650mm3 ), the tumor inhibitory effect of the mPEG-L5-rhIL-2 group modified by the present invention was increased by 27%, and the administration frequency was greatly reduced. The advantage of greatly improving drug bioavailability and patient compliance.
本发明实施例中所述的“偶联度”是指每个IL-2分子上偶联的PEG分子数目(即本发明中通式Ⅸ中所述的n值)，如，偶联度为4的mPEG-L5-rhIL-2，是指每个rhIL-2分子上偶联4个mPEG-L5链段；偶联度为4-7的mPEG-L5-rhIL-2，是指含有偶联度为4的mPEG-L5-rhIL-2、偶联度为5的mPEG-L5-rhIL-2、偶联度为6的mPEG-L5-rhIL-2和偶联度为7的mPEG-L5-rhIL-2的混合物。The "coupling degree" described in the examples of the present invention refers to the number of PEG molecules coupled to each IL-2 molecule (that is, the n value described in the general formula IX in the present invention), for example, the coupling degree is 4 mPEG-L5-rhIL-2 refers to the coupling of 4 mPEG-L5 segments on each rhIL-2 molecule; mPEG-L5-rhIL-2 with a coupling degree of 4-7 refers to the mPEG-L5-rhIL-2 with a degree of 4, mPEG-L5-rhIL-2 with a degree of conjugation of 5, mPEG-L5-rhIL-2 with a degree of conjugation of 6, and mPEG-L5- rhIL-2 mixture.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. within.
Claims (15)
Translated fromChinese
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710919466 | 2017-09-30 | ||
| CN2017109194665 | 2017-09-30 | ||
| CN201811043884.3ACN109589415B (en) | 2017-09-30 | 2018-09-07 | Conjugate of polyethylene glycol-polypeptide and protein medicine |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811043884.3ADivisionCN109589415B (en) | 2017-09-30 | 2018-09-07 | Conjugate of polyethylene glycol-polypeptide and protein medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114668852Atrue CN114668852A (en) | 2022-06-28 |
Family
ID=65900673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210270700.7APendingCN114668852A (en) | 2017-09-30 | 2018-09-07 | A combination of polyethylene glycol-polypeptide and protein drugs |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114668852A (en) |
| WO (1) | WO2019062665A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117603335A (en)* | 2024-01-19 | 2024-02-27 | 北京春雷杰创生物科技有限公司 | Human serum amyloid A mutant |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6180095B1 (en)* | 1997-12-17 | 2001-01-30 | Enzon, Inc. | Polymeric prodrugs of amino- and hydroxyl-containing bioactive agents |
| WO2006116948A1 (en)* | 2005-04-30 | 2006-11-09 | Chengdu Institute Of Biological Products | Interleukin-6 polyethylene glycol conjugate and its preparing method and use |
| CN103965458A (en)* | 2013-01-28 | 2014-08-06 | 天津键凯科技有限公司 | Polyethylene glycol-amino acid oligopeptide-dasatinib conjugate and pharmaceutical composition thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104078B (en)* | 2006-07-11 | 2011-11-02 | 北京美倍他药物研究有限公司 | Polypeptide and protein medicine polyethylene coupling compound |
| CN101104077A (en)* | 2006-07-12 | 2008-01-16 | 北京紫辰医药生物技术研究所 | Recombinant human interleukin-2 and polyethylene coupling compound |
| PL2396036T3 (en)* | 2009-02-13 | 2017-12-29 | Immunomedics, Inc. | Immunoconjugates with an intracellularly-cleavable linkage |
| CN103083680B (en)* | 2011-11-07 | 2014-12-24 | 北京键凯科技有限公司 | Polyethylene glycol (PEG)-amino acid oligopeptide-irinotecan combo and its medicinal composition |
- 2018
- 2018-09-07CNCN202210270700.7Apatent/CN114668852A/enactivePending
- 2018-09-21WOPCT/CN2018/106972patent/WO2019062665A1/ennot_activeCeased
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6180095B1 (en)* | 1997-12-17 | 2001-01-30 | Enzon, Inc. | Polymeric prodrugs of amino- and hydroxyl-containing bioactive agents |
| WO2006116948A1 (en)* | 2005-04-30 | 2006-11-09 | Chengdu Institute Of Biological Products | Interleukin-6 polyethylene glycol conjugate and its preparing method and use |
| CN103965458A (en)* | 2013-01-28 | 2014-08-06 | 天津键凯科技有限公司 | Polyethylene glycol-amino acid oligopeptide-dasatinib conjugate and pharmaceutical composition thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117603335A (en)* | 2024-01-19 | 2024-02-27 | 北京春雷杰创生物科技有限公司 | Human serum amyloid A mutant |
| CN117603335B (en)* | 2024-01-19 | 2024-04-30 | 北京春雷杰创生物科技有限公司 | Human serum amyloid A mutant |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019062665A1 (en) | 2019-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6620916B1 (en) | Modified physiologically active proteins and medicinal compositions containing the same | |
| CN108452321B (en) | Antibody-drug conjugate with acidic self-stabilizing joint | |
| JP4272537B2 (en) | Y-shaped branched hydrophilic polymer derivatives, methods for their preparation, binding products of said derivatives and drug molecules, and pharmaceutical compositions comprising said binding products | |
| AU749321B2 (en) | Polymeric derivatives of camptothecins | |
| JP5977229B2 (en) | Sustained release from macromolecular conjugates | |
| WO2016026458A1 (en) | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof | |
| WO2024140450A1 (en) | Antibody-conjugated drug of n-alkoxyalkyl substituted camptothecin derivative | |
| CN108727208B (en) | A kind of linker compound, polyethylene glycol-linker combination and derivatives thereof and polyethylene glycol-linker-drug combination | |
| CN103140512B (en) | Polymer-conjugated MetAP2 inhibitors and methods of treatment for their use | |
| WO2022022649A1 (en) | Dual-drug link assembly unit and dual-drug targeting linker-drug conjugate | |
| CN104147612B (en) | Tumor microenvironment specific activated micromolecular targeted conjugate and application thereof | |
| JP2024503508A (en) | Immunomodulatory antibody-drug conjugates | |
| CN111001012A (en) | Hydrophilic carbonate type antibody coupling drug | |
| CN112955434B (en) | Drug conjugates comprising quinoline derivatives | |
| CN109589415B (en) | Conjugate of polyethylene glycol-polypeptide and protein medicine | |
| WO2008052428A1 (en) | Preparation method and conjugate with drug molecule thereof | |
| JP2005514505A (en) | Preparation and use of multi-arm dendritic and functional PEG | |
| WO2019034175A1 (en) | Non-natural amatoxin-type antibody conjugate | |
| US20200247842A1 (en) | Immune-stimulating soluble doxorubicin-conjugated complex | |
| CN114668852A (en) | A combination of polyethylene glycol-polypeptide and protein drugs | |
| CN118401256A (en) | Bienamide linker for antibody drug conjugates | |
| US20030103934A1 (en) | Drugs having long-term retention in target tissue | |
| WO2004029018A1 (en) | Glycerol derivative | |
| KR100330635B1 (en) | Anthracycline conjugates, methods for their preparation and pharmaceutical compositions comprising the same | |
| CA2696478C (en) | Protein-polymer conjugates |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | Application publication date:20220628 |