WO2025007839A1

WO2025007839A1 - Conjugate compound, and preparation method thereof and use thereof

Info

Publication number: WO2025007839A1
Application number: PCT/CN2024/102918
Authority: WO
Inventors: 刘力锋; 戴衍; 陈家煜; 梁超
Original assignee: Wayne Biotechnology Co Ltd
Current assignee: Wayne Biotechnology Co Ltd
Priority date: 2023-07-03
Filing date: 2024-07-01
Publication date: 2025-01-09
Anticipated expiration: 2026-01-03

Abstract

A conjugate compound or a pharmaceutically acceptable salt thereof. The conjugate compound comprises a payload, a linker and a targeting molecule, wherein the targeting molecule has a bicyclic polypeptide ligand.

Description

A coupled compound and its preparation method and use

Technical Field

本发明涉及药物领域，具体涉及一种具有双环多肽靶向分子的偶联体化合物。The present invention relates to the field of medicines, and in particular to a coupled compound with a bicyclic polypeptide targeting molecule.

Background Art

多肽偶联药物(PDC)是一种新型的分子药物递送系统，由有效载荷、连接体和靶向分子三部分构成，是将靶向肽作为靶向给药载体，与有效载荷共价偶联，来增强药物的靶向性。通常药物通过共价连接体与靶向多肽分子的结合后，赋予多肽和药物的双向功能，能够起到促进药物的杀伤或靶向作用，PDC药物是一种有前景的癌症治疗方式。目前Bicycle Therapeutics在开发的BT8009正处于临床II期，是一款双环肽偶联毒素药物。与抗体偶联药物相比，环肽偶联药物具有强的肿瘤穿透性、较低的免疫原性以及更低的生产成本等优势。Peptide-drug conjugates (PDC) are a new type of molecular drug delivery system, which consists of three parts: payload, linker and targeting molecule. The targeting peptide is used as a targeted drug delivery carrier and is covalently coupled to the payload to enhance the targeting of the drug. Usually, after the drug is combined with the targeting peptide molecule through a covalent linker, the peptide and the drug are given a bidirectional function, which can promote the killing or targeting effect of the drug. PDC drugs are a promising cancer treatment method. Currently, BT8009, which is being developed by Bicycle Therapeutics, is in Phase II clinical trials and is a bicyclic peptide-conjugated toxin drug. Compared with antibody-conjugated drugs, cyclic peptide-conjugated drugs have the advantages of strong tumor penetration, lower immunogenicity and lower production costs.

连接子(Linker)在多肽-药物偶联物(PDC)结构中起着至关重要的作用，其会影响PDC的药代动力学参数、治疗指数和药效。Linker需要维持PDC在血流中的稳定性，这样才能防止连接子在血液中被分解提前释放毒素。如果连接子在血液中不够稳定，就会导致PDC在进入肿瘤细胞之前就被分解，药物对肿瘤的治疗作用就会降低，甚至会误杀其他正常细胞带来较大的副作用。而到达肿瘤位置时，Linker应能够保证快速释放细胞毒性药物，起到杀伤肿瘤的效果。由此，连接子的开发对于PDC药品的安全性、有效性具有显著的决定作用。Linkers play a vital role in the structure of peptide-drug conjugates (PDCs), which will affect the pharmacokinetic parameters, therapeutic index and efficacy of PDCs. Linkers need to maintain the stability of PDCs in the bloodstream to prevent linkers from being decomposed in the blood and releasing toxins prematurely. If the linker is not stable enough in the blood, PDCs will be decomposed before entering tumor cells, and the therapeutic effect of the drug on tumors will be reduced, and other normal cells may even be killed by mistake, causing greater side effects. When reaching the tumor site, Linkers should be able to ensure the rapid release of cytotoxic drugs to kill tumors. Therefore, the development of linkers plays a significant role in the safety and effectiveness of PDC drugs.

领域内迫切需要获得改进的多肽偶联药物，拓宽治疗窗口以及增强药效减少药物来的的毒副作用。开发在血液系统循环中稳定且到达肿瘤后能快速释放的，具有更好抗肿瘤效果的多肽偶联药物将具有广泛的应用前景。There is an urgent need to obtain improved peptide-coupled drugs in the field to broaden the therapeutic window, enhance drug efficacy and reduce drug toxicity. The development of peptide-coupled drugs that are stable in the blood system circulation and can be quickly released after reaching the tumor, with better anti-tumor effects, will have broad application prospects.

发明内容Summary of the invention

本发明的目的在于提供一种新的PDC偶联体化合物及其制备方法、含有其的药物组合物、以及其在药物中的用途。具体地，本发明提供了以下技术方案：The object of the present invention is to provide a new PDC conjugate compound and a preparation method thereof, a pharmaceutical composition containing the same, and its use in medicine. Specifically, the present invention provides the following technical solutions:

本发明一方面提供了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物包含有效载荷，连接体和靶向分子，其中所述靶向分子包含肽配体和分子支架，所述肽配体包含被两个环序列隔开的三个氨基酸残基，分子支架通过共价键与三个氨基酸残基相连。On the one hand, the present invention provides a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound comprises a payload, a linker and a targeting molecule, wherein the targeting molecule comprises a peptide ligand and a molecular scaffold, the peptide ligand comprises three amino acid residues separated by two ring sequences, and the molecular scaffold is connected to the three amino acid residues by a covalent bond.

作为优选的方案，所述三个氨基酸残基各自独立地选自Cys、hCys、βCys、Pen、Dap、N-甲基-Dap残基，所述肽配体中的一个或多个天然氨基酸经过化学修饰。 As a preferred embodiment, the three amino acid residues are independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap residues, and one or more natural amino acids in the peptide ligand are chemically modified.

作为优选的方案，所述肽配体包含的两个环序列分别为P(1Nal)(D-Asp)和M(HArg)DWSTP(HyP)W，所述环序列中的一个或多个氨基酸经过化学修饰。As a preferred embodiment, the two loop sequences contained in the peptide ligand are P(1Nal)(D-Asp) and M(HArg)DWSTP(HyP)W, and one or more amino acids in the loop sequences are chemically modified.

作为优选的方案，所述三个氨基酸残基各自独立地选自Cys、hCys、βCys、Pen、Dap、N-甲基-Dap残基，所述肽配体包含的两个环序列分别为P(1Nal)(D-Asp)和M(HArg)DWSTP(HyP)W，所述环序列中的一个或多个氨基酸经过化学修饰。As a preferred embodiment, the three amino acid residues are independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap residues, the two ring sequences contained in the peptide ligand are P(1Nal)(D-Asp) and M(HArg)DWSTP(HyP)W, and one or more amino acids in the ring sequence are chemically modified.

作为优选的方案，所述三个氨基酸残基各自独立地选自Cys、Pen残基，所述肽配体包含的两个环序列分别为P(1Nal)(D-Asp)和M(HArg)DWSTP(HyP)W，所述环序列中的一个或多个氨基酸经过化学修饰。As a preferred embodiment, the three amino acid residues are independently selected from Cys and Pen residues, the two ring sequences contained in the peptide ligand are P(1Nal)(D-Asp) and M(HArg)DWSTP(HyP)W, and one or more amino acids in the ring sequence are chemically modified.

作为优选的方案，上述方案中所述的化学修饰包含烷基基化修饰、卤化修饰、硫代修饰、磷酸化修饰、酰基化修饰、羟基化、羧基化中的一种或任意几种，或本领域常用的化学修饰As a preferred embodiment, the chemical modification described in the above embodiment includes one or more of alkylation, halogenation, thiolation, phosphorylation, acylation, hydroxylation, carboxylation, or chemical modification commonly used in the art.

作为进一步优选的方案，所述烷基化修饰选自甲基修饰、乙基修饰、丙基修饰和异丙基修饰，卤化修饰选自氟修饰、氯修饰和溴修饰。As a further preferred embodiment, the alkylation modification is selected from methyl modification, ethyl modification, propyl modification and isopropyl modification, and the halogenation modification is selected from fluorine modification, chlorine modification and bromine modification.

作为优选的方案，氨基酸残基选自Cys、Pen残基，所述肽配体选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8：As a preferred embodiment, the amino acid residue is selected from Cys and Pen residues, and the peptide ligand is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8:

-Cys-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(HyP)W-Cys-(SEQ ID NO:1)；-Cys-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(HyP)W-Cys-(SEQ ID NO:1);

-Cys-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:2)；-Cys-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:2);

-Pen-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:3)；-Pen-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:3);

-Cys-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:4)；-Cys-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:4);

-Pen-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:5)；-Pen-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:5);

-Pen-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:6)；-Pen-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:6);

-Cys-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:7)；-Cys-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:7);

-Pen-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:8)；-Pen-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Pen-(SEQ ID NO:8);

其中，X选自被化学修饰的脯氨酸，所述化学修饰为甲氧基修饰、氟化修饰或硫代修饰中的一种或任意几种。Wherein, X is selected from chemically modified proline, and the chemical modification is one or any combination of methoxy modification, fluorination modification or thio modification.

作为优选的方案，分子支架选自1，1’,1”-(1，3，5-三嗪烷-1，3，5-三基)三丙-2-烯-1-酮。As a preferred embodiment, the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one.

作为优选的方案，所述靶向分子选自如下结构：
As a preferred solution, the targeting molecule is selected from the following structures:

作为优选的方案，所述连接体选自-PABC-Cit-Val-连接子-β-Ala-Sar10-。其中PABC代表对氨基苄基氨基甲酸酯；Sar10代表10个Sar。As a preferred embodiment, the linker is selected from -PABC-Cit-Val-Linker-β-Ala-Sar10-, wherein PABC represents p-aminobenzylcarbamate; Sar10 represents 10 Sars.

作为优选的方案，所述连接子选自肽类连接子、pH依赖型连接子、二硫化物连接子或者上述连接子的组合。As a preferred embodiment, the linker is selected from a peptide linker, a pH-dependent linker, a disulfide linker or a combination of the above linkers.

作为优选的方案，所述连接子选自：

As a preferred embodiment, the linker is selected from:

本发明另一方面提供了一种靶向分子，选自如下结构：
Another aspect of the present invention provides a targeting molecule selected from the following structures:

本发明另一方面提供了一种偶联体化合物，所述偶联体化合物包含有效载荷，连接体和靶向分子，其中所述靶向分子包含肽配体和分子支架，所述肽配体包含被两个环序列隔开的三个氨基酸残基，分子支架通过共价键与三个氨基酸残基相连，所述偶联体化合物包含如下结构：
Another aspect of the present invention provides a coupled compound, the coupled compound comprising a payload, a connector and a targeting molecule, wherein the targeting molecule comprises a peptide ligand and a molecular scaffold, the peptide ligand comprises three amino acid residues separated by two ring sequences, and the molecular scaffold is connected to the three amino acid residues by a covalent bond, and the coupled compound comprises the following structure:

其中R₁和R₂分别独立地选自氢和C_1-6烷基。wherein _R1 and _R2 are independently selected from hydrogen and _C1-6 alkyl.

作为优选的方案，所述偶联体化合物包含如下结构：
As a preferred embodiment, the coupled compound comprises the following structure:

其中R₁和R₂分别独立地选自氢和C_1-6烷基；wherein R ₁ and R ₂ are independently selected from hydrogen and C _1-6 alkyl;

n选自0，1，2，3，4，5，6，7或8。n is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8.

作为优选的方案，所述三个氨基酸残基各自独立地选自Cys、hCys、βCys、Pen、Dap、N-甲基-Dap残基，所述肽配体中的一个或多个天然氨基酸经过化学修饰，所述连接体包含如下结构： As a preferred embodiment, the three amino acid residues are independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap residues, one or more natural amino acids in the peptide ligand are chemically modified, and the linker comprises the following structure:

作为进一步优选的方案，所述连接体包含如下结构 As a further preferred embodiment, the linker comprises the following structure

作为优选的方案，所述肽配体包含的两个环序列分别为P(1Nal)(D-Asp)和M(HArg)DWSTP(HyP)W，所述环序列中的一个或多个氨基酸经过化学修饰，所述连接体包含如下结构： As a preferred embodiment, the two loop sequences contained in the peptide ligand are P(1Nal)(D-Asp) and M(HArg)DWSTP(HyP)W, one or more amino acids in the loop sequence are chemically modified, and the linker comprises the following structure:

作为优选的方案，偶联体化合物靶向Nectin-4、Kallikrein、MT1-MMP、CD137、Epha2、IL-17、PSMA、PD-L1、αvβ3、CD38、CAIX、OX40、PBP、TSLP、ACE2、TfR1、FAPα、NK细胞或TREM2。 As a preferred embodiment, the conjugate compound targets Nectin-4, Kallikrein, MT1-MMP, CD137, Epha2, IL-17, PSMA, PD-L1, αvβ3, CD38, CAIX, OX40, PBP, TSLP, ACE2, TfR1, FAPα, NK cells or TREM2.

作为进一步优选的方案，所述偶联体化合物包含如下结构：所述氨基酸残基选自Cys、Pen残基，所述肽配体选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8：As a further preferred embodiment, the coupled compound comprises the following structure: The amino acid residue is selected from Cys and Pen residues, and the peptide ligand is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8:

作为进一步优选的方案，分子支架选自1，1’,1”-(1，3，5-三嗪烷-1，3，5-三基)三丙-2-烯-1-酮。As a further preferred embodiment, the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one.

作为进一步优选的方案，所述靶向分子选自如下结构：
As a further preferred embodiment, the targeting molecule is selected from the following structures:

作为进一步优选的方案，所述连接体选自-PABC-Cit-Val-连接子-β-Ala-Sar10-。 As a further preferred embodiment, the linker is selected from -PABC-Cit-Val-linker-β-Ala-Sar10-.

作为进一步优选的方案，为连接子。As a further preferred solution, For the connector.

本发明另一方面提供了一种偶联体化合物，包含有效载荷、连接体和靶向分子，所述靶向分子通过连接体连接至有效载荷，所述连接体和靶向分子具有如下结构：
Another aspect of the present invention provides a conjugate compound, comprising a payload, a linker and a targeting molecule, wherein the targeting molecule is connected to the payload via the linker, and the linker and the targeting molecule have the following structure:

作为优选的方案，上述各方案中有效载荷选自顺铂、卡铂、奥沙利铂、氮芥、环磷酰胺、苯丁酸氮芥、硫唑嘌呤、巯嘌呤、异环磷酰胺、嘧啶类似物、长春新碱、长春花碱、长春瑞滨、长春地辛、依托泊苷、替尼泊苷、伊立替康、拓扑替康、紫杉醇、安丫啶、依托泊苷、磷酸依托泊苷、替尼泊苷、多柔比星、表柔比星、喜树碱及其衍生物、埃博霉素及其衍生物、博来霉素及其衍生物、普卡霉素及其衍生物、更生霉素及其衍生物、美登素及其衍生物、奥瑞他汀及其衍生物。As a preferred scheme, in each of the above schemes, the effective load is selected from cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, ifosfamide, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, acridine, etoposide, etoposide phosphate, teniposide, doxorubicin, epirubicin, camptothecin and its derivatives, epothilone and its derivatives, bleomycin and its derivatives, plicamycin and its derivatives, dactinomycin and its derivatives, maytansine and its derivatives, and auristatin and its derivatives.

作为优选的方案，上述各方案中所述有效载荷选自美登木素生物碱、单甲基奥瑞他汀或喜树碱衍生物。As a preferred embodiment, the effective load in the above embodiments is selected from maytansinoids, monomethyl auristatin or camptothecin derivatives.

作为优选的方案，上述各方案中所述有效载荷选自MMAE(一甲基澳瑞他汀E)。As a preferred embodiment, the effective load in the above embodiments is selected from MMAE (monomethyl auristatin E).

作为最优方案，选自下组：

As the best solution, select from the following group:

本发明另一方面提供了一种药物组合物，其包含上述的化合物或其药学上可接受盐及药学上可接受的载体。 Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

本发明另一方面提供了上述的化合物、其立体异构体、互变异构体或其药学上可接受盐或上述药物组合物在制备治疗和/或预防Nectin-4过度表达的疾病的药物中的用途。Another aspect of the present invention provides use of the above-mentioned compound, its stereoisomer, tautomer or pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for treating and/or preventing a disease with overexpression of nectin-4.

本发明另一方面提供了上述的化合物、其立体异构体、互变异构体或其药学上可接受盐或上述的药物组合物在制备预防和/或治疗肿瘤的药物中的用途。Another aspect of the present invention provides the use of the above-mentioned compound, its stereoisomer, tautomer or pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of drugs for preventing and/or treating tumors.

本发明另一方面提供了上述的化合物、其立体异构体、互变异构体或其药学上可接受盐或上述的药物组合物在制备预防和/或治疗血癌、宫颈癌、肺癌、前列腺癌、间皮瘤、甲状腺癌、肾癌、胆道癌、膀胱癌、乳腺癌、咽癌、喉癌、食道癌、肉瘤、皮肤癌、卵巢癌、肝癌、结直肠癌或胰腺癌的药物中的用途。Another aspect of the present invention provides the use of the above-mentioned compound, its stereoisomer, tautomer or pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and/or treating blood cancer, cervical cancer, lung cancer, prostate cancer, mesothelioma, thyroid cancer, kidney cancer, bile duct cancer, bladder cancer, breast cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, sarcoma, skin cancer, ovarian cancer, liver cancer, colorectal cancer or pancreatic cancer.

应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

DETAILED DESCRIPTION

本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一类结构新颖的偶联体化合物。在此基础上完成了本发明。The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a class of coupled compounds with novel structures, on the basis of which the present invention was completed.

除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

“有效载荷”在本发明中指拟递送至靶细胞或组织的分子或物质。非限制性地，有效载荷可以是旨在用于诊断、治疗或预防对象中的疾病的任意分子或物质。在一些情形中，有效载荷具有小于或等于约5kDa的分子量。在一些情形中，有效载荷具有小于或等于约1.5kDa的分子量。在一些情形中，所述有效载荷是已经被适当的药物审批和注册机构(例如，FDA，EMEA或NMPA)认为使用安全且有效的药物或诊断试剂。"Payload" in the present invention refers to a molecule or substance intended to be delivered to a target cell or tissue. Without limitation, a payload can be any molecule or substance intended for diagnosis, treatment or prevention of a disease in a subject. In some cases, the payload has a molecular weight of less than or equal to about 5 kDa. In some cases, the payload has a molecular weight of less than or equal to about 1.5 kDa. In some cases, the payload is a drug or diagnostic agent that has been deemed safe and effective for use by an appropriate drug approval and registration agency (e.g., FDA, EMEA or NMPA).

有效载荷可以在连接至本申请的偶联体化合物之前具有游离的氨基或者羧基，有效载荷通过上述氨基或者羧基与偶联体化合物的相应部分(例如，连接子)的基团发生酰化反应从而偶联至偶联体化合物。在一些实施方式中，对上述游离的氨基或者羧基的修饰(例如，通过偶联至本申请的偶联体化合物)会显著降低有效载荷的活性(例如，降低至少50％、60％、70％、80％、90％、95％、98％或99％)。The useful load may have a free amino group or a carboxyl group before being connected to the conjugate compound of the present application, and the useful load is coupled to the conjugate compound by an acylation reaction between the above-mentioned amino group or the carboxyl group and the group of the corresponding part (e.g., a linker) of the conjugate compound. In some embodiments, modification of the above-mentioned free amino group or carboxyl group (e.g., by conjugation to the conjugate compound of the present application) can significantly reduce the activity of the useful load (e.g., by reducing at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%).

有效载荷可以是小分子化合物、核苷酸(例如，DNA、质粒DNA、RNA、siRNA、反义寡核苷酸或核酸适体等)、肽或蛋白(例如，酶)。小分子化合物指的是具有小于或等于约2kDa分子量的化合物，例如喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物(例如，MMAE)、美登素及其任何衍生物、环氧化酶-2抑制剂、放射性核素络合物、紫杉醇及其任何衍生物、埃博霉素及其任何衍生物、博来霉素及其任何衍生物、更生霉素及其任何衍生物、普卡霉素及其任何衍生物，以及丝裂霉素C。The payload can be a small molecule compound, a nucleotide (e.g., DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotide or nucleic acid aptamer, etc.), a peptide or a protein (e.g., an enzyme). A small molecule compound refers to a compound having a molecular weight of less than or equal to about 2 kDa, such as camptothecin and any derivative thereof, auristatin and any derivative thereof (e.g., MMAE), maytansine and any derivative thereof, cyclooxygenase-2 inhibitors, radionuclide complexes, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C.

本发明所有方案中的有效载荷均可选自顺铂、卡铂、奥沙利铂、氮芥、环磷酰胺、苯丁酸氮芥、硫唑嘌呤、巯嘌呤、异环磷酰胺、嘧啶类似物、长春新碱、长春花碱、长春瑞滨、长春地辛、依托泊苷、替尼泊苷、伊立替康、拓扑替康、紫杉醇、安丫啶、依托泊苷、磷酸依托泊苷、替尼泊苷、多柔比星、表柔比星、喜树碱及其衍生物、埃博霉素及其衍生物、博来霉素及其衍生物、普卡霉素及其衍生物、更生霉素及其衍生物、美登素及其衍生物、奥瑞他汀及其衍生物。The effective load in all schemes of the present invention can be selected from cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, ifosfamide, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, acetylcholine, irinotecan, cyclophosphamide, chlorambucil, thiopurine, thiopurine, ifosfamide, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, acetylcholine, irinotecan, cyclophosphamide ... Toposide, etoposide phosphate, teniposide, doxorubicin, epirubicin, camptothecin and its derivatives, epothilone and its derivatives, bleomycin and its derivatives, plicamycin and its derivatives, dactinomycin and its derivatives, maytansine and its derivatives, auristatin and its derivatives.

“连接体”在本发明中指将有效载荷与靶向分子共价连接的部分，在特定条件下裂解释放有效载荷，连接体包括用于将有效载荷与至少一个靶向分子连接的官能团。官能团可以含有两个反应性部分，一个用于与有效载荷连接，另一个用于与靶向分子连接。官能团彼此之间可以相同或不同。为控制裂解的速率和伴随的效应物分子的释放，连接体可做适当修饰，如在其与肽配体或有效载荷连接处连接一些基团增加链长度，以及在裂解键周围增加基团修饰控制裂解键的阻碍，本发明的连接体包括基于上述加以修饰的连接体衍生物。本发明的连接体可以选自-PABC-Cit-Val-连接子-β-Ala-Sar10-。"Linker" in the present invention refers to a part that covalently connects the payload to the targeting molecule, and is cleaved under specific conditions to release the payload. The linker includes a functional group for connecting the payload to at least one targeting molecule. The functional group may contain two reactive parts, one for connecting to the payload and the other for connecting to the targeting molecule. The functional groups may be the same or different from each other. In order to control the rate of cleavage and the release of the accompanying effector molecules, the linker may be appropriately modified, such as connecting some groups at the connection with the peptide ligand or the payload to increase the chain length, and adding groups around the cleavage bond to modify and control the hindrance of the cleavage bond. The linker of the present invention includes a linker derivative modified based on the above. The linker of the present invention can be selected from -PABC-Cit-Val-Linker-β-Ala-Sar10-.

本发明的连接体或连接子没有指明其连接方向，其连接方向是任意的，例如，连接体为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接有效载荷和靶向分子也可以按照与从左往右的读取顺序相反的方向连接有效载荷和靶向分子。The linker or linker of the present invention does not specify its connection direction, and its connection direction is arbitrary. For example, the linker is -M-W-. In this case, -M-W- can connect the payload and the targeting molecule in the same direction as the reading order from left to right, or in the opposite direction to the reading order from left to right.

“靶向分子”在本发明中指能够将本申请的偶联体化合物靶向至靶部位、靶组织、靶器官、靶细胞或靶细胞内区域的任意分子或部分。在一些实施方式中，靶向分子使得，相比于非靶部位、非靶组织、非靶器官、非靶细胞或非靶细胞内区域，靶向分子使得本申请的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，靶向分子使得，相比于不带靶向分子，带有靶向分子的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，靶向分子能够触发或促进含有此类靶向分子的偶联体化合物与靶分子的特异性结合，触发或促进靶细胞对偶联体化合物的内吞作用，触发或促进偶联体化合物在靶细胞周围富集和/或进入靶细胞。"Targeting molecule" in the present invention refers to any molecule or part that can target the conjugate compound of the present application to a target site, target tissue, target organ, target cell or target cell intraregional area. In some embodiments, the targeting molecule makes the conjugate compound of the present application more distributed in the target site, target tissue, target organ, target cell or target cell intraregional area compared to the non-target site, non-target tissue, non-target organ, non-target cell or non-target cell intraregional area, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the targeting molecule allows the conjugate compound with the targeting molecule to be distributed more at the target site, target tissue, target organ, target cell or target cell intracellular region than without the targeting molecule, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the targeting molecule can trigger or promote the specific binding of the conjugate compound containing such targeting molecule to the target molecule, trigger or promote the endocytosis of the conjugate compound by the target cell, trigger or promote the enrichment of the conjugate compound around the target cell and/or enter the target cell.

在一些实施方式中，本申请的偶联体化合物包括至少两个靶向分子。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子是相同的或不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子中至少两个靶向分子是不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子彼此之间都是不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子中至少两个靶向分子是能够与不同的细胞表面蛋白或标记物特异性结合。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子能够与不同的细胞表面蛋白或标记物特异性结合。In some embodiments, the conjugate compound of the present application includes at least two targeting molecules. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are the same or different. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application are different. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are different from each other. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application are capable of specifically binding to different cell surface proteins or markers. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are capable of specifically binding to different cell surface proteins or markers.

“肽配体”在本发明中指共价结合至分子支架所形成的含有氨基酸序列(肽结构)的化合物。通常，此类肽包含能够与支架形成共价键(如硫醚键)的两个或更多个反应性基团(如半胱氨酸残基和/或2-氨基-3-巯基-3-甲基丁羧酸残基)、以及在所述反应性基团之间相对的序列(称为环序列)，之所以称为环序列，是因为当肽与支架结合时，其形成环。在本发明的情况下，肽包含至少三个氨基酸残基，其在支架上形成至少两个环，本发明肽配体可以靶向Nectin-4、Kallikrein、MT1-MMP、CD137、Epha2、IL-17、PSMA、PD-L1、αvβ3、CD38、CAIX、OX40、PBP、TSLP、ACE2、TfR1、FAPα、NK细胞或TREM2。"Peptide ligand" in the present invention refers to a compound containing an amino acid sequence (peptide structure) formed by covalently binding to a molecular scaffold. Generally, such peptides contain two or more reactive groups (such as cysteine residues and/or 2-amino-3-mercapto-3-methylbutanecarboxylic acid residues) that can form covalent bonds (such as thioether bonds) with the scaffold, and a sequence (called a ring sequence) relative to the reactive groups, which is called a ring sequence because when the peptide is bound to the scaffold, it forms a ring. In the present invention, In the present invention, the peptide comprises at least three amino acid residues, which form at least two loops on the scaffold, and the peptide ligand of the present invention can target Nectin-4, Kallikrein, MT1-MMP, CD137, Epha2, IL-17, PSMA, PD-L1, αvβ3, CD38, CAIX, OX40, PBP, TSLP, ACE2, TfR1, FAPα, NK cells or TREM2.

肽配体的修饰的衍生物在本发明的范围内。衍生物是指一种化合物中的氢原子或原子团被其他原子或原子团取代而衍生的产物。这种合适的修饰衍生物的示例包含一个或多个选自以下的修饰：N-末端和/或C-末端修饰；用一个或多个非天然氨基酸残基替换一个或多个氨基酸残基(例如用一个或多个等排或等电子氨基酸替换一个或多个极性氨基酸残基；用其它非天然的等排或等电子氨基酸替换一个或多个非极性氨基酸残基)；加入间隔物基团；用一个或多个氧化耐受性氨基酸残基替换一个或多个氧化敏感性氨基酸残基；用丙氨酸替换一个或多个氨基酸残基，用一个或多个D-氨基酸残基替换一个或多个L-氨基酸残基；双环肽配体中一个或多个酰胺键的N-烷基化；用替代键(surrogate bond)替换一个或多个肽键；肽骨架长度修饰；用另一个化学基团取代一个或多个氨基酸残基的α-碳上的氢，用合适的胺、硫醇、羧酸和酚反应性试剂修饰氨基酸如半胱氨酸、赖氨酸、谷氨酸/天冬氨酸和酪氨酸，以功能化所述氨基酸，和引入或替换氨基酸，以引入适合于官能化的正交反应性，例如携带叠氮化物或炔烃基团的氨基酸分别允许用携带炔烃或叠氮化物的部分进行官能化。Modified derivatives of peptide ligands are within the scope of the present invention. Derivatives refer to products derived from the replacement of hydrogen atoms or atomic groups in a compound by other atoms or atomic groups. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and/or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with alanine, and replacement of one or more L-amino acid residues with one or more D-amino acid residues. amino acid residues; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; modification of peptide backbone length; replacement of a hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or replacement of amino acids to introduce orthogonal reactivity suitable for functionalization, for example amino acids carrying azide or alkyne groups allow functionalization with alkyne or azide carrying moieties, respectively.

“分子支架”在本发明中指非芳族分子支架。非芳香族分子支架是指本发明定义的任何不包含芳族碳环或芳族杂环环体系的分子支架。适当的非香芳族分子支架的示例描述于Heinis等人(2014)Angewandte Chemie,International Edition 53(6)1602-1606中。分子支架可以是小分子，例如有机小分子。分子支架也可以是大分子，在一些情形中，分子支架是由氨基酸、核苷酸或碳水化合物组成的大分子。在一些情形中，分子支架包含能够与多肽的官能团反应形成共价键的反应性基团。分子支架可以包括与肽形成连接的化学基团，例如胺、硫醇、醇、酮、醛、腈、羧酸、酯、烯烃、炔烃、叠氮化物、酸酐、琥珀酰亚胺、马来酰亚胺、烷基卤化物和酰基卤化物。含有αβ不饱和羰基的化合物的示例是1,1',1”-(1,3,5-三嗪烷-1,3,5-三基)三丙-2-烯-1-酮(TATA)(Angewandte Chemie,International Edition(2014)，53(6)，1602-1606)。"Molecular scaffold" in the present invention refers to a non-aromatic molecular scaffold. A non-aromatic molecular scaffold refers to any molecular scaffold defined in the present invention that does not contain an aromatic carbocyclic ring or an aromatic heterocyclic ring system. Examples of suitable non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53 (6) 1602-1606. The molecular scaffold can be a small molecule, such as an organic small molecule. The molecular scaffold can also be a macromolecule, and in some cases, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates. In some cases, the molecular scaffold contains reactive groups that can react with functional groups of a polypeptide to form covalent bonds. The molecular scaffold can include chemical groups that form a connection with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides. An example of a compound containing an αβ-unsaturated carbonyl group is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

“氨基酸”在本发明中指天然存在的和合成的氨基酸，以及起到与天然存在的氨基酸类似的作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如，羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构(例如与氢、羧基基团、氨基基团和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这样的类似物可以具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指其结构不同于一般的氨基酸化学结构，但起到与天然存在的氨基酸相似的作用的化学化合物。"Amino acid" in the present invention refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but function similarly to naturally occurring amino acids.

本发明中涉及的氨基酸及三字母、单字母缩写：
Cys      半胱氨酸       (缩写C)
Pro      脯氨酸        (缩写P)
1Nal     萘基丙氨酸
D-Asp    D-天冬氨酸
Met      甲硫氨酸       (缩写M)
HArg     高精氨酸
Asp      天冬氨酸       (缩写D)
Trp      色氨酸        (缩写W)
Ser      丝氨酸        (缩写S)
Thr      苏氨酸        (缩写T)
Ala      丙氨酸        (缩写A)
HyP      羟基脯氨酸
Sar      肌氨酸
Dap      2,3-二氨基丙酸The amino acids and three-letter and one-letter abbreviations involved in the present invention are:
Cys Cysteine (abbreviated as C)
Proline (abbreviated as P)
1Nal naphthylalanine
D-Asp D-Aspartic acid
Met Methionine (abbreviated as M)
HArg Homoarginine
Asp Aspartic acid (abbreviation D)
Trp Tryptophan (abbreviated as W)
Ser Serine (abbreviated S)
Thr Threonine (abbreviated as T)
Ala Alanine (abbreviation A)
HyP Hydroxyproline
Sar Sarcosine
Dap 2,3-Diaminopropionic acid

Pen代表 Pen Representative

βCys代表 βCys represents

hCys代表 hCys represents

N-甲基-Dap代表 N-methyl-Dap represents

本发明中涉及的化合物缩写：Abbreviations of compounds involved in the present invention:

HATU：2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HOBT：1-羟基苯并三唑HOBT: 1-Hydroxybenzotriazole

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

COMU：(1-氰基-2-乙氧基-2-氧代亚乙基氨基氧基)二甲基氨基-吗啉-碳鎓六氟磷酸盐COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinium-carbonium hexafluorophosphate

TFA：三氟乙酸TFA: trifluoroacetic acid

DCM：二氯甲烷DCM: dichloromethane

HBTU：苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯HBTU: Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate

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

TIS：三异丙基硅烷 TIS: Triisopropylsilane

ACN：乙腈ACN: Acetonitrile

如本文所用，“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。As used herein, "comprising" or "including" may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

“药学上可接受盐”在本发明中是指药学上可接受的酸加成盐，包括无机酸盐和有机酸盐，这些盐可通过本专业已知的方法制备。"Pharmaceutically acceptable salt" in the present invention refers to pharmaceutically acceptable acid addition salts, including inorganic acid salts and organic acid salts, which can be prepared by methods known in the art.

“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

本申请的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(³H)，碘-125(¹²⁵I)或C-14(¹⁴C)。又例如，可用重氢取代氢形成氘代药物。本申请的化合物的所有同位素组成的变换，无论放射性与否，都包括在本申请的范围之内。The compounds of the present application may contain unnatural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. All isotopic composition changes of the compounds of the present application, whether radioactive or not, are included in the scope of the present application.

下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention will be further described below in conjunction with specific implementations. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

本发明的化合物结构是通过核磁共振(NMR)或/和液质联用色谱(LC-MS)来确定的。NMR化学位移(δ)以百万分之一(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400/500核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d₆)，氘代甲醇(CD₃OD)和氘代氯仿(CDCl₃)，内标为四甲基硅烷(TMS)。The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shift (δ) is given in parts per million (ppm). The NMR measurement is performed using a Bruker AVANCE-400/500 nuclear magnetic spectrometer, the measurement solvents are deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated methanol (CD ₃ OD) and deuterated chloroform (CDCl ₃ ), and the internal standard is tetramethylsilane (TMS).

液质联用色谱LC-MS的测定用Agilent 6120质谱仪。HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 6120 mass spectrometer. HPLC was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6 mm column).

薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，TLC采用的规格是0.15mm～0.20mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。Thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications used for TLC are 0.15mm-0.20mm, and the specifications used for thin layer chromatography separation and purification products are 0.4mm-0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

本发明实施例中的起始原料是已知的并且可以在市场上买到，或者可以采用或按照本领域已知的方法来合成。The starting materials in the examples of the present invention are known and can be purchased on the market, or can be synthesized by or according to methods known in the art.

在无特殊说明的情况下，本发明的所有反应均在连续的磁力搅拌下，在干燥氮气或氩气氛下进行，溶剂为干燥溶剂，反应温度单位为摄氏度(℃)。Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, the solvent is a dry solvent, and the reaction temperature is in degrees Celsius (°C).

实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

本发明参照化合物BT8009结构参见专利WO2019243833A1说明书第19页。The structure of the reference compound BT8009 of the present invention can be found on page 19 of the patent specification WO2019243833A1.

中间体的制备Preparation of intermediates

中间体INT-A的合成：
Synthesis of intermediate INT-A:

中间体INT-A通过以下路线合成：
The intermediate INT-A was synthesized by the following route:

将起始物料Val-Cit-PAB-MMAE(200mg,0.178mmol)和戊二酸酐(22.33mg,0.195mmol)溶解于N，N二甲基甲酰胺(3mL)中，向反应体系中加入N,N-二异丙基乙胺(68.88mg,0.534mmol)，反应置于25℃下搅拌2小时。反应监测起始物料Val-Cit-PAB-MMAE消耗完全后，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(80.36mg,0.267mmol)。反应置于25℃下搅拌3小时。反应液经过反相柱纯化得到中间体INT-A(218mg,0.163mmol,91.9％)。The starting material Val-Cit-PAB-MMAE (200 mg, 0.178 mmol) and glutaric anhydride (22.33 mg, 0.195 mmol) were dissolved in N, N-dimethylformamide (3 mL), and N, N-diisopropylethylamine (68.88 mg, 0.534 mmol) was added to the reaction system, and the reaction was stirred at 25 ° C for 2 hours. After the reaction monitoring starting material Val-Cit-PAB-MMAE was completely consumed, 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (80.36 mg, 0.267 mmol) was added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by reverse phase column to obtain the intermediate INT-A (218 mg, 0.163 mmol, 91.9%).

LCMS(ESI)m/z[M+H]⁺＝1335.0；LCMS(ESI)m/z[M+2H]⁺/2＝668.4。LCMS(ESI)m/z[M+H] ⁺ =1335.0; LCMS(ESI)m/z[M+2H] ⁺ /2=668.4.

中间体INT-B和INT-C的合成：
Synthesis of intermediates INT-B and INT-C:

中间体INT-B和INT-C通过以下路线合成：
Intermediates INT-B and INT-C were synthesized by the following route:

第一步：INT-B1的合成Step 1: Synthesis of INT-B1

取起始物料SM-1(5.0g，16mmol)溶于N,N-二甲基甲酰胺(15mL)，加入N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(9.0g,24mmol)，N,N-二异丙基乙胺(6.2g,48mmol)，室温反应0.5小时，随后加入SM-2(3.4g，19.2mmol)，继续室温反应过夜。反应完毕后反应液加150mL乙酸乙酯稀释，用20mL 0.1M的盐酸水溶液洗涤1次，饱和氯化钠洗涤2次，有机相用无水硫酸钠干燥，过滤，减压旋干，得中间体INT-B1粗品9.0g。Take the starting material SM-1 (5.0g, 16mmol) and dissolve it in N,N-dimethylformamide (15mL), add N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (9.0g, 24mmol), N,N-diisopropylethylamine (6.2g, 48mmol), react at room temperature for 0.5 hours, then add SM-2 (3.4g, 19.2mmol), and continue to react at room temperature overnight. After the reaction is completed, the reaction solution is diluted with 150mL of ethyl acetate, washed once with 20mL 0.1M hydrochloric acid aqueous solution, washed twice with saturated sodium chloride, and the organic phase is dried with anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain 9.0g of the crude intermediate INT-B1.

LCMS(ESI)[M+Na]⁺＝461.5；LCMS(ESI)[M-56]⁺＝383.4。LCMS(ESI)[M+Na] ⁺ =461.5; LCMS(ESI)[M-56] ⁺ =383.4.

第二步：INT-B2的合成Step 2: Synthesis of INT-B2

取INT-B1(9.0g，粗品)溶于DCM(30mL),室温下滴加三氟乙酸(10mL)，室温反应2.5h。反应毕，反应液旋干，所得粗品过反相柱C18纯化(乙腈：水(0.05％三氟乙酸体系)＝20～50％)冻干得INT-B2(4.0g)。INT-B1 (9.0 g, crude product) was dissolved in DCM (30 mL), trifluoroacetic acid (10 mL) was added dropwise at room temperature, and the mixture was reacted at room temperature for 2.5 h. After the reaction, the reaction solution was spin-dried, and the crude product was purified by reverse phase column C18 (acetonitrile: water (0.05% trifluoroacetic acid system) = 20-50%) and freeze-dried to obtain INT-B2 (4.0 g).

LCMS(ESI)[M+Na]⁺＝405.4；LCMS(ESI)[M+H]⁺＝383.4。LCMS(ESI)[M+Na] ⁺ =405.4; LCMS(ESI)[M+H] ⁺ =383.4.

第三步：INT-B3的合成Step 3: Synthesis of INT-B3

取INT-B2(4.0g，10mmol)溶于N,N-二甲基甲酰胺(10mL)，加入N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(6.0g,15mmol)，N,N-二异丙基乙胺(3.9g,48mmol),室温反应0.5h,随后加入SM2(2.1g，12mmol)，继续室温反应过夜。反应毕，反应液加乙酸乙酯稀释，0.1M的盐酸水溶液洗涤1次，饱和氯化钠洗涤2次，有机相用无水硫酸钠干燥，过滤，旋干，得INT-B3粗品6.0g。Take INT-B2 (4.0 g, 10 mmol) and dissolve it in N, N-dimethylformamide (10 mL), add N, N, N', N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (6.0 g, 15 mmol), N, N-diisopropylethylamine (3.9 g, 48 mmol), react at room temperature for 0.5 h, then add SM2 (2.1 g, 12 mmol), and continue to react at room temperature overnight. After the reaction, the reaction solution is diluted with ethyl acetate, washed once with 0.1 M hydrochloric acid aqueous solution, washed twice with saturated sodium chloride, and the organic phase is dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain 6.0 g of crude INT-B3.

LCMS(ESI)[M+Na]⁺＝532.6；LCMS(ESI)[M-56]⁺＝454.4；LCMS(ESI)[M+H]⁺＝510.5。LCMS(ESI)[M+Na] ⁺ =532.6; LCMS(ESI)[M-56] ⁺ =454.4; LCMS(ESI)[M+H] ⁺ =510.5.

第四步：INT-B的合成Step 4: Synthesis of INT-B

取中间体INT-B3(6.0g，粗品)溶于DCM(15mL)，室温下滴加三氟乙酸(5mL)，室温反应2.5h。反应毕，反应液旋干，所得粗品通过反相柱层析(C18，乙腈：水(0.05％三氟乙酸体系＝20～50％)纯化冻干得INT-B(3.0g)。 The intermediate INT-B3 (6.0 g, crude product) was dissolved in DCM (15 mL), and trifluoroacetic acid (5 mL) was added dropwise at room temperature, and the reaction was carried out at room temperature for 2.5 h. After the reaction, the reaction solution was spin-dried, and the crude product was purified by reverse phase column chromatography (C18, acetonitrile: water (0.05% trifluoroacetic acid system = 20-50%) and lyophilized to obtain INT-B (3.0 g).

LCMS(ESI)[M+H]⁺＝454.4。LCMS (ESI) [M+H] ⁺ = 454.4.

第五步：INT-C1的合成Step 5: Synthesis of INT-C1

取INT-B(1.0g，2.35mmol)溶于N,N-二甲基甲酰胺(5mL)，加入N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(1.34g,3.5mmol),N,N-二异丙基乙胺(911mg,7mmol)，室温反应0.5小时，随后加入SM2(428mg，2.35mmol)，继续室温反应过夜。反应毕，反应液加乙酸乙酯稀释，0.1M的盐酸水溶液洗涤1次，饱和氯化钠洗涤2次，有机相用无水硫酸钠干燥，过滤，旋干，得INT-C1粗品560mg。Take INT-B (1.0 g, 2.35 mmol) and dissolve it in N, N-dimethylformamide (5 mL), add N, N, N', N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (1.34 g, 3.5 mmol), N, N-diisopropylethylamine (911 mg, 7 mmol), react at room temperature for 0.5 hours, then add SM2 (428 mg, 2.35 mmol), and continue to react at room temperature overnight. After the reaction, the reaction solution is diluted with ethyl acetate, washed once with 0.1 M hydrochloric acid aqueous solution, washed twice with saturated sodium chloride, and the organic phase is dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain 560 mg of crude INT-C1.

LCMS(ESI)[M-56]⁺＝454.4；LCMS(ESI)[M+H]⁺＝510.5。LCMS(ESI)[M-56] ⁺ =454.4; LCMS(ESI)[M+H] ⁺ =510.5.

第六步：INT-C的合成Step 6: Synthesis of INT-C

取INT-C1(560mg，粗品)溶于DCM(5mL)，室温下滴加三氟乙酸(2mL)，室温反应2.5h。反应毕，反应液旋干，得粗品INT-C(850mg)。INT-C1 (560 mg, crude product) was dissolved in DCM (5 mL), trifluoroacetic acid (2 mL) was added dropwise at room temperature, and the mixture was reacted at room temperature for 2.5 h. After the reaction, the reaction solution was dried by rotary evaporation to obtain crude INT-C (850 mg).

LCMS(ESI)[M+H]⁺＝454.4。LCMS (ESI) [M+H] ⁺ = 454.4.

中间体环肽INT-D的合成
Synthesis of Intermediate Cyclic Peptide INT-D

中间体环肽INT-D通过以下路线合成：

The intermediate cyclic peptide INT-D was synthesized by the following route:

表1：原料投料表

Table 1: Raw material feeding table

多肽使用标准的Fmoc化学逐步合成方法合成。Peptides were synthesized using standard step-wise Fmoc chemistry.

第一步：溶胀树脂Step 1: Swelling the resin

取Rink Amide MBHAResin树脂(800mg,0.4mmol)于100mL固相合成管中，加60mL二氯甲烷溶胀2h，抽去溶剂，用N,N-二甲基甲酰胺洗涤3次，抽去溶剂。Take Rink Amide MBHAResin resin (800 mg, 0.4 mmol) in a 100 mL solid phase synthesis tube, add 60 mL of dichloromethane to swell for 2 hours, remove the solvent, wash with N,N-dimethylformamide three times, and remove the solvent.

第二步：将Fmoc-S-Trt-L-Cys载到Rink Amide MBHA Resin树脂Step 2: Load Fmoc-S-Trt-L-Cys onto Rink Amide MBHA Resin

往固相合成管中加50mL 20％哌啶/N,N-二甲基甲酰胺溶液，鼓氮气反应5分钟，抽去溶剂，重复反应一次；分别用二氯甲烷，甲醇，二氯甲烷洗涤，用印三酮显色显示深蓝色，再次用N,N-二甲基甲酰胺洗涤。取Fmoc-S-Trt-L-CYS(936mg，1.6mmol)，N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(608mg，1.6mmol)，1-羟基苯并三唑(216mg，1.6mmol)，2,4,6-三甲基吡啶(290mg，2.4mmol)溶于N,N-二甲基甲酰胺(15mL)，震荡1分钟后加至固相合成管中，摇床反应过夜。Add 50mL 20% piperidine/N,N-dimethylformamide solution to the solid phase synthesis tube, react with nitrogen for 5 minutes, remove the solvent, and repeat the reaction once; wash with dichloromethane, methanol, and dichloromethane respectively, develop with phenhydramine to show dark blue, and wash again with N,N-dimethylformamide. Take Fmoc-S-Trt-L-CYS (936mg, 1.6mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (608mg, 1.6mmol), 1-hydroxybenzotriazole (216mg, 1.6mmol), 2,4,6-trimethylpyridine (290mg, 2.4mmol) and dissolve in N,N-dimethylformamide (15mL), shake for 1 minute, add to the solid phase synthesis tube, and shake overnight.

第三步：脱N端Fmoc保护Step 3: Remove N-terminal Fmoc protection

抽去溶剂，分别用二氯甲烷/甲醇/二氯甲烷洗涤树脂，用印三酮显色，显示无色，反应完毕，再次用N,N-二甲基甲酰胺洗涤树脂，往固相合成管中加25mL 20％哌啶/N,N- 二甲基甲酰胺溶液，鼓氮气反应5分钟，抽去溶剂，重复反应一次；分别用二氯甲烷，甲醇，二氯甲烷洗涤，用印三酮显色显示深蓝色，Fmoc基团已脱除，再次用N,N-二甲基甲酰胺洗涤。The solvent was removed, and the resin was washed with dichloromethane/methanol/dichloromethane respectively. The color was developed with pyridinone, and it showed colorless. After the reaction was completed, the resin was washed again with N,N-dimethylformamide. 25 mL of 20% piperidine/N,N- The dimethylformamide solution was reacted with nitrogen for 5 minutes, the solvent was removed, and the reaction was repeated once; it was washed with dichloromethane, methanol, and dichloromethane respectively, and the color was developed with phenhydramine to show dark blue, indicating that the Fmoc group had been removed, and it was washed again with N,N-dimethylformamide.

第四步：将Fmoc-5-羟基-L-色氨酸偶联到肽树脂Step 4: Coupling Fmoc-5-hydroxy-L-tryptophan to the peptide resin

取Fmoc-L-色氨酸(Boc)-OH(840mg,1.6mmol)，N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(608mg，1.6mmol)，1-羟基苯并三唑(216mg，1.6mmol)，N,N-二异丙基乙胺(312mg，2.4mmol)溶于N,N-二甲基甲酰胺(15mL),震荡1分钟后加至固相合成管中，鼓氮气反应1小时。Take Fmoc-L-tryptophan (Boc) -OH (840 mg, 1.6 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (608 mg, 1.6 mmol), 1-hydroxybenzotriazole (216 mg, 1.6 mmol), N,N-diisopropylethylamine (312 mg, 2.4 mmol) and dissolve them in N,N-dimethylformamide (15 mL). After shaking for 1 minute, add them to the solid phase synthesis tube and react with nitrogen for 1 hour.

第五步：后续氨基酸偶联Step 5: Subsequent amino acid coupling

参照Fmoc-5-羟基-L-色氨酸偶联到树脂的方法以及脱N端Fmoc保护方法依次将Fmoc-4-叔丁氧基-L-脯氨酸、Fmoc-L-脯氨酸、Fmoc-O-叔丁基-L-苏氨酸、Fmoc-O-叔丁基-L-丝氨酸、Fmoc-L-色氨酸(Boc)-OH、N-Fmoc-L-天冬氨酸4-叔-丁基酯、N-Fmoc-N'-Pbf-L-高精氨酸、Fmoc-L-蛋氨酸偶联至树脂。Referring to the method for coupling Fmoc-5-hydroxy-L-tryptophan to a resin and the method for removing N-terminal Fmoc protection, Fmoc-4-tert-butoxy-L-proline, Fmoc-L-proline, Fmoc-O-tert-butyl-L-threonine, Fmoc-O-tert-butyl-L-serine, Fmoc-L-tryptophan (Boc)-OH, N-Fmoc-L-aspartic acid 4-tert-butyl ester, N-Fmoc-N'-Pbf-L-homoarginine, and Fmoc-L-methionine were coupled to the resin in sequence.

参照Fmoc-S-Trt-L-Cys载到Rink Amide MBHAResin树脂的方法以及脱N端Fmoc保护方法将Fmoc-S-Trt-L-青霉胺偶联至树脂。Fmoc-S-Trt-L-penicillamine was coupled to the resin by referring to the method of loading Fmoc-S-Trt-L-Cys onto Rink Amide MBHAResin resin and removing N-terminal Fmoc protection.

参照参照Fmoc-5-羟基-L-色氨酸偶联到树脂的方法以及脱N端Fmoc保护方法依次将N-Fmoc-D-天冬氨酸-4-叔丁酯、Fmoc-3-(1-萘基)-L-丙氨酸、(2S,4S)-FMOC-4-氟吡咯烷-2-甲酸偶联至树脂。Referring to the method of coupling Fmoc-5-hydroxy-L-tryptophan to the resin and the method of removing N-terminal Fmoc protection, N-Fmoc-D-aspartic acid-4-tert-butyl ester, Fmoc-3-(1-naphthyl)-L-alanine and (2S,4S)-FMOC-4-fluoropyrrolidine-2-carboxylic acid were coupled to the resin in sequence.

参照Fmoc-S-Trt-L-Cys载到Rink Amide MBHAResin树脂的方法以及脱N端Fmoc保护方法将Fmoc-S-Trt-L-Cys偶联至树脂，得WB004-290-15树脂粗品(16g,0.4mmol)。Referring to the method of loading Fmoc-S-Trt-L-Cys onto Rink Amide MBHA Resin resin and removing N-terminal Fmoc protection, Fmoc-S-Trt-L-Cys was coupled to the resin to obtain crude WB004-290-15 resin (16 g, 0.4 mmol).

参照Fmoc-5-羟基-L-色氨酸偶联到树脂的方法以及脱N端Fmoc保护方法依次将Fmoc(Sar)₃OH、Fmoc(Sar)₃OH、Fmoc(Sar)₄OH、Boc-Βeta-Ala-OH偶联至树脂。Referring to the method of coupling Fmoc-5-hydroxy-L-tryptophan to the resin and the method of removing the N-terminal Fmoc protection, Fmoc(Sar) ₃ OH, Fmoc(Sar) ₃ OH, Fmoc(Sar) ₄ OH and Boc-Βeta-Ala-OH were coupled to the resin in sequence.

第六步：将线性肽从肽树脂切除Step 6: Cleavage of the linear peptide from the peptide resin

将所得线性肽树脂粗品加至250mL单口瓶，0-5℃下加入40mL裂解液，缓慢升值室温，低速搅拌2小时。反应毕，过滤，用二氯甲烷洗涤，合并滤液，30℃下旋去大部分溶剂，母液加160mL甲基叔丁基醚稀释，打浆5分钟，离心，收集固体，得线性肽粗品。所得粗品过反相C18柱纯化，流动相：乙腈：水(0.5％)＝25％～40％冲出产品，得INT-D20线性肽500mg。Add the obtained crude linear peptide resin to a 250mL single-mouth bottle, add 40mL lysate at 0-5°C, slowly warm to room temperature, and stir at low speed for 2 hours. After the reaction, filter, wash with dichloromethane, combine the filtrate, spin off most of the solvent at 30°C, dilute the mother liquor with 160mL methyl tert-butyl ether, beat for 5 minutes, centrifuge, collect the solid, and obtain the crude linear peptide. The obtained crude product is purified by a reverse phase C18 column, and the mobile phase: acetonitrile: water (0.5%) = 25% to 40% flushes out the product to obtain 500mg of INT-D20 linear peptide.

第七步：中间体INT-D的合成Step 7: Synthesis of intermediate INT-D

取INT-D20(500mg,0.18mmol)溶于乙腈(200mL)和水(200mL)，加入碳酸氢钠(152mg，1.8mmol)，TATA(45mg,0.18mmol)，室温反应3小时。LCMS显示原料消失，加入半胱氨酸盐酸盐(157mg,0.9mmol)淬灭反应，搅拌1小时，反应液冻干，得800mg粗品，所得粗品过反相纯化得中间体环肽INT-D(340mg,收率：62.3％)。INT-D20 (500 mg, 0.18 mmol) was dissolved in acetonitrile (200 mL) and water (200 mL), and sodium bicarbonate (152 mg, 1.8 mmol) and TATA (45 mg, 0.18 mmol) were added, and the reaction was carried out at room temperature for 3 hours. LCMS showed that the raw material disappeared, and cysteine hydrochloride (157 mg, 0.9 mmol) was added to quench the reaction, and the mixture was stirred for 1 hour. The reaction solution was lyophilized to obtain 800 mg of crude product, which was then purified by reverse phase to obtain the intermediate cyclic peptide INT-D (340 mg, yield: 62.3%).

LCMS(ESI)[M+2H]⁺/2＝1500.9；LCMS(ESI)[M+3H]⁺/3＝1001.2。LCMS(ESI)[M+2H] ⁺ /2=1500.9; LCMS(ESI)[M+3H] ⁺ /3=1001.2.

具体化合物的制备Preparation of specific compounds

实施例1：化合物1的合成
Example 1: Synthesis of Compound 1

化合物1通过以下路线合成：
Compound 1 was synthesized via the following route:

第一步：中间体1-1合成：Step 1: Synthesis of intermediate 1-1:

该多肽使用标准的Fmoc化学逐步合成方法合成。The peptide was synthesized using standard step-wise Fmoc chemistry.

将DMF加入含有Rink Amide MBHA树脂(浓度:0.3mmol/g,0.5mmol,1.67g)的容器中，膨胀2小时。加入20％哌啶/DMF，混合30分钟。用茚三酮试验监测偶联反应，沥干树脂，然后用DMF清洗5次(每次30秒)。加入Fmoc-氨基酸溶液混合30秒，再加入缩合剂。反应持续1小时，氮气冒泡。沥干树脂，然后用DMF清洗5次(每次30秒)。重复步骤2到步骤5进行下一个氨基酸偶联。Add DMF to a container containing Rink Amide MBHA resin (concentration: 0.3mmol/g, 0.5mmol, 1.67g) and swell for 2 hours. Add 20% piperidine/DMF and mix for 30 minutes. Monitor the coupling reaction with the ninhydrin test, drain the resin, and then wash it with DMF 5 times (30 seconds each). Add the Fmoc-amino acid solution and mix for 30 seconds, then add the condensing agent. The reaction continues for 1 hour, bubbling with nitrogen. Drain the resin, then wash it with DMF 5 times (30 seconds each). Repeat steps 2 to 5 for the next amino acid coupling.

合成中间体1-1所用到的氨基酸及缩合试剂的加料顺序如表2所示。The order of adding the amino acids and condensation reagents used in the synthesis of intermediate 1-1 is shown in Table 2.

表2：原料投料表

Table 2: Raw material feeding table

肽裂解和纯化:将裂解缓冲液(2.5％H₂O/2.5％TIS/95％TFA)加入到含有侧链保护肽的烧瓶中，室温下搅拌3小时。用40毫升TFA过滤洗净。联合滤液用冷甲基叔丁基醚(MTBE)沉淀。将混合物离心(3000转/分，3分钟)，倒出。固体用MTBE洗涤并离心。将残余物冻干后给予1.40g粗肽。Peptide cleavage and purification: Add lysis buffer (2.5% _H2O /2.5% TIS/95% TFA) to the flask containing the side chain protected peptide and stir at room temperature for 3 hours. Filter and wash with 40 ml TFA. The combined filtrate is precipitated with cold methyl tert-butyl ether (MTBE). The mixture is centrifuged (3000 rpm, 3 minutes) and poured off. The solid is washed with MTBE and centrifuged. The residue is lyophilized to give 1.40 g of crude peptide.

第二步中间体1-2的合成： The second step is the synthesis of intermediate 1-2:

将粗肽中间体1-1(1.40g,0.50mmol)溶于50％ MeCN/H₂O(0.5L)中，然后将肽搅拌后的溶液中加入1a(1.3eq)，并加入NH₄HCO₃(1M)，使pH为8。4小时后LCMS显示反应完成。用预处理高效液相色谱法(A:0.075％ TFA in H₂O,B:CH₃CN)纯化得到中间体1-2(35.0mg，纯度～90％，产率2.13％，TFA盐)。The crude peptide intermediate 1-1 (1.40 g, 0.50 mmol) was dissolved in 50% MeCN/H ₂ O (0.5 L), and then 1a (1.3 eq) was added to the stirred solution of the peptide, and NH ₄ HCO ₃ (1 M) was added to adjust the pH to 8. After 4 hours, LCMS showed that the reaction was complete. The intermediate 1-2 (35.0 mg, purity ~90%, yield 2.13%, TFA salt) was purified by pretreatment HPLC (A: 0.075% TFA in H ₂ O, B: CH ₃ CN).

第三步化合物1的合成：Step 3 Synthesis of Compound 1:

将原料1-b(1.9mg,0.0048mmol)和中间体1-2(10.5mg,0.0035mmol)溶解于N,N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(1.29mg,0.01mmol)，反应置于25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)，得到化合物1(5.30mg,0.0015mmol,46.9％)。The raw material 1-b (1.9 mg, 0.0048 mmol) and the intermediate 1-2 (10.5 mg, 0.0035 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (1.29 mg, 0.01 mmol) was added to the reaction system, and the reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150mm, 5um, mobile phase: [A: water (10mM TFA)-B: ACN], gradient B%: 37%-50%, 13min) to obtain compound 1 (5.30 mg, 0.0015 mmol, 46.9%).

LCMS(ESI)m/z[M+2H]⁺/2＝1672.65；LCMS(ESI)m/z[M+3H]⁺/3＝1115.43；LCMS(ESI)m/z[M+4H]⁺/4＝836.83。LCMS(ESI)m/z[M+2H] ⁺ /2＝1672.65; LCMS(ESI)m/z[M+3H] ⁺ /3＝1115.43; LCMS(ESI)m/z[M+4H] ⁺ / 4=836.83.

实施例2：化合物2的合成
Example 2: Synthesis of Compound 2

化合物2通过以下路线合成：
Compound 2 was synthesized by the following route:

第一步：中间体2-3的合成Step 1: Synthesis of intermediate 2-3

首先将起始物料2-1(461mg，1mmol)、2-1A(290mg，1mmol)用N，N-二甲基甲酰胺(5mL)溶解，加入N,N-二异丙基乙胺(378mg，3mmol)、COMU(428mg，1mmol)，室温下反应1小时。LCMS监测反应结束，直接向反应液中滴加20％哌啶N，N二甲基甲酰胺溶液，20分钟后LCMS显示反应结束，反应液经反相柱(水：乙腈＝35％：65％)纯化后冻干得300mg中间体2-3，收率：40％。First, the starting materials 2-1 (461 mg, 1 mmol) and 2-1A (290 mg, 1 mmol) were reacted with N,N-dimethyl Dissolve in formamide (5 mL), add N, N-diisopropylethylamine (378 mg, 3 mmol) and COMU (428 mg, 1 mmol), and react at room temperature for 1 hour. After LCMS monitoring, the reaction is completed, and 20% piperidine N, N dimethylformamide solution is directly added to the reaction solution. After 20 minutes, LCMS shows that the reaction is complete. The reaction solution is purified by reverse phase column (water: acetonitrile = 35%: 65%) and freeze-dried to obtain 300 mg of intermediate 2-3, with a yield of 40%.

LCMS(ESI)[M+H]⁺＝475.14。LCMS (ESI) [M+H] ⁺ = 475.14.

第二步：中间体2-5的合成Step 2: Synthesis of intermediate 2-5

将中间体2-3(300mg，0.63mmol)、起始物料2-1B(144mg，1.26mmol)用N，N-二甲基甲酰胺(5mL)溶解，加入N,N-二异丙基乙胺(244mg，1.89mmol)。反应液室温下反应30分钟。LCMS监测反应结束，直接向反应液中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(379mg，1.26mmol)，室温反应1.5小时，LCMS显示反应结束，反应液经反相柱(水：乙腈＝60％：40％)纯化冻干得200mg中间体2-5，收率：50％。The intermediate 2-3 (300 mg, 0.63 mmol) and the starting material 2-1B (144 mg, 1.26 mmol) were dissolved in N, N-dimethylformamide (5 mL), and N, N-diisopropylethylamine (244 mg, 1.89 mmol) was added. The reaction solution was reacted at room temperature for 30 minutes. LCMS monitored the completion of the reaction, and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (379 mg, 1.26 mmol) was directly added to the reaction solution, and the reaction was carried out at room temperature for 1.5 hours. LCMS showed that the reaction was completed. The reaction solution was purified by reverse phase column (water: acetonitrile = 60%: 40%) and lyophilized to obtain 200 mg of intermediate 2-5, with a yield of 50%.

LCMS(ESI)[M+H]⁺＝686.19。LCMS (ESI) [M+H] ⁺ = 686.19.

第三步：中间体2-6的合成Step 3: Synthesis of intermediate 2-6

将中间体2-5(200mg，0.29mmol)、起始物料2-1C(71mg，0.29mmol)用N，N-二甲基甲酰胺(2mL)溶解，加入N,N-二异丙基乙胺(112mg，0.87mmol).反应液室温下反应2小时。LCMS监测反应结束，反应液经反相柱(水：乙腈＝55％：45％)纯化冻干得100mg中间体2-6，收率：45％。Dissolve the intermediate 2-5 (200 mg, 0.29 mmol) and the starting material 2-1C (71 mg, 0.29 mmol) in N, N-dimethylformamide (2 mL), add N, N-diisopropylethylamine (112 mg, 0.87 mmol). The reaction solution was reacted at room temperature for 2 hours. The reaction was monitored by LCMS, and the reaction solution was purified by reverse phase column (water: acetonitrile = 55%: 45%) and lyophilized to obtain 100 mg of intermediate 2-6, with a yield of 45%.

LCMS(ESI)[M+H]⁺＝717.32。LCMS (ESI) [M+H] ⁺ = 717.32.

第四步：中间体2-7的合成Step 4: Synthesis of intermediate 2-7

将中间体2-6(50mg，0.06mmol)、2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(36mg，0.12mmol)用N，N-二甲基甲酰(1.5ml)溶解，加入N,N-二异丙基乙胺(23mg，0.18mmol)。反应液在室温下反应1.5小时。LCMS监测反应结束，反应液经反相柱(水：乙腈＝60％：40％)纯化冻干得30mg中间体2-7，收率：52％。Dissolve intermediate 2-6 (50 mg, 0.06 mmol) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (36 mg, 0.12 mmol) in N,N-dimethylformamide (1.5 ml), and add N,N-diisopropylethylamine (23 mg, 0.18 mmol). The reaction solution was reacted at room temperature for 1.5 hours. LCMS monitored the completion of the reaction, and the reaction solution was purified and freeze-dried by a reverse phase column (water: acetonitrile = 60%: 40%) to obtain 30 mg of intermediate 2-7, with a yield of 52%.

LCMS(ESI)[M+H]⁺＝814.34。LCMS (ESI) [M+H] ⁺ = 814.34.

第五步：中间体2-8的合成Step 5: Synthesis of intermediate 2-8

将中间体2-7(14mg，0.015mmol)、环肽中间体INT-D(45mg，0.015mmol)溶于N，N-二甲基甲酰胺(5ml)中，加入N,N-二异丙基乙胺(6mg，0.0045mmol)室温搅拌5h。LCMS显示无原料，反应液经反相柱分离纯化，冻干得50mg中间体2-8，收率：87％。Intermediate 2-7 (14 mg, 0.015 mmol) and cyclopeptide intermediate INT-D (45 mg, 0.015 mmol) were dissolved in N, N-dimethylformamide (5 ml), and N, N-diisopropylethylamine (6 mg, 0.0045 mmol) was added and stirred at room temperature for 5 h. LCMS showed no starting material, and the reaction solution was separated and purified by reverse phase column and freeze-dried to obtain 50 mg of intermediate 2-8, with a yield of 87%.

LCMS(ESI)[M+3H⁺]/3＝1267.40，[M-Boc+3H⁺]/3＝1234.10，[M-Boc+4H⁺]/4＝925.70。LCMS (ESI) [M+3H ⁺ ]/3=1267.40, [M-Boc+3H ⁺ ]/3=1234.10, [M-Boc+4H ⁺ ]/4=925.70.

第五步：中间体2-9的合成Step 5: Synthesis of intermediate 2-9

将中间体2-8(50mg，0.013mmol)用三氟乙酸(3mL)溶解。反应液室温下反应1小时，反应液加入到甲叔醚中，离心，固体加水和乙腈冻干后得到40mg中间体2-9，收率不计。Intermediate 2-8 (50 mg, 0.013 mmol) was dissolved in trifluoroacetic acid (3 mL). The reaction solution was reacted at room temperature for 1 hour, and then added to methyl tert-ether, centrifuged, and the solid was added with water and acetonitrile and freeze-dried to obtain 40 mg of intermediate 2-9. The yield was not counted.

LCMS(ESI)[M+3H⁺]/3＝1215.40，[M+4H⁺]/4＝911.80。LCMS (ESI) [M+3H ⁺ ]/3=1215.40, [M+4H ⁺ ]/4=911.80.

第六步：化合物2的合成 Step 6: Synthesis of Compound 2

将中间体2-9(40mg，0.011mmol)、中间体INT-A(20mg，0.015mmol)溶于N，N二甲基甲酰胺(2mL)中，加入N,N-二异丙基乙胺(4mg，0.03mmol)。反应液室温下搅拌17小时。LCMS监测反应结束，送制备高效液相纯化(制备条件：色谱柱：Welch Xtimate C18 150mm*21.2mm,5um流速：15ml/min流动相：A:NH₄HCO₃水B:ACN波长：214nm梯度:25-55(B)13min)冻干得7mg化合物2。Intermediate 2-9 (40 mg, 0.011 mmol) and intermediate INT-A (20 mg, 0.015 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4 mg, 0.03 mmol) was added. The reaction solution was stirred at room temperature for 17 hours. LCMS monitored the completion of the reaction and sent to preparative high performance liquid phase for purification (preparation conditions: chromatographic column: Welch Xtimate C18 150 mm*21.2 mm, 5 um flow rate: 15 ml/min mobile phase: A: NH ₄ HCO ₃ water B: ACN wavelength: 214 nm gradient: 25-55 (B) 13 min) and freeze-dried to obtain 7 mg of compound 2.

LCMS(ESI)[M+3H⁺]/3＝1620.84，[M+4H⁺]/4＝1216.31。LCMS (ESI) [M+3H ⁺ ]/3=1620.84, [M+4H ⁺ ]/4=1216.31.

实施例3：化合物3的合成
Example 3: Synthesis of Compound 3

化合物3通过以下路线合成：

Compound 3 was synthesized via the following route:

第一步：中间体3-2的合成Step 1: Synthesis of intermediate 3-2

将起始物料3-1(108.1mg,0.271mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(97.8mg,0.325mmol)和N,N-二异丙基乙胺(174.7mg,1.35mmol)。反应置于25℃下搅拌0.5小时后，向反应液中加入起始物料3-1a(100mg,0.271mmol)，继续反应3小时。混合物经过反相柱纯化得到中间体3-2(154mg,0.059mmol,收率75.7％)。The starting material 3-1 (108.1 mg, 0.271 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (97.8 mg, 0.325 mmol) and N, N-diisopropylethylamine (174.7 mg, 1.35 mmol) were added to the reaction system. After the reaction was stirred at 25 ° C for 0.5 hours, the starting material 3-1a (100 mg, 0.271 mmol) was added to the reaction solution, and the reaction was continued for 3 hours. The mixture was purified by reverse phase column to obtain intermediate 3-2 (154 mg, 0.059 mmol, yield 75.7%).

LCMS(ESI)m/z[M+H]⁺＝749.4LCMS (ESI) m/z [M+H] ⁺ = 749.4

第二步：中间体3-3的合成Step 2: Synthesis of intermediate 3-3

将中间体3-2(154mg,0.205mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(92.9mg,0.308mmol)和N,N-二异丙基乙胺(79.4mg,0.615mmol)。反应置于25℃下搅拌3小时。混合物经过反相柱纯化得到中间体3-3(102mg,0.059mmol,收率58.8％)。Intermediate 3-2 (154 mg, 0.205 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (92.9 mg, 0.308 mmol) and N, N-diisopropylethylamine (79.4 mg, 0.615 mmol) were added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. The mixture was purified by reverse phase column to obtain intermediate 3-3 (102 mg, 0.059 mmol, yield 58.8%).

LCMS(ESI)m/z[M+H]⁺＝856.4。LCMS (ESI) m/z[M+H] ⁺ =856.4.

第三步：中间体3-4的合成Step 3: Synthesis of intermediate 3-4

将中间体3-3(14.9mg,0.017mmol)和环肽中间体INT-D(35.0mg,0.011mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(7.09mg,0.055mmol)。反应置于25℃下搅拌3小时。混合物经过反相柱纯化得到中间体3-4(32mg,0.008mmol)。Intermediate 3-3 (14.9 mg, 0.017 mmol) and cyclopeptide intermediate INT-D (35.0 mg, 0.011 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (7.09 mg, 0.055 mmol) was added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. The mixture was purified by reverse phase column to obtain intermediate 3-4 (32 mg, 0.008 mmol).

LCMS(ESI)m/z[M+2H]⁺/2＝1866.8；LCMS(ESI)m/z[M+3H]⁺/3＝1244.7。LCMS(ESI)m/z[M+2H] ⁺ /2=1866.8; LCMS(ESI)m/z[M+3H] ⁺ /3=1244.7.

第四步：中间体3-5的合成Step 4: Synthesis of intermediate 3-5

将中间体3-4(20mg,0.0053mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(4.0mg，0.026mmol)。反应置于25℃下搅拌1小时。混合物经过反相柱纯化得到中间体3-5(15mg,0.006mmol)。Intermediate 3-4 (20 mg, 0.0053 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (4.0 mg, 0.026 mmol) was added to the reaction system. The reaction was stirred at 25°C for 1 hour. The mixture was purified by reverse phase column to obtain intermediate 3-5 (15 mg, 0.006 mmol).

LCMS(ESI)m/z[M+2H]⁺/2＝1755.3；LCMS(ESI)m/z[M+3H]⁺/3＝1170.7。LCMS(ESI)m/z[M+2H] ⁺ /2=1755.3; LCMS(ESI)m/z[M+3H] ⁺ /3=1170.7.

第五步：化合物3的合成Step 5: Synthesis of compound 3

将中间体3-5(15mg,0.0042mmol)和中间体INT-A(8.5mg,0.0064mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(2.7mg,0.021mmol)，反应置于25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)，得到化合物3(7.84mg,0.0016mmol,收率38.4％)。Intermediate 3-5 (15 mg, 0.0042 mmol) and intermediate INT-A (8.5 mg, 0.0064 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (2.7 mg, 0.021 mmol) was added to the reaction system. The reaction was stirred at 25°C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150mm, 5um, mobile phase: [A: water (10mM TFA)-B: ACN], gradient B%: 37%-50%, 13min) to obtain compound 3 (7.84mg, 0.0016mmol, yield 38.4%).

LCMS(ESI)m/z[M+4H]⁺/4＝1182.58；LCMS(ESI)m/z[M+3H]⁺/3＝1576.61。LCMS(ESI)m/z[M+4H] ⁺ /4=1182.58; LCMS(ESI)m/z[M+3H] ⁺ /3=1576.61.

实施例4：化合物4的合成
Example 4: Synthesis of Compound 4

化合物4通过以下路线合成：

Compound 4 was synthesized via the following route:

第一步：中间体4-1b的合成Step 1: Synthesis of intermediate 4-1b

将原料SM-1(2.5g,7.3mmol)溶解于四氢呋喃(20mL)中。0℃下向反应体系中加入NaH(400mg,60％纯度，11.9mmol)，搅拌30min。缓慢加入溴乙酸叔丁酯(1.8ml,8.5mmol)。反应缓慢升温到25℃搅拌3小时。反应液用饱和氯化铵溶液(20mL)淬灭，二氯甲烷(20mL×3)萃取，合并有机层。有机层使用无水硫酸钠干燥，过滤，减压浓缩。所得残渣经过柱层析(A：乙酸乙酯-B:甲醇,梯度B％:0％-5％)纯化得到中间体4-1a(2.5g,5.4mmol)。The raw material SM-1 (2.5 g, 7.3 mmol) was dissolved in tetrahydrofuran (20 mL). NaH (400 mg, 60% purity, 11.9 mmol) was added to the reaction system at 0°C and stirred for 30 min. Tert-butyl bromoacetate (1.8 ml, 8.5 mmol) was slowly added. The reaction was slowly heated to 25°C and stirred for 3 hours. The reaction solution was quenched with saturated ammonium chloride solution (20 mL), extracted with dichloromethane (20 mL × 3), and the organic layers were combined. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (A: ethyl acetate-B: methanol, gradient B%: 0%-5%) to obtain intermediate 4-1a (2.5 g, 5.4 mmol).

将中间体4-1a(2.5g,5.4mmol)溶于二氯甲烷(20mL)中，加入三氟乙酸(5mL)。反应置于25℃下搅拌3小时。反应完全后，反应液浓缩得中间体4-1b(2.05g,5.1mmol)。Dissolve intermediate 4-1a (2.5 g, 5.4 mmol) in dichloromethane (20 mL), add trifluoroacetic acid (5 mL), and stir the reaction at 25°C for 3 hours. After the reaction is complete, the reaction solution is concentrated to obtain intermediate 4-1b (2.05 g, 5.1 mmol).

第二步：中间体4-2的合成Step 2: Synthesis of intermediate 4-2

将原料4-1(250mg,0.51mmol)溶解于二氯甲烷(4mL)中，向反应体系加入三氟乙酸(1ml)。反应置于25℃下搅拌3小时。反应完全后，反应液浓缩，加入水和乙腈冻干得粗品中间体4-2(200mg)。The raw material 4-1 (250 mg, 0.51 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (1 ml) was added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. After the reaction was complete, the reaction solution was concentrated, and water and acetonitrile were added and freeze-dried to obtain the crude intermediate 4-2 (200 mg).

LCMS(ESI)m/z[M+H]⁺＝383.3。LCMS (ESI) m/z[M+H] ⁺ =383.3.

第三步：中间体4-3的合成Step 3: Synthesis of intermediate 4-3

将中间体4-1b(100mg,0.251mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(75.5mg,0.251mmol)和N,N-二异丙基乙胺(97.1mg,0.753mmol)。反应置于25℃下搅拌0.5小时。向反应液中加入中间体4-2(92.3mg,0.251mmol)，继续搅拌3小时。反应液经过反相C18柱纯化(A：H₂O(0.05％TFA)-B：ACN,梯度B％:30％-60％)得到中间体4-3(80mg,0.106mmol,收率：42.5％)。Intermediate 4-1b (100 mg, 0.251 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (75.5 mg, 0.251 mmol) and N,N-diisopropylethylamine (97.1 mg, 0.753 mmol) were added to the reaction system. The reaction was stirred at 25°C for 0.5 hours. Intermediate 4-2 (92.3 mg, 0.251 mmol) was added to the reaction solution and continued to stir for 3 hours. The reaction solution was purified by reverse phase C18 column (A: H ₂ O (0.05% TFA)-B: ACN, gradient B%: 30%-60%) to obtain intermediate 4-3 (80 mg, 0.106 mmol, yield: 42.5%).

LCMS(ESI)m/z[M+H]⁺＝721.3。LCMS (ESI) m/z[M+H] ⁺ =721.3.

第四步：中间体4-4的合成Step 4: Synthesis of intermediate 4-4

将中间体4-3(80mg,0.11mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(50.1mg,0.16mmol)和N,N-二异丙基乙胺(42.5mg,0.33mmol)。反应置于25℃下搅拌3小时。反应液经过反相C18柱纯化(A：水(0.05％TFA)-B：ACN,梯度B％:30％-60％)得到中间体4-4(30mg,0.036mmol,33.3％)。Intermediate 4-3 (80 mg, 0.11 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (50.1 mg, 0.16 mmol) and N,N-diisopropylethylamine (42.5 mg, 0.33 mmol) were added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by reverse phase C18 column (A: water (0.05% TFA)-B: ACN, gradient B%: 30%-60%) to obtain intermediate 4-4 (30 mg, 0.036 mmol, 33.3%).

LCMS(ESI)m/z[M+H]⁺＝818.4。 LCMS (ESI) m/z[M+H] ⁺ =818.4.

第五步：中间体4-6的合成Step 5: Synthesis of intermediate 4-6

将中间体4-4(15.0mg,0.018mmol)和环肽中间体INT-D(20.0mg,0.0066mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(4.29mg,0.033mmol)。反应置于25℃下搅拌3小时。反应监测环肽消耗完全后，向反应液中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(4.60mg,0.03mmol)，继续搅拌1小时。反应液经过反相C18柱纯化(A：水(0.05％TFA)-B：ACN],梯度B％:30％-60％)得到中间体4-6(18mg,0.0051mmol,77.9％)。Intermediate 4-4 (15.0 mg, 0.018 mmol) and cyclopeptide intermediate INT-D (20.0 mg, 0.0066 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4.29 mg, 0.033 mmol) was added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. After the reaction monitoring cyclopeptide was completely consumed, 1,8-diazabicyclo[5.4.0]undec-7-ene (4.60 mg, 0.03 mmol) was added to the reaction solution and continued to stir for 1 hour. The reaction solution was purified by reverse phase C18 column (A: water (0.05% TFA)-B: ACN], gradient B%: 30%-60%) to obtain intermediate 4-6 (18 mg, 0.0051 mmol, 77.9%).

LCMS(ESI)m/z[M+2H]⁺/2＝1741.0；LCMS(ESI)m/z[M+3H]⁺/3＝1161.2。LCMS(ESI)m/z[M+2H] ⁺ /2=1741.0; LCMS(ESI)m/z[M+3H] ⁺ /3=1161.2.

第六步：化合物4的合成Step 6: Synthesis of compound 4

将中间体4-6(18mg,0.0051mmol)和中间体INT-A(10.3mg,0.0077mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(3.2mg,0.025mmol)，反应置于25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)，得到化合物4(5.24mg,0.0011mmol,21.56％)。Intermediate 4-6 (18 mg, 0.0051 mmol) and intermediate INT-A (10.3 mg, 0.0077 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (3.2 mg, 0.025 mmol) was added to the reaction system, and the reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150mm, 5um, mobile phase: [A: water (10mM TFA)-B: ACN], gradient B%: 37%-50%, 13min) to obtain compound 4 (5.24 mg, 0.0011 mmol, 21.56%).

LCMS(ESI)m/z[M+4H]⁺/4＝1175.48；LCMS(ESI)m/z[M+3H]⁺/3＝1567.18。LCMS(ESI)m/z[M+4H] ⁺ /4=1175.48; LCMS(ESI)m/z[M+3H] ⁺ /3=1567.18.

实施例5：化合物5的合成
Example 5: Synthesis of Compound 5

化合物5通过以下路线合成：
Compound 5 was synthesized via the following route:

第一步：中间体5-1a的合成Step 1: Synthesis of intermediate 5-1a

将二甲基亚砜(244mg，3.12mmol)溶于二氯甲烷(5ml)中，在-78℃滴加草酰氯(363mg,2.86mmol)，反应液搅拌1小时，然后将起始原料SM1(1g，2.6mmol)溶于二氯甲烷(5ml)滴加到上述反应液中。该反应液搅拌1小时后加入三乙胺(795mg，7.8mmol)，并在-78℃反应2小时。在低温下滴加水淬灭反应，用二氯甲烷萃取。有机相用无水硫酸钠干燥，过滤，滤液浓缩，得到中间体5-1a(1g，粗品)Dimethyl sulfoxide (244 mg, 3.12 mmol) was dissolved in dichloromethane (5 ml), and oxalyl chloride (363 mg, 2.86 mmol) was added dropwise at -78 °C. The reaction solution was stirred for 1 hour, and then the starting material SM1 (1 g, 2.6 mmol) was dissolved in dichloromethane (5 ml) and added dropwise to the above reaction solution. After stirring the reaction solution for 1 hour, triethylamine (795 mg, 7.8 mmol) was added and reacted at -78 °C for 2 hours. Water was added dropwise at low temperature to quench the reaction and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain intermediate 5-1a (1 g, crude product)

LCMS(ESI)m/z[M+H]+＝395。LCMS (ESI) m/z [M+H]+=395.

第二步：中间体5-2的合成Step 2: Synthesis of intermediate 5-2

将原料5-1(500mg,1.01mmol)溶于乙酸乙酯(5ml)，加入氯化氢乙酸乙酯溶液(3M/L，5ml)。反应液在室温下反应2小时。LCMS监测反应完全，将直接旋干，得到中间体5-2(500mg，粗品)。The raw material 5-1 (500 mg, 1.01 mmol) was dissolved in ethyl acetate (5 ml), and a hydrogen chloride ethyl acetate solution (3 M/L, 5 ml) was added. The reaction solution was reacted at room temperature for 2 hours. LCMS monitored the reaction to be complete, and the mixture was directly spin-dried to obtain Intermediate 5-2 (500 mg, crude).

LCMS(ESI)m/z[M+H]⁺＝395.2LCMS (ESI) m/z [M+H] ⁺ = 395.2

第三步：中间体5-3的合成Step 3: Synthesis of intermediate 5-3

将中间体5-2(395mg,1.0mmol)溶于甲醇(5ml)，加入5-1a(1g，粗品)和一滴醋酸。反应液在室温下搅拌0.5小时，然后加入氰基硼氢化钠(252mg,4.0mmol)。监测反应结束后，反应液用水(10mL)淬灭，乙酸乙酯(10mL×3)萃取，合并有机相。有机相用无水硫酸钠干燥，过滤，减压浓缩得到粗品中间体5-3(270mg)。Dissolve intermediate 5-2 (395 mg, 1.0 mmol) in methanol (5 ml), add 5-1a (1 g, crude) and a drop of acetic acid. Stir the reaction solution at room temperature for 0.5 hours, then add sodium cyanoborohydride (252 mg, 4.0 mmol). After monitoring the reaction, quench the reaction solution with water (10 mL), extract with ethyl acetate (10 mL × 3), and combine the organic phases. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude intermediate 5-3 (270 mg).

LCMS(ESI)m/z[M+H]⁺＝761.9。LCMS (ESI) m/z[M+H] ⁺ =761.9.

第四步：中间体5-4的合成Step 4: Synthesis of intermediate 5-4

将中间体5-3(140mg,0.18mmol)溶解于N，N-二甲基甲酰胺(3mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(66.5mg,0.22mmol)和N,N-二异丙基乙胺(69.6mg,0.54mmol)。反应置于25℃下搅拌3小时。反应液经过反相C18柱纯化(流动相：水(0.05％TFA)-ACN,梯度B％:30％-60％)得到中间体5-4(85mg,0.036mmol，收率：53.8％)。Intermediate 5-3 (140 mg, 0.18 mmol) was dissolved in N, N-dimethylformamide (3 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (66.5 mg, 0.22 mmol) and N, N-diisopropylethylamine (69.6 mg, 0.54 mmol) were added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by reverse phase C18 column (mobile phase: water (0.05% TFA)-ACN, gradient B%: 30%-60%) to obtain intermediate 5-4 (85 mg, 0.036 mmol, yield: 53.8%).

LCMS(ESI)m/z[M+H]⁺＝858.5。LCMS (ESI) m/z[M+H] ⁺ =858.5.

第五步：中间体5-6的合成Step 5: Synthesis of intermediate 5-6

将中间体5-4(20.0mg,0.023mmol)和环肽中间体INT-D(20.0mg,0.006mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(3.87mg,0.03mmol)。反应置于25℃下搅拌3小时。反应监测环肽消耗完全后，向反应液中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(4.60mg,0.03mmol)，继续搅拌1小时。反应液经过反相C18柱纯化(流动相：水(0.05％TFA)-ACN,梯度B％:30％-60％)得到中间体5-6(17mg,0.0045mmol，收率：72.6％)。Intermediate 5-4 (20.0 mg, 0.023 mmol) and cyclopeptide intermediate INT-D (20.0 mg, 0.006 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (3.87 mg, 0.03 mmol) was added to the reaction system. The reaction was stirred at 25 ° C for 3 hours. After the reaction monitoring cyclopeptide was completely consumed, 1,8-diazabicyclo[5.4.0]undec-7-ene (4.60 mg, 0.03 mmol) was added to the reaction solution and continued to stir for 1 hour. The reaction solution was purified by reverse phase C18 column (mobile phase: water (0.05% TFA)-ACN, gradient B%: 30%-60%) to obtain intermediate 5-6 (17 mg, 0.0045 mmol, yield: 72.6%).

LCMS(ESI)m/z[M+3H]⁺/3＝1174.8；LCMS(ESI)m/z[M+4H]⁺/4＝881.5。LCMS(ESI)m/z[M+3H] ⁺ /3=1174.8; LCMS(ESI)m/z[M+4H] ⁺ /4=881.5.

第六步：化合物5的合成Step 6: Synthesis of Compound 5

将中间体5-6(17mg,0.0048mmol)和中间体INT-A(9.66mg,0.0072mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(4.6mg,0.036mmol)，反应置于25℃下搅拌3小时。反应液经过制备分离纯化(Prep HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)。Intermediate 5-6 (17 mg, 0.0048 mmol) and intermediate INT-A (9.66 mg, 0.0072 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4.6 mg, 0.036 mmol) was added to the reaction system, and the reaction was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (Prep HPLC (chromatographic column: Welch Xtimate, 21.2*150mm, 5um, mobile phase: [A: water (10mM TFA)-B: ACN], gradient B%: 37%-50%, 13min).

LCMS(ESI)m/z[M+4H]⁺/4＝1185.43；LCMS(ESI)m/z[M+3H]⁺/3＝1580.43。LCMS(ESI)m/z[M+4H] ⁺ /4=1185.43; LCMS(ESI)m/z[M+3H] ⁺ /3=1580.43.

实施例6：化合物6的合成
Example 6: Synthesis of Compound 6

化合物6通过以下路线合成：
Compound 6 was synthesized via the following route:

第一步：中间体6-1的合成Step 1: Synthesis of intermediate 6-1

取起始物料6-1a(80mg,0.21mmol)溶于四氢呋喃(2mL)，加入三乙胺(530mg，5.25mmol)，溴乙酸叔丁酯(806mg,4.1mmol)，室温反应3小时。监测反应结束后，反应液减压浓缩，得粗品6-1(150mg)。The starting material 6-1a (80 mg, 0.21 mmol) was dissolved in tetrahydrofuran (2 mL), and triethylamine (530 mg, 5.25 mmol) and tert-butyl bromoacetate (806 mg, 4.1 mmol) were added, and the mixture was reacted at room temperature for 3 hours. After the reaction was monitored, the reaction solution was concentrated under reduced pressure to obtain a crude product 6-1 (150 mg).

LCMS(ESI)[M+H]⁺＝612.8。LCMS (ESI) [M+H] ⁺ = 612.8.

第二步：中间体6-2的合成Step 2: Synthesis of intermediate 6-2

取中间体6-1(150mg,粗品)溶于二氯甲烷(1mL),滴加三氟乙酸(2mL)，反应液室温下反应2小时。监测反应结束后，反应液减压浓缩。所得粗品加二氯甲烷(5mL)溶解，再次旋干，得中间体6-2(120mg,粗品)。Take intermediate 6-1 (150 mg, crude product) and dissolve it in dichloromethane (1 mL), add trifluoroacetic acid (2 mL) dropwise, and react the reaction solution at room temperature for 2 hours. After monitoring the reaction, the reaction solution is concentrated under reduced pressure. The obtained crude product is dissolved in dichloromethane (5 mL) and dried again to obtain intermediate 6-2 (120 mg, crude product).

LCMS(ESI)[M+H]⁺＝500.6。LCMS (ESI) [M+H] ⁺ = 500.6.

第三步：中间体6-3的合成Step 3: Synthesis of intermediate 6-3

取中间体6-2(27mg,粗品)溶于N,N-二甲基甲酰胺(1mL),加入Val-Cit-PAB-MMAE (30mg，0.041mmol),N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(15mg,0.135mmol)，N,N-二异丙基乙胺(17.5mg,0.135mmol)，室温反应3小时。监测反应完毕后，反应液直接过反相C18柱纯化(乙腈：水(0.05％三氟乙酸体系)＝35～60％)冲出产品，冻干得20mg中间体6-3。Intermediate 6-2 (27 mg, crude product) was dissolved in N,N-dimethylformamide (1 mL) and Val-Cit-PAB-MMAE was added. (30 mg, 0.041 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)uronium hexafluorophosphate (15 mg, 0.135 mmol), N,N-diisopropylethylamine (17.5 mg, 0.135 mmol), react at room temperature for 3 hours. After the reaction is completed, the reaction solution is directly purified by reverse phase C18 column (acetonitrile: water (0.05% trifluoroacetic acid system) = 35-60%) to flush out the product, and freeze-dried to obtain 20 mg of intermediate 6-3.

LCMS(ESI)[M+2H]⁺/2＝803.6。LCMS(ESI)[M+2H] ⁺ /2=803.6.

第四步：化合物6的合成Step 4: Synthesis of compound 6

取中间体6-3(12mg,0.075mmol)溶于N,N-二甲基甲酰胺(1mL),加入环肽中间体INT-D(315mg，0.005mmol),N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲(3mg,0.008mmol)，N,N-二异丙基乙胺(3mg,0.023mmol)，室温反应3小时。监测反应结束后，反应液直接过反相C18柱纯化(流动相：乙腈：水(0.05％三氟乙酸体系)＝35～60％)冲出产品，得化合物6(9mg)。Take the intermediate 6-3 (12 mg, 0.075 mmol) and dissolve it in N, N-dimethylformamide (1 mL), add the cyclic peptide intermediate INT-D (315 mg, 0.005 mmol), N, N, N', N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate (3 mg, 0.008 mmol), N, N-diisopropylethylamine (3 mg, 0.023 mmol), and react at room temperature for 3 hours. After the reaction is monitored, the reaction solution is directly purified by a reverse phase C18 column (mobile phase: acetonitrile: water (0.05% trifluoroacetic acid system) = 35-60%) to flush out the product to obtain compound 6 (9 mg).

LCMS(ESI)[1/4M+H]⁺＝1148.0；LCMS(ESI)[1/3M+H]⁺＝1530.3。LCMS(ESI)[1/4M+H] ⁺ =1148.0; LCMS(ESI)[1/3M+H] ⁺ =1530.3.

实施例7：化合物7的合成
Example 7: Synthesis of Compound 7

化合物7通过以下路线合成：

Compound 7 was synthesized via the following route:

第一步：中间体7-1a的合成Step 1: Synthesis of intermediate 7-1a

称取起始物料SM(m-PEG7-CH₂COOH)(100mg,0.25mmol)溶解于N,N-二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(90mg，0.30mmol，1.2eq)，N,N-二异丙基乙胺(161mg,1.25mmol,5eq)，室温搅拌2小时。然后在向反应体系中加入(S)-2-((((9H-芴-9-基)甲氧基)羰基)氨基)-5-氨基戊酸(133mg,0.38mmol,1.5eq)，室温搅拌2小时，反应液经过反相柱分离纯化，冻干后得到透明油状物中间体7-1a(70mg,0.10mmol,收率：38.1％)。The starting material SM (m-PEG7-CH ₂ COOH) (100 mg, 0.25 mmol) was weighed and dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (90 mg, 0.30 mmol, 1.2 eq) and N, N-diisopropylethylamine (161 mg, 1.25 mmol, 5 eq) were added to the reaction system, and stirred at room temperature for 2 hours. Then (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-aminopentanoic acid (133 mg, 0.38 mmol, 1.5 eq) was added to the reaction system, and stirred at room temperature for 2 hours. The reaction solution was separated and purified by reverse phase column, and after freeze-drying, a transparent oily intermediate 7-1a (70 mg, 0.10 mmol, yield: 38.1%) was obtained.

LCMS(ESI)[M+H]⁺＝735.7。LCMS (ESI) [M+H] ⁺ = 735.7.

第二步：中间体7-1b的合成Step 2: Synthesis of intermediate 7-1b

称取中间体7-1a(70mg,0.095mmol)溶解于N,N-二甲基甲酰胺(1.5mL)中，向反应体系中加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(37mg,0.191mmol,2.0eq)，N-羟基丁二酰亚胺(22mg,0.191mmol,2.0e)。反应液室温搅拌2小时。反应液经过反相柱纯化，冻干后得到中间体7-1b(25mg,0.030mmol,收率：31.6％)。Weigh the intermediate 7-1a (70 mg, 0.095 mmol) and dissolve it in N,N-dimethylformamide (1.5 mL). Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (37 mg, 0.191 mmol, 2.0 eq) and N-hydroxysuccinimide (22 mg, 0.191 mmol, 2.0 eq) to the reaction system. Stir the reaction solution at room temperature for 2 hours. The reaction solution is purified by reverse phase column and freeze-dried to obtain intermediate 7-1b (25 mg, 0.030 mmol, yield: 31.6%).

LCMS(ESI)[M+H]⁺＝832.8。LCMS (ESI) [M+H] ⁺ = 832.8.

第三步：中间体7-1的合成Step 3: Synthesis of intermediate 7-1

称取起始物料7-1b(17mg,0.02mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(5mg,0.04mmol,3.0eq),环肽中间体INT-D(40mg, 0.013mmol,1.0eq)，室温搅拌3小时。反应液经过反相柱纯化，冻干后得到中间体7-1(41mg,0.011mmol,收率：82.7％)。The starting material 7-1b (17 mg, 0.02 mmol) was weighed and dissolved in N, N-dimethylformamide (2 mL). N, N-diisopropylethylamine (5 mg, 0.04 mmol, 3.0 eq) and the cyclopeptide intermediate INT-D (40 mg, 0.013mmol, 1.0eq) and stirred at room temperature for 3 hours. The reaction solution was purified by reverse phase column and freeze-dried to obtain intermediate 7-1 (41mg, 0.011mmol, yield: 82.7%).

LCMS(ESI)[M+3H]⁺/3＝1240.20。LCMS(ESI)[M+3H] ⁺ /3=1240.20.

第四步：中间体7-2的合成Step 4: Synthesis of intermediate 7-2

称取中间体7-1(41mg,0.011mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入3滴1,8-二氮杂二环[5.4.0]十一碳-7-烯，室温搅拌3小时。反应液经过反相柱纯化，冻干后得到7-2(28mg,0.008mmol,收率：72.3％)。Weigh the intermediate 7-1 (41 mg, 0.011 mmol) and dissolve it in N, N-dimethylformamide (2 mL). Add 3 drops of 1,8-diazabicyclo[5.4.0]undec-7-ene to the reaction system and stir at room temperature for 3 hours. The reaction solution is purified by reverse phase column and freeze-dried to obtain 7-2 (28 mg, 0.008 mmol, yield: 72.3%).

LCMS(ESI)[M+3H]⁺/3＝1165.60；[M+4H]⁺/4＝874.40。LCMS (ESI) [M+3H] ⁺ /3 = 1165.60; [M+4H] ⁺ /4 = 874.40.

第五步：化合物7的合成Step 5: Synthesis of compound 7

称取中间体7-2(28mg,0.008mmol)溶解于N，N-二甲基甲酰胺(2mL)中，分别向反应体系中加入3滴N,N-二异丙基乙胺，中间体INT-A(21mg,0.016mmol,2.0eq)，室温搅拌18小时。反应液经过制备色谱(色谱柱：Welch pfp C18 150mm*21.2mm,5um流速：15ml/min流动相：A:0.1％ FA水B:ACN波长：214nm梯度:35-45(B)13min)纯化，冻干，得到化合物7(8.3mg,0.00176mmol,收率22％)。Weigh the intermediate 7-2 (28 mg, 0.008 mmol) and dissolve it in N, N-dimethylformamide (2 mL). Add 3 drops of N, N-diisopropylethylamine and intermediate INT-A (21 mg, 0.016 mmol, 2.0 eq) to the reaction system respectively, and stir at room temperature for 18 hours. The reaction solution is purified by preparative chromatography (chromatographic column: Welch pfp C18 150 mm*21.2 mm, 5 um flow rate: 15 ml/min mobile phase: A: 0.1% FA water B: ACN wavelength: 214 nm gradient: 35-45 (B) 13 min) and freeze-dried to obtain compound 7 (8.3 mg, 0.00176 mmol, yield 22%).

LCMS(ESI)[M+3H]⁺/3＝1572.40，LCMS(ESI)[M+4H]⁺/4＝1179.80。LCMS(ESI)[M+3H] ⁺ /3=1572.40, LCMS(ESI)[M+4H] ⁺ /4=1179.80.

实施例8：化合物8的合成
Example 8: Synthesis of Compound 8

化合物8通过以下路线合成：

Compound 8 was synthesized via the following route:

第一步：中间体8-2的合成Step 1: Synthesis of intermediate 8-2

将原料8-1(150mg,0.31mmol)溶解于二氯甲烷(3mL)中，在0℃下向反应体系滴加三氟乙酸(1mL)，5分钟后将反应体系转移到室温下搅拌5小时。反应完全后，反应液减压浓缩，粗品直接投下一步。The raw material 8-1 (150 mg, 0.31 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added dropwise to the reaction system at 0°C, and after 5 minutes, the reaction system was transferred to room temperature and stirred for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was directly used for the next step.

LCMS(ESI)m/z[M+H]⁺＝383.4。LCMS (ESI) m/z[M+H] ⁺ =383.4.

第二步：中间体8-3的合成Step 2: Synthesis of intermediate 8-3

称取羧酸8-2a(m-PEG7-CH₂COOH，50mg，0.13mmol)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(42mg,0.14mmol,1.1eq)，N,N-二异丙基乙胺(49mg,0.38mmol,3eq)，室温搅拌4小时。然后向反应体系中加入中间体8-2(118mg，0.31mmol，2.4eq)，室温搅拌18小时。反应液经过反相柱分离纯化，冻干后得到中间体8-3(84mg,0.11mmol,收率：78.6％)。Carboxylic acid 8-2a (m-PEG7-CH ₂ COOH, 50 mg, 0.13 mmol) was weighed and dissolved in N, N-dimethylformamide (2 mL), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (42 mg, 0.14 mmol, 1.1 eq) and N, N-diisopropylethylamine (49 mg, 0.38 mmol, 3 eq) were added to the reaction system, and stirred at room temperature for 4 hours. Then, intermediate 8-2 (118 mg, 0.31 mmol, 2.4 eq) was added to the reaction system, and stirred at room temperature for 18 hours. The reaction solution was separated and purified by reverse phase column, and intermediate 8-3 (84 mg, 0.11 mmol, yield: 78.6%) was obtained after freeze drying.

LCMS(ESI)[M+H]+＝763.50。LCMS (ESI) [M+H]+ = 763.50.

第三步：中间体8-4的合成Step 3: Synthesis of intermediate 8-4

将中间体8-3(84mg,0.11mmol)溶解于N，N-二甲基甲酰胺(3mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(66mg,0.22mmol)和N,N-二异丙基乙胺(43mg,0.33mmol)，搅拌3小时。反应液经过反相柱纯化，得到中间体8-4(70mg,0.081mmol,收率：74.1％)。Intermediate 8-3 (84 mg, 0.11 mmol) was dissolved in N, N-dimethylformamide (3 mL), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (66 mg, 0.22 mmol) and N, N-diisopropylethylamine (43 mg, 0.33 mmol) were added to the reaction system and stirred for 3 hours. The reaction solution was purified by reverse phase column to obtain intermediate 8-4 (70 mg, 0.081 mmol, yield: 74.1%).

LCMS(ESI)m/z[M+H]⁺＝860.50。 LCMS (ESI) m/z[M+H] ⁺ =860.50.

第四步：中间体8-6的合成Step 4: Synthesis of intermediate 8-6

将中间体8-4(17mg,0.02mmol)和环肽中间体INT-D(40mg,0.013mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(5mg,0.039mmol)，反应18小时。反应液经过反相柱纯化得到白色固体。将白色固体溶于N，N-二甲基甲酰胺(2mL)中，加入3滴1,8-二氮杂二环[5.4.0]十一碳-7-烯。反应液室温下搅拌3小时。监测反应结束后，该反应液经过反相柱纯化，得到中间体8-6(20mg,0.0057mmol,收率：28.5％)。The intermediate 8-4 (17 mg, 0.02 mmol) and the cyclic peptide intermediate INT-D (40 mg, 0.013 mmol) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (5 mg, 0.039 mmol) was added to the reaction system and reacted for 18 hours. The reaction solution was purified by reverse phase column to obtain a white solid. The white solid was dissolved in N, N-dimethylformamide (2 mL), and 3 drops of 1,8-diazabicyclo[5.4.0]undec-7-ene were added. The reaction solution was stirred at room temperature for 3 hours. After the reaction was monitored, the reaction solution was purified by reverse phase column to obtain intermediate 8-6 (20 mg, 0.0057 mmol, yield: 28.5%).

LCMS(ESI)m/z[M+3H]⁺/3＝1249.30；LCMS(ESI)m/z[M+3H]⁺/3＝1175.30。LCMS(ESI)m/z[M+3H] ⁺ /3=1249.30; LCMS(ESI)m/z[M+3H] ⁺ /3=1175.30.

第五步：化合物8的合成Step 5: Synthesis of Compound 8

将中间体8-6(20mg，0.0057mmol)和中间体INT-A(15mg,0.0114mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入3滴N,N-二异丙基乙胺。反应液室温搅拌18小时。反应液经过反向制备(色谱柱：Welch pfp C18，150mm*21.2mm,5um；流速：15ml/min；流动相：A:(0.1％甲酸)水B:乙腈；波长：214nm；梯度B％:35％-45％；13min)分离纯化得到化合物8(9.32mg,0.00196mmol,收率：34.5％)。Intermediate 8-6 (20 mg, 0.0057 mmol) and intermediate INT-A (15 mg, 0.0114 mmol) were dissolved in N, N-dimethylformamide (2 mL), and 3 drops of N, N-diisopropylethylamine were added to the reaction system. The reaction solution was stirred at room temperature for 18 hours. The reaction solution was separated and purified by reverse preparation (chromatographic column: Welch pfp C18, 150 mm*21.2 mm, 5 um; flow rate: 15 ml/min; mobile phase: A: (0.1% formic acid) water B: acetonitrile; wavelength: 214 nm; gradient B%: 35%-45%; 13 min) to obtain compound 8 (9.32 mg, 0.00196 mmol, yield: 34.5%).

LCMS(ESI)m/z[M+4H]⁺/4＝1186.80；[M+3H]⁺/3＝1582.00。LCMS (ESI) m/z [M+4H] ⁺ /4=1186.80; [M+3H] ⁺ /3=1582.00.

实施例9：化合物9的合成
Example 9: Synthesis of Compound 9

化合物9通过以下路线合成：

Compound 9 was synthesized via the following route:

第一步：中间体9-1的合成Step 1: Synthesis of intermediate 9-1

称取化合物9-1a(m-PEG7-CH₂COOH，110mg,0.276mmol)溶解于N，N二甲基甲酰胺(2mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(83mg,0.276mmol,1.0eq)，N，N二异丙基乙胺(178mg,1.38mmol,5.0eq)，室温搅拌2小时。反应液无需处理，粗品中间体9-1直接用于下一步。Weigh compound 9-1a (m-PEG7-CH ₂ COOH, 110 mg, 0.276 mmol) and dissolve it in N, N dimethylformamide (2 mL). Add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (83 mg, 0.276 mmol, 1.0 eq) and N, N diisopropylethylamine (178 mg, 1.38 mmol, 5.0 eq) to the reaction system and stir at room temperature for 2 hours. The reaction solution does not need to be treated, and the crude intermediate 9-1 is directly used in the next step.

第二步：中间体9-2的合成Step 2: Synthesis of intermediate 9-2

称取化合物9-1b(102mg,0.276mmol)溶解于中间体9-1的反应液中，室温搅拌2小时。反应液经过反相柱纯化，冻干后得到中间体9-2(95mg,0.127mmol,收率46.0％)。Compound 9-1b (102 mg, 0.276 mmol) was weighed and dissolved in the reaction solution of intermediate 9-1, and stirred at room temperature for 2 hours. The reaction solution was purified by reverse phase column and freeze-dried to obtain intermediate 9-2 (95 mg, 0.127 mmol, yield 46.0%).

LCMS(ESI)[M+H]⁺＝749.7。LCMS (ESI) [M+H] ⁺ = 749.7.

第三步：中间体9-3的合成Step 3: Synthesis of intermediate 9-3

称取中间体9-2(95mg,0.127mmol)溶解于N，N-二甲基甲酰胺(2.0mL)中，向反应体系中加入N，N二异丙基乙胺(49mg,0.381mmol,3.0eq),2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(57mg,0.190mmol,1.5eq)。室温搅拌2小时。反应液经过反相柱纯化，冻干后得到中间体9-3(25mg,0.030mmol,收率23.6％)。Weigh the intermediate 9-2 (95 mg, 0.127 mmol) and dissolve it in N, N-dimethylformamide (2.0 mL). Add N, N-diisopropylethylamine (49 mg, 0.381 mmol, 3.0 eq) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (57 mg, 0.190 mmol, 1.5 eq) to the reaction system. Stir at room temperature for 2 hours. The reaction solution is purified by reverse phase column and freeze-dried to obtain intermediate 9-3 (25 mg, 0.030 mmol, yield 23.6%).

LCMS(ESI)[M+H]⁺＝846.6。LCMS (ESI) [M+H] ⁺ = 846.6.

第四步：中间体9-4的合成Step 4: Synthesis of intermediate 9-4

称取环肽中间体INT-D(25mg,0.008mmol,1eq)溶解于N，N二甲基甲酰胺(1mL) 中，向反应体系中加入N，N-二异丙基乙胺(4.16mg,0.032mmol,4.0eq)，中间体9-3(10.5mg,0.012mmol,1.5eq)，室温搅拌2.5小时。反应液直接过反相C18柱纯化(流动相：乙腈：水(0.05％三氟乙酸体系)＝35～60％)冲出产品，冻干后得到中间体9-4(15mg)。Weigh the cyclic peptide intermediate INT-D (25 mg, 0.008 mmol, 1 eq) and dissolve it in N, N-dimethylformamide (1 mL). N, N-diisopropylethylamine (4.16 mg, 0.032 mmol, 4.0 eq) and intermediate 9-3 (10.5 mg, 0.012 mmol, 1.5 eq) were added to the reaction system and stirred at room temperature for 2.5 hours. The reaction solution was directly purified by reverse phase C18 column (mobile phase: acetonitrile: water (0.05% trifluoroacetic acid system) = 35-60%) to flush out the product, and then freeze-dried to obtain intermediate 9-4 (15 mg).

LCMS(ESI)[M+3H]⁺/3＝1244.8。LCMS(ESI)[M+3H] ⁺ /3=1244.8.

第五步：中间体9-5的合成Step 5: Synthesis of intermediate 9-5

称取中间体9-4(15mg,0.004mmol,1.0eq)溶解于N，N-二甲基甲酰胺(1mL)中，向反应体系中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(6mg,0.04mmol,10.0eq)。室温搅拌30分钟。反应液反相C18柱纯化(乙腈：水(0.05％三氟乙酸体系)＝30～50％)冲出产品，冻干后得到中间体9-5(8mg)。Weigh the intermediate 9-4 (15 mg, 0.004 mmol, 1.0 eq) and dissolve it in N, N-dimethylformamide (1 mL). Add 1,8-diazabicyclo[5.4.0]undec-7-ene (6 mg, 0.04 mmol, 10.0 eq) to the reaction system. Stir at room temperature for 30 minutes. The reaction solution is purified by reverse phase C18 column (acetonitrile: water (0.05% trifluoroacetic acid system) = 30-50%) to flush out the product, and freeze-dry to obtain the intermediate 9-5 (8 mg).

LCMS(ESI)[M+3H]⁺/3＝1171.00。LCMS(ESI)[M+3H] ⁺ /3=1171.00.

第六步：化合物9的合成Step 6: Synthesis of compound 9

称取中间体9-5(8mg,0.0022mmol)溶解于N，N-二甲基甲酰胺(1mL)中，分别向反应体系中加入N，N-二异丙基乙胺(1.1mg,0.009mmol),中间体INT-A(4.3mg,0.0033mmol)。反应液室温下搅拌2小时。反应液经过Pre-HPLC(色谱柱：Welch pfp C18 150mm*21.2mm,5um；流速：15ml/min；流动相：A:0.1％ TFA水B:ACN；波长：214nm；梯度B％:30-55％；保留时间：1.43min)制备得到化合物9(5mg)。Weigh the intermediate 9-5 (8 mg, 0.0022 mmol) and dissolve it in N, N-dimethylformamide (1 mL). Add N, N-diisopropylethylamine (1.1 mg, 0.009 mmol) and intermediate INT-A (4.3 mg, 0.0033 mmol) to the reaction system respectively. Stir the reaction solution at room temperature for 2 hours. The reaction solution was subjected to Pre-HPLC (chromatographic column: Welch pfp C18 150 mm*21.2 mm, 5 um; flow rate: 15 ml/min; mobile phase: A: 0.1% TFA water B: ACN; wavelength: 214 nm; gradient B%: 30-55%; retention time: 1.43 min) to prepare compound 9 (5 mg).

LCMS(ESI)[M+3H]+/3＝1576.66；LCMS(ESI)[M+4H]+/4＝1182.53。LCMS(ESI)[M+3H]+/3=1576.66; LCMS(ESI)[M+4H]+/4=1182.53.

实施例10：化合物10的合成
Example 10: Synthesis of Compound 10

化合物10通过以下路线合成：
Compound 10 was synthesized via the following route:

第一步：中间体10-2的合成Step 1: Synthesis of intermediate 10-2

称取化合物10-1(250mg,0.42mmol,1eq)溶解于N，N-二甲基甲酰胺(5mL)中，向反应体系中加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(150mg,0.50mmol,1.2eq)，N，N-二异丙基乙胺(165mg,1.26mmol,3eq)，室温搅拌2.5小时。向反应体系中加入N-(叔丁氧羰基)-D-赖氨酸(135mg,0.55mmol,1.3eq)，室温搅拌18小时。反应液经过反相柱纯化，冻干后得到中间体10-2(283mg,0.34mmol,收率80.9％)。Weigh compound 10-1 (250 mg, 0.42 mmol, 1 eq) and dissolve it in N, N-dimethylformamide (5 mL). Add 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (150 mg, 0.50 mmol, 1.2 eq) and N, N-diisopropylethylamine (165 mg, 1.26 mmol, 3 eq) to the reaction system and stir at room temperature for 2.5 hours. Add N-(tert-butyloxycarbonyl)-D-lysine (135 mg, 0.55 mmol, 1.3 eq) to the reaction system and stir at room temperature for 18 hours. The reaction solution is purified by reverse phase column and freeze-dried to obtain intermediate 10-2 (283 mg, 0.34 mmol, yield 80.9%).

LCMS(ESI)[M+H-Boc]⁺＝724.40，[M+Na]⁺＝846.50。LCMS (ESI) [M+H-Boc] ⁺ =724.40, [M+Na] ⁺ =846.50.

第二步：中间体10-3的合成Step 2: Synthesis of intermediate 10-3

称取中间体10-2(283mg,0.34mmol,1eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(157mg,1.03mmol,3eq)，室温搅拌4小时。反应液经过反相柱纯化，冻干后得到中间体10-3(186mg,0.31mmol,收率91.2％)。Intermediate 10-2 (283 mg, 0.34 mmol, 1 eq) was weighed and dissolved in N,N-dimethylformamide (2 mL). 1,8-diazabicyclo[5.4.0]undec-7-ene (157 mg, 1.03 mmol, 3 eq) was added to the reaction system and stirred at room temperature for 4 hours. The reaction solution was purified by reverse phase column and freeze-dried to obtain intermediate 10-3 (186 mg, 0.31 mmol, yield 91.2%).

LCMS(ESI)[M+H]⁺＝602.40。LCMS (ESI) [M+H] ⁺ = 602.40.

第三步：中间体10-4的合成Step 3: Synthesis of intermediate 10-4

称取中间体10-3(183mg,0.31mmol,1.0eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中滴加醋酸酐(38mg,0.37mmol,1.2eq)的N，N-二甲基甲酰胺(1mL)溶液，N，N-二异丙基乙胺(120mg,0.93mmol,3eq)，室温搅拌3小时。反应液经过反相柱纯化，冻干后得到中间体10-4(200mg,0.31mmol,收率99.9％)。Weigh the intermediate 10-3 (183 mg, 0.31 mmol, 1.0 eq) and dissolve it in N, N-dimethylformamide (2 mL). Add acetic anhydride (38 mg, 0.37 mmol, 1.2 eq) in N, N-dimethylformamide (1 mL) and N, N-diisopropylethylamine (120 mg, 0.93 mmol, 3 eq) to the reaction system and stir at room temperature for 3 hours. The reaction solution is purified by reverse phase column and freeze-dried to obtain the intermediate 10-4 (200 mg, 0.31 mmol, yield 99.9%).

LCMS(ESI)[M+H]⁺＝644.40，[M+Na]⁺＝666.50,[M+H-Boc]⁺＝544.40。LCMS (ESI) [M+H] ⁺ =644.40, [M+Na] ⁺ =666.50, [M+H-Boc] ⁺ =544.40.

第四步：中间体10-5的合成Step 4: Synthesis of intermediate 10-5

称取中间体10-4(13mg,0.02mmol,1.5eq)溶解于N，N-二甲基甲酰胺(1mL)中，向反应体系中加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(7.5mg,0.02mmol,1.5eq)和N，N-二异丙基乙胺(6.9mg,0.053mmol,4.0eq)，室温搅拌0.5小时。然后向反应体系中加入环肽中间体INT-D(40mg,0.013mmol,1.0eq)，室温搅拌1小时。反应液经过反相柱纯化，冻干后得到中间体10-5(13mg)。Weigh the intermediate 10-4 (13 mg, 0.02 mmol, 1.5 eq) and dissolve it in N, N-dimethylformamide (1 mL). Add 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (7.5 mg, 0.02 mmol, 1.5 eq) and N, N-diisopropylethylamine (6.9 mg, 0.053 mmol, 4.0 eq) to the reaction system and stir at room temperature for 0.5 hours. Then add the cyclic peptide intermediate INT-D (40 mg, 0.013 mmol, 1.0 eq) to the reaction system and stir at room temperature for 1 hour. The reaction solution is purified by reverse phase column and freeze-dried to obtain intermediate 10-5 (13 mg).

LCMS(ESI)[M+3H]⁺/3＝1209.8,[M-100+3H]⁺/3＝1176.6。LCMS (ESI) [M+3H] ⁺ /3 = 1209.8, [M-100 + 3H] ⁺ /3 = 1176.6.

第五步：中间体10-6的合成Step 5: Synthesis of intermediate 10-6

称取中间体10-5(25mg,0.0069mmol)溶解于乙腈(2mL)和水(1mL)中，向反应体系中加入三氟乙酸(1mL)。反应液室温下搅拌1小时。反应液经过反相柱纯化，冻干后得到中间体10-6(13mg)。Weigh the intermediate 10-5 (25 mg, 0.0069 mmol) and dissolve it in acetonitrile (2 mL) and water (1 mL), and add trifluoroacetic acid (1 mL) to the reaction system. Stir the reaction solution at room temperature for 1 hour. The reaction solution is purified by reverse phase column and freeze-dried to obtain intermediate 10-6 (13 mg).

LCMS(ESI)[M+3H]⁺/3＝1176.6。LCMS(ESI)[M+3H] ⁺ /3=1176.6.

第六步：化合物10的合成Step 6: Synthesis of compound 10

称取中间体10-6(13mg,0.0037mmol,1eq)溶解于N，N-二甲基甲酰胺(1mL)中，向反应体系中加入中间体INT-A(9.8mg,0.0074mmol,2eq)和1滴N，N-二异丙基乙胺。反应液室温下搅拌8小时。反应液经过反向制备(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:30％-60％,13min)分离纯化得到化合物10(7.7mg)。Intermediate 10-6 (13 mg, 0.0037 mmol, 1 eq) was weighed and dissolved in N, N-dimethylformamide (1 mL), and intermediate INT-A (9.8 mg, 0.0074 mmol, 2 eq) and 1 drop of N, N-diisopropylethylamine were added to the reaction system. The reaction solution was stirred at room temperature for 8 hours. The reaction solution was separated and purified by reverse preparation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150 mm, 5 um, mobile phase: [A: water (10 mM TFA)-B: ACN], gradient B%: 30%-60%, 13 min) to obtain compound 10 (7.7 mg).

LCMS(ESI)[M+4H]⁺/4＝1186.83,[M+3H]⁺/3＝1582.41。LCMS (ESI) [M+4H] ⁺ /4 = 1186.83, [M + 3H] ⁺ /3 = 1582.41.

实施例11：化合物11的合成
Example 11: Synthesis of Compound 11

化合物11通过以下路线合成：
Compound 11 was synthesized via the following route:

第一步：中间体11-3的合成Step 1: Synthesis of intermediate 11-3

将原料化合物11-1(333mg,0.783mmol，1.0eq)和原料化合物11-2(300mg,0.783mmol，1.0eq)溶于N，N-二甲基甲酰胺(10ml)中，加入2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(596mg,1.567mmol，2.0eq)和N,N-二异丙基乙胺(304mg,2.350mmol,3.0eq)。反应液在室温下反应0.5小时。反应液经过C18反相柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:49％-51％)得到中间体11-3(145mg,收率：23％)。The raw material compound 11-1 (333 mg, 0.783 mmol, 1.0 eq) and the raw material compound 11-2 (300 mg, 0.783 mmol, 1.0 eq) were dissolved in N, N-dimethylformamide (10 ml), and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (596 mg, 1.567 mmol, 2.0 eq) and N, N-diisopropylethylamine (304 mg, 2.350 mmol, 3.0 eq) were added. The reaction solution was reacted at room temperature for 0.5 hours. The reaction solution was purified by C18 reverse phase column (A: water (0.05% TFA)-B: ACN, gradient B%: 49%-51%) to obtain intermediate 11-3 (145 mg, yield: 23%).

LCMS(ESI)m/z[M+H]⁺＝791.5。LCMS (ESI) m/z[M+H] ⁺ =791.5.

第二步：中间体11-4的合成Step 2: Synthesis of intermediate 11-4

将中间体11-3(135mg,0.171mmol)溶于二氯甲烷(1.2ml)中，加入三氟乙酸(0.3ml)。反应液在室温下搅拌过夜。反应液减压浓缩得粗产物中间体11-4。(190mg,粗品)Dissolve intermediate 11-3 (135 mg, 0.171 mmol) in dichloromethane (1.2 ml) and add trifluoroacetic acid (0.3 ml). Stir the reaction mixture at room temperature overnight. Concentrate the reaction mixture under reduced pressure to obtain the crude product intermediate 11-4. (190 mg, crude product)

LCMS(ESI)m/z[M+H]+＝735.4。LCMS (ESI) m/z [M+H]+ = 735.4.

第三步：中间体11-5的合成Step 3: Synthesis of intermediate 11-5

将中间体11-4(180mg,0.245mmol，1.0eq)溶于N，N-二甲基甲酰胺(2ml)，加入2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(111mg,0.368mmol，1.5eq)和N,N-二异丙基乙胺(95mg,0.736mmol，3.0eq)。反应液在室温下反应过夜。然后反应液经过C18反相柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:5％-95％)得到中间体11-5(90mg,收率:48％)。Intermediate 11-4 (180 mg, 0.245 mmol, 1.0 eq) was dissolved in N, N-dimethylformamide (2 ml), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (111 mg, 0.368 mmol, 1.5 eq) and N, N-diisopropylethylamine (95 mg, 0.736 mmol, 3.0 eq) were added. The reaction solution was reacted at room temperature overnight. The reaction solution was then purified by C18 reverse phase column (A: water (0.05% TFA)-B: ACN, gradient B%: 5%-95%) to obtain intermediate 11-5 (90 mg, yield: 48%).

LCMS(ESI)m/z[M+H]⁺＝832.4。LCMS (ESI) m/z[M+H] ⁺ =832.4.

第四步：中间体11-6的合成Step 4: Synthesis of intermediate 11-6

将中间体INT-D(20mg,0.0066mmol，1.0eq)和中间体11-5(20mg,0.024mmol，3.6eq)溶于N，N二甲基甲酰胺(3ml)，加入N,N-二异丙基乙胺(4.2mg,0.033mmol，5.0eq)。反应液在25℃下反应3小时。反应液经过C18反相柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:5％-95％)得到中间体11-6(19mg，收率：79％)。The intermediate INT-D (20 mg, 0.0066 mmol, 1.0 eq) and the intermediate 11-5 (20 mg, 0.024 mmol, 3.6 eq) were dissolved in N, N-dimethylformamide (3 ml), and N, N-diisopropylethylamine (4.2 mg, 0.033 mmol, 5.0 eq) was added. The reaction solution was reacted at 25 ° C for 3 hours. The reaction solution was purified by C18 reverse phase column (A: water (0.05% TFA)-B: ACN, gradient B%: 5%-95%) to obtain intermediate 11-6 (19 mg, yield: 79%).

LCMS(ESI)m/z[M+2H]⁺/2＝1240.2。LCMS (ESI) m/z[M+2H] ⁺ /2=1240.2.

第五步：中间体11-7的合成Step 5: Synthesis of intermediate 11-7

将中间体11-6(19mg,0.0051mmol，1.0eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应液中加入1,8-二氮杂二环[5.4.0]十一碳-7-烯(4.60mg,0.03mmol，6eq)。反应液在25℃下反应1小时。反应液经过反相C18柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:5％-95％)得到中间体11-7(14mg，收率：80％)。Intermediate 11-6 (19 mg, 0.0051 mmol, 1.0 eq) was dissolved in N, N-dimethylformamide (2 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (4.60 mg, 0.03 mmol, 6 eq) was added to the reaction solution. The reaction solution was reacted at 25°C for 1 hour. The reaction solution was purified by reverse phase C18 column (A: water (0.05% TFA)-B: ACN, gradient B%: 5%-95%) to obtain intermediate 11-7 (14 mg, yield: 80%).

LCMS(ESI)m/z[M+3H]⁺/3＝1165.8；LCMS(ESI)m/z[M+2H]⁺/2＝1748.3。LCMS(ESI)m/z[M+3H] ⁺ /3=1165.8; LCMS(ESI)m/z[M+2H] ⁺ /2=1748.3.

第六步：化合物11的合成Step 6: Synthesis of compound 11

将中间体11-7(14mg，0.0040mmol，1.0eq)和中间体INT-A(8.0mg,0.0060mmol，1.5eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(4.6mg,0.036mmol)。反应液在25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)得到化合物11(16.54mg，收率：86.6％)。 Intermediate 11-7 (14 mg, 0.0040 mmol, 1.0 eq) and intermediate INT-A (8.0 mg, 0.0060 mmol, 1.5 eq) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4.6 mg, 0.036 mmol) was added to the reaction system. The reaction solution was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150 mm, 5 um, mobile phase: [A: water (10 mM TFA)-B: ACN], gradient B%: 37%-50%, 13 min) to obtain compound 11 (16.54 mg, yield: 86.6%).

LCMS(ESI)m/z[M+4H]⁺/4＝1179.24；LCMS(ESI)m/z[M+3H]⁺/3＝1572.26。LCMS(ESI)m/z[M+4H] ⁺ /4=1179.24; LCMS(ESI)m/z[M+3H] ⁺ /3=1572.26.

实施例12：化合物12的合成
Example 12: Synthesis of Compound 12

化合物12通过以下路线合成：
Compound 12 was synthesized via the following route:

第一步：中间体12-1的合成Step 1: Synthesis of intermediate 12-1

称取N-Boc-β-丙氨酸(15g,79.28mmol，1.0eq)和3-溴丙烯(10.55g,87.2mmol，1.1eq)溶于N，N-二甲基甲酰胺(300ml)，加入碳酸铯(54.24g,166.49mmol，2.1eq)。反应液在10℃下反应12小时。监测反应结束后，反应液减压浓缩得到粗品。反相柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)得中间体12-1(14g,收率:77％)。Weigh N-Boc-β-alanine (15 g, 79.28 mmol, 1.0 eq) and 3-bromopropylene (10.55 g, 87.2 mmol, 1.1 eq) and dissolve in N, N-dimethylformamide (300 ml), add cesium carbonate (54.24 g, 166.49 mmol, 2.1 eq). The reaction solution was reacted at 10 ° C for 12 hours. After the reaction was monitored, the reaction solution was concentrated under reduced pressure to obtain a crude product. Reverse phase column purification (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 12-1 (14 g, yield: 77%).

LCMS(ESI)m/z[M+H]⁺＝174.2。LCMS (ESI) m/z[M+H] ⁺ =174.2.

第二步：中间体12-2的合成Step 2: Synthesis of intermediate 12-2

称取中间体12-1(12g,52.34mmol)置于二氯甲烷中(120ml)，加入三氟乙酸(12ml)。反应液在10℃下搅拌12小时。反应液减压浓缩得中间体12-2(6.75g，crude)。Weigh the intermediate 12-1 (12 g, 52.34 mmol) in dichloromethane (120 ml), add trifluoroacetic acid (12 ml). Stir the reaction solution at 10°C for 12 hours. Concentrate the reaction solution under reduced pressure to obtain the intermediate 12-2 (6.75 g, crude).

LCMS(ESI)m/z[M+H]+＝130.2。LCMS (ESI) m/z [M+H]+ = 130.2.

第三步：中间体12-3的合成Step 3: Synthesis of intermediate 12-3

称取中间体12-2(6.75g,52.34mmol，1.0eq)和丙烯酸叔丁酯(6.7g,52.34mmol，1.0eq)溶于二甲基亚砜(100ml)中，加入N,N-二异丙基乙胺(20.29g,157.02mmol，3.0eq)。反应液在60℃下反应2小时。监测反应完成后，反应液用水(200mL)稀释。用甲基叔丁醚(100mL)萃取三次，合并有机相。有机相用无水硫酸钠干燥，过滤，减压浓缩得到粗品。该粗品通过柱层析(A:石油醚-B:乙酸乙酯,梯度B％:0％-60％)纯化得中间体12-3(6.5g,收率:48％)。Weigh the intermediate 12-2 (6.75g, 52.34mmol, 1.0eq) and tert-butyl acrylate (6.7g, 52.34mmol, 1.0eq) and dissolve them in dimethyl sulfoxide (100ml), and add N,N-diisopropylethylamine (20.29g, 157.02mmol, 3.0eq). The reaction solution was reacted at 60°C for 2 hours. After the reaction was monitored, the reaction solution was diluted with water (200mL). Extracted three times with methyl tert-butyl ether (100mL), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (A: petroleum ether-B: ethyl acetate, gradient B%: 0%-60%) to obtain intermediate 12-3 (6.5g, yield: 48%).

LCMS(ESI)m/z[M+H]⁺＝258.0。LCMS (ESI) m/z[M+H] ⁺ =258.0.

第四步：中间体12-4的合成Step 4: Synthesis of intermediate 12-4

称取中间体12-3(350mg,1.36mmol，1.0eq)和甲基-七聚乙二醇-乙酸(482mg,1.36mmol，1.0eq)溶于N，N-二甲基甲酰胺(3ml)，加入N,N-二异丙基乙胺(527mg,4.08mmol，3.0eq)和2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(568mg,1.496mmol，1.1eq)。反应液在10℃下反应12小时。监测反应完成后，反应液减压浓缩得到粗品。该粗品通过柱层析(A:二氯甲烷-B:甲醇,梯度B％:0％-30％)纯化得中间体12-4(700mg,收率:86％)。Weigh the intermediate 12-3 (350 mg, 1.36 mmol, 1.0 eq) and methyl-heptapeptide glycol-acetic acid (482 mg, 1.36 mmol, 1.0 eq) and dissolve them in N, N-dimethylformamide (3 ml), add N, N-diisopropylethylamine (527 mg, 4.08 mmol, 3.0 eq) and 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (568 mg, 1.496 mmol, 1.1 eq). The reaction solution was reacted at 10 ° C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (A: dichloromethane-B: methanol, gradient B%: 0%-30%) to obtain intermediate 12-4 (700 mg, yield: 86%).

LCMS(ESI)m/z[M+H]⁺＝594.4。LCMS (ESI) m/z[M+H] ⁺ =594.4.

第五步：中间体12-5的合成Step 5: Synthesis of intermediate 12-5

称取中间体12-4(670mg,1.13mmol)溶于二氯甲烷(3ml)中，加入三氟乙酸(1mL)。反应液在10℃下反应12小时。监测反应完成后，反应液减压浓缩得到中间体12-5(606mg,收率:100％)。Intermediate 12-4 (670 mg, 1.13 mmol) was weighed and dissolved in dichloromethane (3 ml), and trifluoroacetic acid (1 mL) was added. The reaction solution was reacted at 10° C. for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain intermediate 12-5 (606 mg, yield: 100%).

LCMS(ESI)m/z[M+H]⁺＝538.3。LCMS (ESI) m/z[M+H] ⁺ =538.3.

第六步：中间体12-6的合成Step 6: Synthesis of intermediate 12-6

称取中间体12-5(30mg,0.0558mmol，1.0eq)和Val-Cit-PAB-MMAE(60mg,0.0558mmol，1.0eq)溶于N，N-二甲基甲酰胺(2ml)中，加入N,N-二异丙基乙胺(21mg,0.167mmol，3.0eq)，1-羟基苯并三唑(15mg,0.0112mmol，2.0eq)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(21mg,0.067mmol，1.2eq)。反应液在10℃下搅拌12小时。监测反应完成后，反应液经过反相柱纯化(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)得到中间体12-6(80mg,收率:87％)。Intermediate 12-5 (30 mg, 0.0558 mmol, 1.0 eq) and Val-Cit-PAB-MMAE (60 mg, 0.0558 mmol, 1.0 eq) were weighed and dissolved in N,N-dimethylformamide (2 ml), and N,N-diisopropylethylamine (21 mg, 0.167 mmol, 3.0 eq), 1-hydroxybenzotriazole (15 mg, 0.0112 mmol, 2.0 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (21 mg, 0.067 mmol, 1.2 eq) were added. The reaction solution was stirred at 10°C for 12 hours. After the reaction was completed, the reaction solution was purified by reverse phase column (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 12-6 (80 mg, yield: 87%).

LCMS(ESI)[M+H]⁺＝1641.9。LCMS (ESI) [M+H] ⁺ = 1641.9.

第七步：中间体12-7的合成Step 7: Synthesis of intermediate 12-7

称取中间体12-6(70mg,0.0426mmol，1.0eq)置于四氢呋喃(3ml)中，加入N-甲基吗啉(21mg,0.213mmol，5.0eq)和四三苯基磷钯(10mg，0.0085mmol，0.2eq)。反应液在10℃下搅拌12小时。监测反应完成后，反应液减压浓缩得到粗品。该粗品通过反相柱层析(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)纯化得到中间体12-7(38mg,收率:51％)。Weigh the intermediate 12-6 (70 mg, 0.0426 mmol, 1.0 eq) in tetrahydrofuran (3 ml), add N-methylmorpholine (21 mg, 0.213 mmol, 5.0 eq) and tetrakis triphenylphosphine palladium (10 mg, 0.0085 mmol, 0.2 eq). The reaction solution was stirred at 10 ° C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 12-7 (38 mg, yield: 51%).

LCMS(ESI)[M+H]⁺＝1602.9；LCMS(ESI)m/z[M+2H]⁺/2＝802.2。LCMS(ESI)[M+H] ⁺ =1602.9; LCMS(ESI) m/z[M+2H] ⁺ /2=802.2.

第八步：中间体12-8的合成Step 8: Synthesis of Intermediate 12-8

称取中间体12-7(38mg,0.0237mmol，1.0eq)溶于N，N-二甲基甲酰胺(3ml)中，加入N,N-二异丙基乙胺(12mg，0.0948mmol，4.0eq)和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(21mg，0.0711mmol，3.0eq)。反应液在10℃下搅拌6小时。监测反应完成后，反应液减压浓缩得到粗品。该粗品通过反相柱(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)纯化分离得到中间体12-8(40mg,收率:95％)。Weigh the intermediate 12-7 (38 mg, 0.0237 mmol, 1.0 eq) and dissolve it in N, N-dimethylformamide (3 ml), add N, N-diisopropylethylamine (12 mg, 0.0948 mmol, 4.0 eq) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (21 mg, 0.0711 mmol, 3.0 eq). The reaction solution was stirred at 10 ° C for 6 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by a reverse phase column (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 12-8 (40 mg, yield: 95%).

LCMS(ESI)[M+H]⁺＝1699.9；LCMS(ESI)m/z[M+2H]⁺/2＝850.8。LCMS(ESI)[M+H] ⁺ =1699.9; LCMS(ESI) m/z[M+2H] ⁺ /2=850.8.

第九步：化合物12的合成Step 9: Synthesis of compound 12

将中间体12-8(16.9mg,0.0099mmol，1.5eq)和环肽中间体INT-D(20mg,0.0066mmol，1.0eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(4.2mg,0.033mmol)。反应液在25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)，得到化合物12(24.26mg,收率：80％)。Intermediate 12-8 (16.9 mg, 0.0099 mmol, 1.5 eq) and cyclopeptide intermediate INT-D (20 mg, 0.0066 mmol, 1.0 eq) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4.2 mg, 0.033 mmol) was added to the reaction system. The reaction solution was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150 mm, 5 um, mobile phase: [A: water (10 mM TFA)-B: ACN], gradient B%: 37%-50%, 13 min) to obtain compound 12 (24.26 mg, yield: 80%).

LCMS(ESI)m/z[M+4H]⁺/4＝1146.43；LCMS(ESI)m/z[M+3H]⁺/3＝1527.88。LCMS(ESI)m/z[M+4H] ⁺ /4=1146.43; LCMS(ESI)m/z[M+3H] ⁺ /3=1527.88.

实施例13：化合物13的合成
Example 13: Synthesis of Compound 13

化合物13通过以下路线合成：
Compound 13 was synthesized by the following route:

第一步：中间体13-1的合成Step 1: Synthesis of intermediate 13-1

往中间体12-3(500mg,1.94mmol，1.0eq)的二氯甲烷(5mL)溶液里加入丁二酸酐(214mg,2.14mmol，1.1eq)和N,N-二异丙基乙胺(753mg,5.83mmol，3.0eq)。反应液在常温下搅拌一小时。监测反应完成后，反应液减压浓缩得到中间体13-1(690mg，粗品)。Add succinic anhydride (214 mg, 2.14 mmol, 1.1 eq) and N,N-diisopropylethylamine (753 mg, 5.83 mmol, 3.0 eq) to a solution of intermediate 12-3 (500 mg, 1.94 mmol, 1.0 eq) in dichloromethane (5 mL). Stir the reaction solution at room temperature for one hour. After monitoring the reaction, the reaction solution was concentrated under reduced pressure to obtain intermediate 13-1 (690 mg, crude product).

LCMS(ESI)m/z[M-56+H]⁺＝302.2。LCMS (ESI) m/z[M-56+H] ⁺ =302.2.

第二步：中间体13-2的合成Step 2: Synthesis of intermediate 13-2

把粗品中间体13-1(690mg，1.94mmol，1.0eq)溶解到N，N-二甲基甲酰胺(5mL)里，往其中加入氨基六甘醇单甲醚(1.14g，3.88mmol，2.0eq)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(2.21g,5.82mmol，3.0eq)，N,N-二异丙基乙胺(750mg,5.82mmol，3.0eq)。反应液常温下搅拌2小时。监测反应结束后，反应液加水(200mL)稀释，乙酸乙酯(200mL×3)萃取，合并有机层。有机层无水硫酸钠干燥，过滤，减压浓缩得到残渣。将残渣溶于二氯甲烷(3mL)和三氟乙酸(3mL)的混合溶液里，常温下继续搅拌1小时。然后反应液减压浓缩，所得残渣用反相柱层析纯化(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)得到中间体13-2(180mg,收率：16％)。The crude intermediate 13-1 (690 mg, 1.94 mmol, 1.0 eq) was dissolved in N, N-dimethylformamide (5 mL), and aminohexaglycol monomethyl ether (1.14 g, 3.88 mmol, 2.0 eq), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (2.21 g, 5.82 mmol, 3.0 eq), and N, N-diisopropylethylamine (750 mg, 5.82 mmol, 3.0 eq) were added thereto. The reaction solution was stirred at room temperature for 2 hours. After the reaction was monitored, the reaction solution was diluted with water (200 mL), extracted with ethyl acetate (200 mL×3), and the organic layers were combined. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was dissolved in a mixed solution of dichloromethane (3 mL) and trifluoroacetic acid (3 mL), and stirred at room temperature for 1 hour. The reaction solution was then concentrated under reduced pressure, and the resulting residue was purified by reverse phase column chromatography (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 13-2 (180 mg, yield: 16%).

LCMS(ESI)m/z[M+H]⁺＝579.4。 LCMS (ESI) m/z[M+H] ⁺ =579.4.

第三步：中间体13-3的合成Step 3: Synthesis of intermediate 13-3

向中间体13-2(44mg,0.076mmol，1.0eq)的N，N-二甲基甲酰胺(3mL)溶液里加入Val-Cit-PAB-MMAE(85.5mg,0.076mmol，1.0eq)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(29mg,0.15mmol，2.0eq)、1-羟基苯并三唑(21mg,0.15mmol，2.0eq)和N,N-二异丙基乙胺(29mg,0.23mmol)。反应液在常温下搅拌2小时。反应液用乙酸乙酯稀释(50mL)，水(50ml)洗涤，有机层用无水硫酸钠干燥，过滤，减压浓缩得到中间体13-3(120mg，粗品)。To a solution of intermediate 13-2 (44 mg, 0.076 mmol, 1.0 eq) in N, N-dimethylformamide (3 mL) were added Val-Cit-PAB-MMAE (85.5 mg, 0.076 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (29 mg, 0.15 mmol, 2.0 eq), 1-hydroxybenzotriazole (21 mg, 0.15 mmol, 2.0 eq) and N, N-diisopropylethylamine (29 mg, 0.23 mmol). The reaction solution was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with water (50 ml), and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 13-3 (120 mg, crude product).

LCMS(ESI)m/z[M+H]⁺＝1684.1；LCMS(ESI)m/z[M+2H]⁺/2＝842.9。LCMS(ESI)m/z[M+H] ⁺ =1684.1; LCMS(ESI)m/z[M+2H] ⁺ /2=842.9.

第四步：中间体13-4的合成Step 4: Synthesis of intermediate 13-4

称取中间体13-3(120mg,0.073mmol，1.0eq)溶于四氢呋喃(3mL)中，加入N-甲基吗啉(21mg,0.213mmol，3.0eq)和四三苯基磷钯(10mg，0.0085mmol，0.11eq)。反应液在10℃下搅拌12小时。反应液经过反相柱层析纯化(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)得到中间体13-4(110mg,收率：90.4％)。Weigh the intermediate 13-3 (120 mg, 0.073 mmol, 1.0 eq) and dissolve it in tetrahydrofuran (3 mL). Add N-methylmorpholine (21 mg, 0.213 mmol, 3.0 eq) and tetrakistriphenylphosphine palladium (10 mg, 0.0085 mmol, 0.11 eq). The reaction solution was stirred at 10 ° C for 12 hours. The reaction solution was purified by reverse phase column chromatography (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 13-4 (110 mg, yield: 90.4%).

LCMS(ESI)[M+H]⁺＝1644.9；LCMS(ESI)m/z[M+2H]⁺/2＝822.9。LCMS(ESI)[M+H] ⁺ =1644.9; LCMS(ESI) m/z[M+2H] ⁺ /2=822.9.

第五步：中间体13-5的合成Step 5: Synthesis of intermediate 13-5

称取中间体13-4(38mg,0.0237mmol，1.0eq)溶于N，N-二甲基甲酰胺(3ml)，加入N,N-二异丙基乙胺(12mg，0.0948mmol，4.0eq)和2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯(21mg，0.0711mmol，3.0eq)。反应液在10℃下反应6小时。反应液减压浓缩得到粗品。该粗品通过反相柱层析(A:water(0.05％TFA)-B:ACN,梯度B％:0％-60％)纯化得到中间体13-5(40mg,收率:95％)Weigh the intermediate 13-4 (38 mg, 0.0237 mmol, 1.0 eq) and dissolve it in N, N-dimethylformamide (3 ml), add N, N-diisopropylethylamine (12 mg, 0.0948 mmol, 4.0 eq) and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (21 mg, 0.0711 mmol, 3.0 eq). The reaction solution was reacted at 10 ° C for 6 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography (A: water (0.05% TFA)-B: ACN, gradient B%: 0%-60%) to obtain intermediate 13-5 (40 mg, yield: 95%)

LCMS(ESI)[M+H]⁺＝1741.9；LCMS(ESI)m/z[M+2H]⁺/2＝871.3。LCMS(ESI)[M+H] ⁺ =1741.9; LCMS(ESI) m/z[M+2H] ⁺ /2=871.3.

第六步：化合物13的合成Step 6: Synthesis of compound 13

将中间体13-5(17.4mg,0.0099mmol，1.5eq)和环肽中间体INT-D(8.0mg,0.0066mmol，1.0eq)溶解于N，N-二甲基甲酰胺(2mL)中，向反应体系中加入N,N-二异丙基乙胺(4.3mg,0.033mmol)。反应液在25℃下搅拌3小时。反应液经过制备分离纯化(制备HPLC(色谱柱:Welch Xtimate，21.2*150mm,5um，流动相:[A：水(10mM TFA)-B：ACN],梯度B％:37％-50％,13min)，得到化合物13(24.78mg,收率80.1％)。Intermediate 13-5 (17.4 mg, 0.0099 mmol, 1.5 eq) and cyclopeptide intermediate INT-D (8.0 mg, 0.0066 mmol, 1.0 eq) were dissolved in N, N-dimethylformamide (2 mL), and N, N-diisopropylethylamine (4.3 mg, 0.033 mmol) was added to the reaction system. The reaction solution was stirred at 25 ° C for 3 hours. The reaction solution was purified by preparative separation (preparative HPLC (chromatographic column: Welch Xtimate, 21.2*150 mm, 5 um, mobile phase: [A: water (10 mM TFA)-B: ACN], gradient B%: 37%-50%, 13 min) to obtain compound 13 (24.78 mg, yield 80.1%).

LCMS(ESI)m/z[M+4H]⁺/4＝1157.11；LCMS(ESI)m/z[M+3H]⁺/3＝1541.95。LCMS(ESI)m/z[M+4H] ⁺ /4=1157.11; LCMS(ESI)m/z[M+3H] ⁺ /3=1541.95.

生物学测试评价Biological test evaluation

一、对人Nectin-4蛋白的亲和力测试1. Affinity test for human nectin-4 protein

1.实验材料1. Experimental Materials

Human Nectin-4,His-avi tag(厂家：Kactus，货号：NEC-HM404)Human Nectin-4, His-avi tag (Manufacturer: Kactus, Product No.: NEC-HM404)

1xAssay Buffer(1xPBS with 0.01％ TWEEN 20)1xAssay Buffer(1xPBS with 0.01% TWEEN 20)

探针(FITC标记的Nectin-4靶向环肽，即化合物1由内部合成) Probe (FITC-labeled Nectin-4 targeting cyclic peptide, i.e., compound 1 synthesized in-house)

384孔板(厂家：Corning，货号：4514)384-well plate (Manufacturer: Corning, Catalog No.: 4514)

2.实验方法2. Experimental Methods

首先，用1X Assay Buffer配制1.33X Human Nectin-4，浓度为13.3nM，按照layout，384孔板每孔加入15ul。随后，用1X Assay Buffer配制5X探针，浓度为5nM，按照layout，384孔板每孔加入4ul。接下来，用1X Assay Buffer配制20X本发明化合物，Top浓度为20uM，按照1:3梯度稀释10个浓度，按照layout，384孔板每孔加入1ul。使得Final DMSO浓度为0.1％。然后，300rpm，室温离心1min后，室温孵育2hrs后，Spectramax i3酶标仪读板，酶标仪参数设置为FP 485 520 optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm。First, 1.33X Human Nectin-4 was prepared with 1X Assay Buffer at a concentration of 13.3nM. According to the layout, 15ul was added to each well of a 384-well plate. Subsequently, 5X probe was prepared with 1X Assay Buffer at a concentration of 5nM. According to the layout, 4ul was added to each well of a 384-well plate. Next, 20X compound of the present invention was prepared with 1X Assay Buffer at a Top concentration of 20uM. Ten concentrations were diluted in a 1:3 gradient. According to the layout, 1ul was added to each well of a 384-well plate. The Final DMSO concentration was 0.1%. Then, after centrifugation at 300rpm at room temperature for 1min and incubation at room temperature for 2hrs, the plate was read with a Spectramax i3 microplate reader. The microplate reader parameters were set to FP 485 520 optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm.

3.数据分析3. Data Analysis

使用GraphPad Prism 9分析数据，绘制四参数拟合曲线计算IC₅₀，公式为：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC₅₀-X)*HillSlope))。再根据以下公式换算出Ki。GraphPad Prism 9 was used to analyze the data, and a four-parameter fitting curve was drawn to calculate IC ₅₀ , using the formula: Y=Bottom+(Top-Bottom)/(1+10^((LogIC ₅₀ -X)*HillSlope)). Ki was then calculated using the following formula.

[Bound]at equilibrium

[Bound] at equilibrium

为了将观察到的IC₅₀转换为Ki，求解该方程时添加抑制剂的条件：浓度I等于IC₅₀。结果见表3。To convert the observed _IC50 to Ki, the equation was solved for the condition where the inhibitor was added: concentration I equaled _IC50 . The results are shown in Table 3.

表3：本发明化合物对人Nectin-4蛋白的亲和力测试
Table 3: Affinity test of the compounds of the present invention for human Nectin-4 protein

从表3结果可以看出；本发明的化合物对人Nectin-4蛋白的亲和力与阳性化合物有显著提高。 From the results in Table 3, it can be seen that the affinity of the compounds of the present invention to human Nectin-4 protein is significantly improved compared with the positive compounds.

二、对小鼠Nectin-4蛋白的亲和力测试2. Affinity test for mouse nectin-4 protein

1.实验材料1. Experimental Materials

Mouse Nectin-4,His-avi tag(厂家：Kactus，货号：NEC-MM104)Mouse Nectin-4, His-avi tag (Manufacturer: Kactus, Product No.: NEC-MM104)

探针(FITC标记的Nectin-4靶向环肽，即化合物1，由内部合成)Probe (FITC-labeled nectin-4 targeting cyclic peptide, i.e., compound 1, synthesized in-house)

2.实验方法2. Experimental Methods

首先，用1X Assay Buffer配制1.33X Mouse Nectin-4，浓度为13.3nM，按照layout，384孔板每孔加入15ul。随后，用1X Assay Buffer配制5X探针，浓度为5nM，按照layout，384孔板每孔加入4ul。接下来，用1X Assay Buffer配制20X本发明化合物，Top浓度为20uM，按照1:3梯度稀释10个浓度，按照layout，384孔板每孔加入1ul。使得Final DMSO浓度为0.1％。然后，300rpm，室温离心1min后，室温孵育2hrs后，Spectramax i3酶标仪读板，酶标仪参数设置为FP 485 520 optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm。First, 1.33X Mouse Nectin-4 was prepared with 1X Assay Buffer at a concentration of 13.3nM. According to the layout, 15ul was added to each well of a 384-well plate. Subsequently, 5X probe was prepared with 1X Assay Buffer at a concentration of 5nM. According to the layout, 4ul was added to each well of a 384-well plate. Next, 20X compound of the present invention was prepared with 1X Assay Buffer at a Top concentration of 20uM. Ten concentrations were diluted in a 1:3 gradient. According to the layout, 1ul was added to each well of a 384-well plate. The Final DMSO concentration was 0.1%. Then, after centrifugation at 300rpm at room temperature for 1min and incubation at room temperature for 2hrs, the plate was read by a Spectramax i3 microplate reader. The microplate reader parameters were set to FP 485 520 optic module which excites at 485nm and detects parallel and perpendicular emission at 520nm.

3.数据分析3. Data Analysis

[Bound]at equilibrium

[Bound] at equilibrium

为了将观察到的IC₅₀转换为Ki，求解该方程时添加抑制剂的条件：浓度I等于IC₅₀。结果见表4。To convert the observed _IC50 to Ki, the equation was solved for the condition where the inhibitor was added: concentration I equaled _IC50 . The results are shown in Table 4.

表4：本发明化合物对小鼠Nectin-4蛋白的亲和力测试
Table 4: Affinity test of the compounds of the present invention for mouse Nectin-4 protein

从表4结果可以看出；本发明的化合物对小鼠Nectin-4蛋白的亲和力优于阳性化合物。From the results in Table 4, it can be seen that the affinity of the compounds of the present invention to mouse Nectin-4 protein is superior to that of the positive compounds.

三、对MDA-MB-468细胞的亲和力测试3. Affinity test for MDA-MB-468 cells

1.实验材料1. Experimental Materials

Mouse Anti-MMAE Antibody(厂家：Acrobiosystem，货号：MME-M5252)Mouse Anti-MMAE Antibody (Manufacturer: Acrobiosystem, Product No.: MME-M5252)

PBA(1xPBS with 1％ BSA)PBA (1xPBS with 1％ BSA)

anti-mouse IgG 488(厂家：碧云天，货号：A0428)anti-mouse IgG 488 (Manufacturer: Biyuntian, Product No.: A0428)

2.实验方法2. Experimental Methods

首先，用PBS稀释本发明化合物，将化合物配制成Top为100nM，两倍梯度稀释，DMSO终浓度为0.5％的工作液。用胰酶消化液消化MDA-MB-468细胞，计数，深孔板每孔加入～1M细胞，2500rpm，1min，弃上清。然后，每孔对应加入200uL不同浓度的本发明化合物工作液，4℃孵育1h后加入200uL/孔PBS，2500rpm，1min，洗2遍后弃上清。接下来，用PBA配制anti-MMAE 1:100，每孔加入50ul，重悬细胞，4℃避光孵育1h。随后加入200uL/孔PBS，2500rpm，1min，洗2遍后弃上清。然后用PBA配制anti-mouse IgG 488 1:500，每孔加入50ul，重悬细胞，4℃避光孵育1h。随后加入200uL/孔PBS，2500rpm，1min，First, the compound of the present invention was diluted with PBS, and the compound was prepared into a working solution with a Top of 100nM, two-fold gradient dilution, and a final DMSO concentration of 0.5%. MDA-MB-468 cells were digested with trypsin digestion solution, counted, and ~1M cells were added to each well of the deep-well plate, 2500rpm, 1min, and the supernatant was discarded. Then, 200uL of the working solution of the compound of the present invention of different concentrations was added to each well, incubated at 4°C for 1h, and then 200uL/well PBS was added, 2500rpm, 1min, and the supernatant was discarded after washing twice. Next, anti-MMAE 1:100 was prepared with PBA, 50ul was added to each well, the cells were resuspended, and incubated at 4°C in the dark for 1h. Subsequently, 200uL/well PBS was added, 2500rpm, 1min, and the supernatant was discarded after washing twice. Then anti-mouse IgG 488 1:500 was prepared with PBA, 50ul was added to each well, the cells were resuspended, and incubated at 4°C in the dark for 1h. Then add 200uL/well PBS, 2500rpm, 1min,

洗2遍后弃上清。每孔加入200ul PBS重悬细胞，FACS上机。After washing twice, discard the supernatant. Add 200ul PBS to each well to resuspend the cells and load on FACS machine.

3.数据分析3. Data Analysis

使用Flow Jo和GraphPad Prism 9分析数据，绘制四参数拟合曲线计算EC₅₀，公式为：Y＝Bottom+(Top-Bottom)/(1+10^((LogEC₅₀-X)*HillSlope))。绘制One site-Total曲线计算Kd，公式为：Y＝Bmax*X/(Kd+X)+NS*X+Background。结果见表5。Data were analyzed using Flow Jo and GraphPad Prism 9, and a four-parameter fitting curve was plotted to calculate EC ₅₀ , with the formula: Y=Bottom+(Top-Bottom)/(1+10^((LogEC ₅₀ -X)*HillSlope)). A One site-Total curve was plotted to calculate Kd, with the formula: Y=Bmax*X/(Kd+X)+NS*X+Background. The results are shown in Table 5.

表5：本发明化合物对MDA-MB-468细胞的亲和力测试
Table 5: Affinity test of the compounds of the present invention for MDA-MB-468 cells

从表5结果可以看出，本发明的化合物对人乳腺癌细胞系MDA-MB-468细胞结合能力较强，优于阳性化合物BT8009。 From the results in Table 5, it can be seen that the compounds of the present invention have a stronger ability to bind to the human breast cancer cell line MDA-MB-468 cells, which is better than the positive compound BT8009.

四、细胞增殖抑制实验IV. Cell Proliferation Inhibition Experiment

1.实验材料1. Experimental Materials

细胞系信息如下表：
The cell line information is as follows:

384孔板(厂家：Corning，货号：3765)384-well plate (Manufacturer: Corning, Catalog No.: 3765)

CCK8试剂(厂家：Invigentech，货号：IV08-2000T)CCK8 reagent (Manufacturer: Invigentech, Catalog No.: IV08-2000T)

MMAE(厂家：MCE，货号：HY-15162)MMAE (Manufacturer: MCE, Part No.: HY-15162)

2.实验方法2. Experimental Methods

第一天，细胞消化后计数，每种细胞用相应培液重悬：T47D：0.025M/mL，NCI-H460：0.025M/mL，LnCap：0.075M/mL，MDA-MB-468：0.05M/mL。按照layout每孔加入20uL细胞悬液，Min孔加入各细胞相应培液的空白培液，不加细胞的空白孔加入25uL PBS，300rpm，室温离心1min后，37度培养过夜。On the first day, cells were digested and counted, and each cell type was resuspended in the corresponding culture medium: T47D: 0.025M/mL, NCI-H460: 0.025M/mL, LnCap: 0.075M/mL, MDA-MB-468: 0.05M/mL. According to the layout, 20uL of cell suspension was added to each well, and the blank culture medium of the corresponding culture medium of each cell was added to the Min well, and 25uL PBS was added to the blank well without cells. After centrifugation at 300rpm at room temperature for 1min, the cells were cultured overnight at 37 degrees.

第二天，化合物粉末用DMSO配置成4mM的储液，用1640空白培液配置成5X工作液，Top浓度为100uM，1:4梯度稀释8个浓度，按照layout，每孔加入5ul工作液，Min和Max孔则加入5ul 2.5％ DMSO的1640空白培液，使得所有孔DMSO终浓度为0.5％。300rpm，室温离心1min后，37度继续培养3天或6天。在第五天和第八天分别处理NCI-H460/LnCap/MDA-MB-468细胞组和T47D细胞组，每孔加入2.5uL CCK8试剂，300rpm离心1min，37度孵育一定时间后，酶标仪OD450读数。On the second day, the compound powder was prepared into a 4mM stock solution with DMSO, and a 5X working solution was prepared with 1640 blank culture solution. The Top concentration was 100uM, and 8 concentrations were diluted in a 1:4 gradient. According to the layout, 5ul of working solution was added to each well, and 5ul of 2.5% DMSO 1640 blank culture solution was added to the Min and Max wells, so that the final DMSO concentration of all wells was 0.5%. After centrifugation at 300rpm for 1min at room temperature, the cells were cultured at 37 degrees for 3 or 6 days. On the fifth and eighth days, the NCI-H460/LnCap/MDA-MB-468 cell group and the T47D cell group were treated respectively, 2.5uL of CCK8 reagent was added to each well, centrifuged at 300rpm for 1min, and incubated at 37 degrees for a certain period of time, and the OD450 reading was taken by the microplate reader.

3.数据分析3. Data Analysis

最后，根据Min和Max读值计算inhibition％，公式为100*(ODMax孔-OD测试孔)/(ODMax孔-ODMin孔)。再使用GraphPad Prism 9分析数据，绘制四参数拟合曲线计算IC₅₀，公式为：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC₅₀-X)*HillSlope))。结果见表6。Finally, inhibition% was calculated based on the Min and Max readings, using the formula 100*(ODMax well-OD test well)/(ODMax well-ODMin well). GraphPad Prism 9 was used to analyze the data, and a four-parameter fitting curve was drawn to calculate _IC50 , using the formula: Y=Bottom+(Top-Bottom)/(1+10^(( _LogIC50 -X)*HillSlope)). The results are shown in Table 6.

表6：本发明化合物对细胞增殖的抑制效应(IC₅₀,nM)

Table 6: Inhibitory effect of the compounds of the present invention on cell proliferation (IC ₅₀ , nM)

从表6数据可以看出，本发明化合物对不同肿瘤细胞的杀伤效果存在较好的靶点依赖性和选择性。It can be seen from the data in Table 6 that the killing effect of the compounds of the present invention on different tumor cells has good target dependence and selectivity.

五、本发明化合物不同种属血浆稳定性分析V. Analysis of Plasma Stability of the Compounds of the Invention in Different Species

1.实验材料1. Experimental Materials

SD大鼠、CD-1小鼠、食蟹猴血浆采集于天津有济医药科技发展有限公司；Plasma from SD rats, CD-1 mice, and cynomolgus monkeys was collected from Tianjin Youji Pharmaceutical Technology Development Co., Ltd.;

人空白血浆购买于山东致臻医药服务有限公司Human blank plasma was purchased from Shandong Zhizhen Pharmaceutical Service Co., Ltd.

2.实验方法2. Experimental Methods

用DMSO溶解化合物粉末，将其配制成2mM储液，再用DMSO将化合物储液进一步稀释成100uM的工作溶液备用。将198μL的SD大鼠、CD-1小鼠和人空白血浆加入到对应的孵育板中，一共7块孵育板，包括T0、T10、T30、T60、T120、T240和空白孵育板，每个样品准备三个平行孔，37度平衡10min。然后在已经加入血浆的孵育板中加入2.00μL的待测化合物工作溶液。所有的样品在37℃水浴锅中进行孵育。每一个孵育时间点结束时，取出相应的孵育板，加入终止液，沉淀蛋白，离心20分钟，取上清液用LC-MS/MS的方法分析。样品中待测化合物的浓度采用液相色谱-串联质谱(LC-MS/MS)方法进行半定量测定。结果见表7。Dissolve the compound powder with DMSO and prepare it into a 2mM stock solution, and then further dilute the compound stock solution with DMSO to a 100uM working solution for standby use. 198μL of SD rat, CD-1 mouse and human blank plasma was added to the corresponding incubation plates, a total of 7 incubation plates, including T0, T10, T30, T60, T120, T240 and blank incubation plates, and three parallel wells were prepared for each sample, and 37 degrees were balanced for 10min. Then 2.00μL of the working solution of the compound to be tested was added to the incubation plate to which plasma had been added. All samples were incubated in a 37℃ water bath. At the end of each incubation time point, the corresponding incubation plate was removed, the stop solution was added, the protein was precipitated, centrifuged for 20 minutes, and the supernatant was analyzed by LC-MS/MS. The concentration of the compound to be tested in the sample was semi-quantitatively determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results are shown in Table 7.

表7：本发明化合物分别在SD大鼠、CD-1小鼠、食蟹猴和人血浆中的稳定性结果
Table 7: Stability results of the compounds of the present invention in SD rats, CD-1 mice, cynomolgus monkeys and human plasma

从表7数据可以看出，本发明的化合物在SD大鼠、CD-1小鼠和人血浆中具有优异的稳定性。It can be seen from the data in Table 7 that the compounds of the present invention have excellent stability in SD rats, CD-1 mice and human plasma.

六、本发明化合物在小鼠体内的药代动力学分析VI. Pharmacokinetic Analysis of the Compounds of the Invention in Mice

1.实验材料1. Experimental Materials

雄性CD-1小鼠，30g左右，6～9周龄，3只/化合物，购于维通利华。Male CD-1 mice, about 30 g, 6-9 weeks old, 3 mice/compound, were purchased from Vitallife.

2.实验方法2. Experimental Methods

以标准方案测试化合物静脉注射后小鼠药代特征，待测化合物配成澄清溶液，溶媒：称取1.00mg为试验化合物的粉末，首先加入0.0500mL DMSO，搅拌至视觉均匀。随后，加入4.95mL 25.0mM Histidine，10％ Sucrose pH＝7.0的水溶液，制得浓度为0.200mg/mL 的药物溶液。给予3只小鼠单次静脉注射1mg/kg待测化合物。按照给药后0.083,0.25,0.5,1,2,4,8,24小时为时间点，收集全血，制备得到血浆，以LC-MS/MS方法分析本发明化合物和其潜在代谢产物MMAE的浓度。分析检测前，所有样品存于-80℃。结果见表8。The pharmacokinetic characteristics of the test compound after intravenous injection in mice were tested using a standard protocol. The test compound was prepared into a clear solution. Solvent: 1.00 mg of the test compound powder was weighed, and 0.0500 mL of DMSO was first added and stirred until visually uniform. Subsequently, 4.95 mL of 25.0 mM Histidine, 10% Sucrose pH = 7.0 aqueous solution was added to obtain a concentration of 0.200 mg/mL. The drug solution was prepared. Three mice were given a single intravenous injection of 1 mg/kg of the test compound. Whole blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration to prepare plasma, and the concentration of the compound of the present invention and its potential metabolite MMAE was analyzed by LC-MS/MS. All samples were stored at -80°C before analysis and detection. The results are shown in Table 8.

表8：本发明化合物在小鼠体内的药代动力学
Table 8: Pharmacokinetics of the compounds of the present invention in mice

从表8数据可以看出，本发明的化合物在血液中清除速率低，暴露量高，代谢产物MMAE的暴露量仅为阳性化合物的8％，有望具有更好的药效和安全性。It can be seen from the data in Table 8 that the compounds of the present invention have a low clearance rate in the blood and a high exposure amount, and the exposure amount of the metabolite MMAE is only 8% of that of the positive compound, and are expected to have better efficacy and safety.

七、本发明化合物在大鼠血浆药代动力学分析VII. Pharmacokinetic Analysis of the Compounds of the Invention in Rat Plasma

1.实验材料1. Experimental Materials

雄性SD大鼠，200～250g左右，6～9周龄，3只/化合物，购于维通利华。Male SD rats, about 200-250 g, 6-9 weeks old, 3 rats/compound, were purchased from Vital River.

动物给药溶液配制:称取化合物粉末3.00mg，首先加入0.100mL DMSO，搅拌至视觉均匀。随后，加入1.90mL 25.0mM组氨酸和10％蔗糖水溶液(pH＝7.0)，制得浓度为1.50mg/mL的动物给药澄清溶液。Preparation of animal dosing solution: Weigh 3.00 mg of compound powder, first add 0.100 mL DMSO, and stir until visually uniform. Then, add 1.90 mL 25.0 mM histidine and 10% sucrose aqueous solution (pH = 7.0) to prepare a clear animal dosing solution with a concentration of 1.50 mg/mL.

2.实验方法2. Experimental Methods

以标准方案测试化合物静脉注射后的药代特征。首先，采用随机选取动物、单剂量(3.00mg/kg)以及单次静脉注射的给药方式，将1.50mg/mL的化合物澄清溶液进行动物给药；其次，按照给药后0.083、0.25、0.5、1、2、4、8和24小时为时间点，通过颈静脉采血而得到全血样品；然后，将一定体积的全血样品中加入到一定体积的终止液中，沉淀蛋白，混匀并离心20分钟，取上清液并用稀释液进行稀释后用用液相色谱-串联质谱(LC-MS/MS)方法分析本发明化合物和其潜在代谢产物MMAE的浓度；最后，采用Phoenix WinNonlin软件，非房室模型统计矩法计算药代动力学(PK)参数。分析检测前，所有样品均保存于-60～-90℃冰箱中。结果见表9。The pharmacokinetic characteristics of the compound after intravenous injection were tested by standard protocol. First, animals were randomly selected, single dose (3.00 mg/kg) and single intravenous injection were used to administer 1.50 mg/mL of the compound clear solution to animals; secondly, whole blood samples were obtained by blood sampling from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration; then, a certain volume of whole blood sample was added to a certain volume of stop solution, protein was precipitated, mixed and centrifuged for 20 minutes, the supernatant was taken and diluted with diluent, and the concentration of the compound of the present invention and its potential metabolite MMAE was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS); finally, Phoenix WinNonlin software was used to calculate the pharmacokinetic (PK) parameters by non-compartmental statistical moment method. Before analysis and detection, all samples were stored in a refrigerator at -60 to -90 °C. The results are shown in Table 9.

表9：本发明化合物在大鼠体内的药代动力学
Table 9: Pharmacokinetics of the compounds of the present invention in rats

NC：无法计算NC: Cannot calculate

从表9数据可以看出，本发明的化合物在血液中清除速率低，暴露量高，代谢产物MMAE的暴露量保持在非常低的水平，有望具有更好的药效和安全性。It can be seen from the data in Table 9 that the compounds of the present invention have a low clearance rate in the blood, a high exposure amount, and the exposure amount of the metabolite MMAE is kept at a very low level, and are expected to have better efficacy and safety.

八、本发明化合物在不同种属的肝细胞稳定性分析8. Analysis of the stability of the compounds of the present invention in different species of hepatocytes

1.实验材料1. Experimental Materials

SD大鼠、CD-1小鼠肝细胞购买于XenoTech；SD rat and CD-1 mouse hepatocytes were purchased from XenoTech;

食蟹猴肝细胞购买于瑞德肝脏疾病研究(上海)有限公司；Cynomolgus monkey hepatocytes were purchased from Reed Liver Disease Research (Shanghai) Co., Ltd.;

人肝细胞购买于上海权阳生物科技有限公司。Human hepatocytes were purchased from Shanghai Quanyang Biotechnology Co., Ltd.

2.实验方法2. Experimental Methods

首先，用DMSO溶解化合物粉末，将其配制成2.00mM储备液，再用20％乙腈/水将化合物储备液进一步稀释成25.0μM的工作溶液备用；其次，用已预热的WEM将复苏的肝细胞稀释成每毫升1.04×10⁶细胞悬浮液；然后，分别将48.0μL的SD大鼠、CD-1小鼠、食蟹猴和人的细胞悬浮液加入到对应的孵育板中(包括T0、T15、T30、T60、T90和T120)和48.0μL的WEM加入到对应的孵育板中(包括T0-MC和T120-MC)，每个样品准备2个平行孔；最后，在已经加入细胞悬浮液或WEM的孵育板中对应的加入2.00μL的化合物工作液，并将相应的孵育板置于37℃二氧化碳培养箱中进行孵育，化合物的最终孵育浓度为1.00μM。在每一个孵育时间点结束时，取出相应的孵育板，加入终止液，沉淀蛋白，混匀并离心20分钟，取上清液并用稀释液进行稀释后用液相色谱-串联质谱(LC-MS/MS)方法分析。样品中待测化合物的浓度采用LC-MS/MS法进行半定量测定。结果见表10。First, the compound powder was dissolved in DMSO to prepare a 2.00 mM stock solution, and then the compound stock solution was further diluted with 20% acetonitrile/water to a 25.0 μM working solution for use; secondly, the revived hepatocytes were diluted with preheated WEM to a 1.04×10 ⁶ cell suspension per ml; then, 48.0 μL of SD rat, CD-1 mouse, crab-eating monkey and human cell suspension were added to the corresponding incubation plates (including T0, T15, T30, T60, T90 and T120) and 48.0 μL of WEM were added to the corresponding incubation plates (including T0-MC and T120-MC), and 2 parallel wells were prepared for each sample; finally, 2.00 μL of the compound working solution was added to the incubation plate to which the cell suspension or WEM had been added, and the corresponding incubation plate was placed in a 37°C carbon dioxide incubator for incubation, and the final incubation concentration of the compound was 1.00 μM. At the end of each incubation time point, the corresponding incubation plate was removed, the stop solution was added, the protein was precipitated, the mixture was mixed and centrifuged for 20 minutes, and the supernatant was taken and diluted with the diluent and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentration of the test compound in the sample was semi-quantitatively determined by LC-MS/MS. The results are shown in Table 10.

表10：本发明化合物在不同种属的肝细胞稳定性
Table 10: Stability of the compounds of the present invention in different species of hepatocytes

从表10数据可以看出，本发明的化合物在SD大鼠、CD-1小鼠、食蟹猴和人肝细胞中具有优异的稳定性。 It can be seen from the data in Table 10 that the compounds of the present invention have excellent stability in SD rats, CD-1 mice, cynomolgus monkeys and human hepatocytes.

Claims

A conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound comprises a payload, a linker and a targeting molecule, wherein the targeting molecule comprises a peptide ligand and a molecular scaffold, the peptide ligand comprises three amino acid residues separated by two ring sequences, and the molecular scaffold is connected to the three amino acid residues through a covalent bond.

The conjugate compound according to claim 1, characterized in that the three amino acid residues are independently selected from Cys, hCys, βCys, Pen, Dap and N-methyl-Dap residues, and one or more natural amino acids in the peptide ligand are chemically modified, and the chemical modification is one or any combination of methoxylation, fluorination, thiolation, phosphorylation, acylation, hydroxylation and carboxylation.

The conjugate compound according to claim 1, characterized in that the two ring sequences contained in the peptide ligand are P(1Nal)(D-Asp) and M(HArg)DWSTP(HyP)W, respectively, and one or more amino acids in the ring sequence are chemically modified, and the chemical modification is one or any combination of methoxylation, fluorination, thiolation, phosphorylation, acylation, hydroxylation, and carboxylation.

The conjugate compound according to claim 1, characterized in that the amino acid residue is selected from Cys and Pen residues, and the peptide ligand is selected from SEQ ID NO: 1 or SEQ ID NO: 2:
-Cys-X(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(HyP)W-Cys-(SEQ ID NO:1);
-Cys-X(1Nal)(D-Asp)-Pen-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:2);

Wherein, X is selected from chemically modified proline, and the chemical modification is one or any combination of methoxylation, fluorination, thiolation, phosphorylation, acylation, hydroxylation, and carboxylation.

The coupled compound according to any one of claims 1 to 4, characterized in that the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one.

The conjugate compound according to any one of claims 1 to 5, characterized in that the targeting molecule is selected from the following structures:

The conjugate compound according to any one of claims 1 to 6, characterized in that the linker is selected from -PABC-Cit-Val-linker-β-Ala-Sar10-.

The conjugate compound according to any one of claims 1 to 7, characterized in that the linker is selected from a peptide linker, a pH-dependent linker, a disulfide linker or a combination of the above linkers.

The conjugate compound according to any one of claims 1 to 8, characterized in that the linker is selected from:

The coupled compound according to any one of claims 1 to 8, characterized in that the linker comprises the following structure:

wherein _R1 and _R2 are independently selected from hydrogen and _C1-6 alkyl.

wherein R ₁ and R ₂ are independently selected from hydrogen and C _1-6 alkyl;

n is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8.

The conjugate compound according to claim 1, characterized in that the three amino acid residues are independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap residues; one or more natural amino acids in the peptide ligand are chemically modified, and the chemical modification comprises one or any of methoxylation, fluorination, thiolation, phosphorylation, acylation, hydroxylation, and carboxylation; and the linker comprises the following structure:

wherein _R1 and _R2 are independently selected from hydrogen and _C1-6 alkyl.

The conjugate compound according to claim 14, characterized in that the conjugate compound targets Nectin-4, Kallikrein, MT1-MMP, CD137, Epha2, IL-17, PSMA, PD-L1, αvβ3, CD38, CAIX, OX40, PBP, TSLP, ACE2, TfR1, FAPα, NK cells or TREM2.

The conjugate compound according to claim 1, characterized in that the targeting molecule is connected to the payload via a linker, and the linker and the targeting molecule have the following structure:

The conjugate compound according to any one of claims 1 to 16, characterized in that the payload is selected from cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, ifosfamide, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, irinotecan, topotecan, paclitaxel, acridine, etoposide, etoposide phosphate, teniposide, doxorubicin, epirubicin, camptothecin and its derivatives, epothilone and its derivatives, bleomycin and its derivatives, plicamycin and its derivatives, dactinomycin and its derivatives, maytansine and its derivatives, auristatin and its derivatives. .

The conjugate compound according to any one of claims 1 to 17, characterized in that the payload is selected from MMAE.

The coupled compound according to any one of claims 1 to 18, characterized in that it is selected from the following group:

A pharmaceutical composition comprising the conjugate compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Use of the conjugate compound according to any one of claims 1 to 19, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating and/or preventing a disease with overexpression of nectin-4.

Use of the coupled compound according to any one of claims 1 to 19, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for preventing and/or treating tumors.

Use of the coupled compound according to any one of claims 1 to 19, its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for preventing and/or treating blood cancer, cervical cancer, lung cancer, prostate cancer, mesothelioma, thyroid cancer, kidney cancer, bile duct cancer, bladder cancer, breast cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, sarcoma, skin cancer, ovarian cancer, liver cancer, colorectal cancer or pancreatic cancer.