WO2025153001A1

Movatterモバイル変換

Info

Publication number: WO2025153001A1
Application number: PCT/CN2025/072661
Authority: WO
Inventors: 王红军; 王彦明; 郭婷婷; 赵焰平; 李维; 张敏; 何景昌; 张琪; 冯泽旺; 张磊
Original assignee: Beijing Tide Pharmaceutical Co Ltd
Current assignee: Beijing Tide Pharmaceutical Co Ltd
Priority date: 2024-01-17
Filing date: 2025-01-16
Publication date: 2025-07-24
Anticipated expiration: 2026-07-17

Abstract

The present invention relates to a new thiol-reactive coupling moiety as shown in formula (I), a linker containing the coupling moiety, a linker-payload conjugate, an antibody-drug conjugate based on the linker and the use thereof, and further relates to a pharmaceutical composition containing the antibody-drug conjugate, and the use of the antibody-drug conjugate for treating and/or preventing a disease.

Description

Translated fromChinese

含有噻唑[5,4-B]吡啶结构的抗体偶联药物及其用途Antibody-conjugated drug containing thiazole [5,4-B] pyridine structure and its use

发明领域Field of the Invention

本发明属于药物化学领域，具体涉及一种新的巯基反应性接头、含有该接头的连接子、连接子-有效荷载(payload)偶联物、基于该连接子的抗体偶联药物及其用途，还涉及包含抗体偶联药物的药物组合物，以及这些抗体偶联药物用于治疗和/或预防疾病的用途。The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a new thiol-reactive linker, a linker containing the linker, a linker-payload conjugate, an antibody-drug conjugate based on the linker and uses thereof, and also relates to a pharmaceutical composition containing the antibody-drug conjugate, and uses of these antibody-drug conjugates for treating and/or preventing diseases.

背景技术Background Art

抗体偶联药物(antibody-drug conjugate，ADC)，利用具有生物靶向性的单克隆抗体(antibody)将强效的细胞毒素(Cytotoxin)靶向输送至病灶部位，其联合了抗体及细胞毒药物两者的优势，具有靶向性强、活性高、毒副作用低、半衰期长等诸多优点。截止目前，全球已有15款ADC药物上市，同时约有200款ADC处于临床在研阶段。Antibody-drug conjugate (ADC) uses biologically targeted monoclonal antibodies (antibodies) to deliver potent cytotoxins to the lesion site. It combines the advantages of both antibodies and cytotoxic drugs, and has many advantages such as strong targeting, high activity, low toxicity and side effects, and long half-life. So far, 15 ADC drugs have been launched in the world, and about 200 ADCs are in clinical research.

ADC在结构上包括抗体、小分子细胞毒素和连接子(linker)三个组成部分，抗体的作用是实现靶向，细胞毒素的作用是发挥疗效杀死靶细胞，而连接子的作用是实现抗体和细胞毒素在结构上的有机结合，从而使之形成一个整体。连接子的性能是确保细胞毒素靶向输送的关键因素，直接决定了ADC的药效、安全性和药代动力学特征，连接子的构建对ADC药物具有重要的意义。ADC consists of three components in structure: antibody, small molecule cytotoxin and linker. The role of antibody is to achieve targeting, the role of cytotoxin is to exert therapeutic effect and kill target cells, and the role of linker is to achieve organic combination of antibody and cytotoxin in structure, so as to form a whole. The performance of linker is the key factor to ensure the targeted delivery of cytotoxin, which directly determines the efficacy, safety and pharmacokinetic characteristics of ADC. The construction of linker is of great significance to ADC drugs.

关于ADC连接子的开发，存在着多种重要的考量因素，其中包括抗体的偶联位点选择、每分子抗体平均偶联细胞毒素分子的数量(drug/antibody ratio，DAR)、连接子的可裂解性、连接子的亲水性等。ADC对连接子的基本要求包括：必须确保ADC给药后的整个治疗周期内在人体循环系统中的稳定性，以及到达靶组织之后可以快速有效地释放出有效荷载，如此ADC方能有效地发挥出抗体靶向性和毒素高效力这两种优势。There are many important considerations for the development of ADC linkers, including the selection of antibody conjugation sites, the average number of cytotoxic molecules conjugated per antibody molecule (drug/antibody ratio, DAR), linker cleavability, linker hydrophilicity, etc. The basic requirements of ADC for linkers include: ensuring the stability of ADC in the human circulatory system throughout the treatment cycle after administration, and quickly and effectively releasing the effective load after reaching the target tissue, so that ADC can effectively play the two advantages of antibody targeting and toxin high efficacy.

另外，连接子的设计还需要匹配所连接的荷载的理化性质，起到调节连接子-有效荷载亲水性的作用。连接子进行合适的亲水性设计，可实现提升偶联过程中的连接子-有效荷载的水溶性、提高偶联收率、降低产品聚合度、提高产品稳定性、改善产品生物利用度和药代动力学等多重作用。In addition, the design of the linker also needs to match the physical and chemical properties of the connected payload to play a role in regulating the hydrophilicity of the linker-payload. Appropriate hydrophilicity design of the linker can achieve multiple effects such as improving the water solubility of the linker-payload during the coupling process, increasing the coupling yield, reducing the degree of polymerization of the product, improving product stability, and improving product bioavailability and pharmacokinetics.

因此，在ADC药物的设计及研发过程中，连接子以及偶联策略的选择极为关键。ADC的连接子必须确保在到达靶组织之前抗体和细胞毒素之间的有效连接，如果细胞毒素在ADC尚未到达靶组织之前提前脱靶，一方面由于荷载率的下降会大大降低ADC向靶组织释放的有效荷载浓度，进而降低ADC的药效，另一方面脱落的强效细胞毒素也会对正常组织造成显著脱靶毒性。Therefore, in the design and development of ADC drugs, the selection of linkers and coupling strategies is extremely critical. The linker of ADC must ensure the effective connection between the antibody and the cytotoxin before reaching the target tissue. If the cytotoxin is off-target before the ADC reaches the target tissue, on the one hand, the decrease in the loading rate will greatly reduce the effective load concentration released by the ADC to the target tissue, thereby reducing the efficacy of the ADC. On the other hand, the detached potent cytotoxin will also cause significant off-target toxicity to normal tissues.

目前，在已上市及临床在研的ADC药物中，绝大多数产品在构建的过程中依旧采用了巯基(thiol)与马来酰亚胺(maleimide)发生迈克尔加成反应(Michael addition reaction)生成硫代琥珀酰亚胺(thiosuccinimide)的过程(图1)。由于该反应具有快速、定量且条件温和等诸多优势，其应用在目前的ADC研究中仍未找到更优的替代。At present, among the ADC drugs that have been marketed or are under clinical development, the vast majority of products still use the Michael addition reaction between thiol and maleimide to generate thiosuccinimide (Figure 1). Due to the many advantages of this reaction, such as rapidity, quantitativeness and mild conditions, its application in current ADC research has not yet found a better alternative.

然而，通过上述反应所构建的ADC产品中生成的硫代琥珀酰亚胺基团是不稳定的，ADC给药后其将在体内即不断发生反迈克尔反应(retro-Michael reaction)从而将导致高效力的细胞毒素源源不断从ADC上发生脱靶并释放于正常组织中(图1)。脱靶生成的马来酰亚胺基团会进一步与血液中的白蛋白、谷胱甘肽、半胱氨酸等含有巯基的物质快速发生反应，进一步加速细胞毒素从ADC上的脱靶过程。根据报道，在体外小鼠血浆中，ADC在3天的时间里细胞毒素的脱靶量就可高达50％，而在小鼠体内模型上ADC给药一周后细胞毒素的脱靶量也高达60％(Nature Biotech.,2014,32(10):1059-1062.)。上述ADC体内代谢将无疑会导致ADC药效的降低和毒性的升高(Nature Biotech.,2012,30(2):184-189.；Bioconjugate Chem.,2008,19(3):759-765.；Bioconjug Chem.,2016,27(7):1588-1598.；J Med Chem.,2014,57(19):7890-7899.)。However, the thiosuccinimide groups generated in the ADC products constructed by the above reaction are unstable. After the ADC is administered, it will continuously undergo a retro-Michael reaction in the body, which will cause the highly effective cytotoxins to continuously go off-target from the ADC and be released into normal tissues (Figure 1). The maleimide groups generated by the off-target will further react rapidly with albumin, glutathione, cysteine and other sulfhydryl-containing substances in the blood, further accelerating the off-target process of the cytotoxins from the ADC. According to reports, in in vitro mouse plasma, the off-target amount of cytotoxins of ADC can be as high as 50% in 3 days, and the off-target amount of cytotoxins in the mouse in vivo model after one week of ADC administration is as high as 60% (Nature Biotech., 2014, 32(10): 1059-1062.). The above-mentioned ADC metabolism in vivo will undoubtedly lead to reduced efficacy and increased toxicity of ADC (Nature Biotech., 2012, 30(2): 184-189.; Bioconjugate Chem., 2008, 19(3): 759-765.; Bioconjug Chem., 2016, 27(7): 1588-1598.; J Med Chem., 2014, 57(19): 7890-7899.).

因此，当前主流的ADC连接子技术尚不能很好地确保ADC在到达靶组织之前的充分稳定性，新一代连接子的构建已经成为ADC发展中亟需解决的重要问题和重大挑战。除稳定性之外，ADC在连接子释药性能优化、亲水性优化以及连接子-有效荷载合理搭配组合等诸多方面也仍然存在着广阔的优化空间。Therefore, the current mainstream ADC linker technology cannot well ensure the full stability of ADC before reaching the target tissue, and the construction of a new generation of linkers has become an important issue and major challenge that needs to be urgently addressed in the development of ADC. In addition to stability, ADC still has a wide range of optimization space in many aspects such as linker drug release performance optimization, hydrophilicity optimization, and reasonable combination of linker-payload.

发明概述SUMMARY OF THE INVENTION

本发明具体涉及新的基于2-(甲基磺酰基)噻唑[5,4-b]吡啶结构(英文名：2-(methylsulfonyl)thiazolo[5,4-b]pyridine)的巯基反应性接头、以及包含该接头的连接子、连接子-有效荷载偶联物、基于该类连接子的抗体偶联药物及连接子的用途，还涉及包含抗体偶联药物的药物组合物，以及这些抗体偶联药物用于治疗和/或预防疾病的用途。The present invention specifically relates to a novel thiol-reactive linker based on a 2-(methylsulfonyl)thiazolo[5,4-b]pyridine structure (English name: 2-(methylsulfonyl)thiazolo[5,4-b]pyridine), a linker comprising the linker, a linker-payload conjugate, an antibody-drug conjugate based on the linker, and uses of the linker, as well as a pharmaceutical composition comprising the antibody-drug conjugate, and uses of the antibody-drug conjugate for treating and/or preventing diseases.

在一方面，本申请提供了一种抗体偶联药物、其中间体、制备方法及应用。本申请的抗体偶联药物，可以实现细胞毒性药物在ADC领域的广泛应用，主要用于治疗肿瘤疾病。本申请的主要技术效果在于：提供的新型巯基反应性接头的连接子，普遍适用于包含有巯基的大分子和小分子的偶联反应，也适用于搭配偶联各种毒素，所得到的偶联物产物具有显著提高的稳定性。例如，本申请中的新型接头应用于ADC领域时，可以通过降低有效荷载的脱靶，一方面可以提高ADC的安全性，另一方面可以提高ADC的药效。On the one hand, the present application provides an antibody-drug conjugate, its intermediate, preparation method and application. The antibody-drug conjugate of the present application can realize the wide application of cytotoxic drugs in the field of ADC, mainly for the treatment of tumor diseases. The main technical effect of the present application is that the connector of the provided novel thiol-reactive linker is generally applicable to the coupling reaction of macromolecules and small molecules containing thiol groups, and is also applicable to the coupling of various toxins, and the resulting conjugate product has significantly improved stability. For example, when the novel linker in the present application is applied to the field of ADC, it can improve the safety of ADC on the one hand by reducing the off-target of the effective load, and on the other hand, it can improve the efficacy of ADC.

2-(甲基磺酰基)噻唑[5,4-b]吡啶模型分子与巯基模型分子的反应如下：
The reaction of the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine model molecule with the thiol model molecule is as follows:

2-(甲基磺酰基)噻唑[5,4-b]吡啶接头(母核)的典型制备方法如下：The typical preparation method of 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker (mother core) is as follows:

制备方法1.
Preparation method 1.

制备方法2.
Preparation method 2.

制备方法3.
Preparation method 3.

制备方法4.
Preparation method 4.

制备方法5.
Preparation method 5.

制备方法6.
Preparation method 6.

基于2-(甲基磺酰基)噻唑[5,4-b]吡啶结构的接头的优势包括：Advantages of linkers based on 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine structures include:

1.与巯基反应后产物较传统接头产物具有更高的稳定性；1. The product after reaction with thiol has higher stability than traditional linker products;

2.较传统接头具有更高的亲水性；2. Higher hydrophilicity than traditional connectors;

3.与巯基反应具有良好的反应动力学；3. It has good reaction kinetics with thiol groups;

4.温和条件下与巯基的反应具有高度的选择性；4. The reaction with thiol groups under mild conditions is highly selective;

5.普遍适用于包含巯基的各类生物大分子及小分子；5. Widely applicable to various biological macromolecules and small molecules containing thiol groups;

6.接头的原料易得，制备工艺简单，反应收率高。6. The raw materials of the joint are easily available, the preparation process is simple, and the reaction yield is high.

因此，在一个方面，本发明提供了式(I)化合物，或其药学上可接受的盐或同位素变体：
Therefore, in one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or isotopic variant thereof:

其中，in,

R₁选自C_1-6烷基或C_1-6卤代烷基；R₁ is selected from C_1-6 alkyl or C_1-6 haloalkyl;

W₁选自-O-、-S-、-NR_b-、-C(O)O-、-C(O)NR_b-、-O-C(O)-、-NR_b-C(O)-、-S(O)_pO-或-O-S(O)_p-；W₁ is selected from -O-, -S-, -NR_b -, -C(O)O-, -C(O)NR_b -, -OC(O)-, -NR_b -C(O)-, -S(O)_p O- or -OS(O)_p -;

其中p＝1或2；Where p = 1 or 2;

L₁为化学键或-(CH₂)_m1-(OCH₂CH₂)_n1-(CH₂CH₂O)_n2-(CH₂)_r1-(L)_q-(CH₂)_r2-(OCH₂CH₂)_n3-(CH₂CH₂O)_n4-(CH₂)_m2-；L₁ is a chemical bond or -(CH₂ )_m1 -(OCH₂ CH₂ )_n1 -(CH₂ CH₂ O)_n2 -(CH₂ )_r1 -(L)_q -(CH₂ )_r2 -(OCH₂ CH₂ )_n3 -(CH₂ CH₂ O)_n4 -(CH₂ )_m2 -;

其中-L-选自-O-、-NR_b-、-C(O)NR_b-、-C(O)O-、-NR_b-C(O)-、-O-C(O)-、-C_3-8亚环烷基-、-3-8元亚杂环基-、-C_6-10亚芳基-或-5-10元亚杂芳基；wherein -L- is selected from -O-, -NR_b -, -C(O)NR_b -, -C(O)O-, -NR_b -C(O)-, -OC(O)-, -C_3-8 cycloalkylene-, -3-8 membered heterocyclylene-, -C_6-10 arylene-, or -5-10 membered heteroarylene;

各个m1、m2、n1、n2、n3、n4、r1和r2独立地选自0、1、2、3、4、5、6、7、8、9或10；Each of m1, m2, n1, n2, n3, n4, r1 and r2 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

q＝0、1或2；q = 0, 1 or 2;

W₂选自化学键、-O-、-S-、-NR_b-或-C(O)-；W₂ is selected from a chemical bond, -O-, -S-, -NR_b - or -C(O)-;

R₂选自H、D、卤素、-OR_a、-NR_bR_c或以下基团：
R₂ is selected from H, D, halogen, -OR_a , -NR_b R_c or the following groups:

R₃选自H、D、卤素、C_1-6烷基或C_1-6卤代烷基；_R3 is selected from H, D, halogen,_C1-6 alkyl or_C1-6 haloalkyl;

s＝0、1或2；s = 0, 1, or 2;

其中R_a、R_b和R_c独立地选自H、C_1-6烷基、C_1-6卤代烷基、C_2-6烯基、C_2-6炔基、C_3-10环烷基、3-10元杂环基、C_6-10芳基或5-10元杂芳基；或者R_b和R_c以及他们连接的N原子一起形成3-10元杂环基；wherein_Ra ,_Rb and_Rc are independently selected from H,_C1-6 alkyl,_C1-6 haloalkyl,_C2-6 alkenyl,_C2-6 alkynyl, C3-10 cycloalkyl,_3-10 membered heterocyclyl,_C6-10 aryl or 5-10 membered heteroaryl; or_Rb and_Rc and the N atom to which they are attached together form a 3-10 membered heterocyclyl;

其中上述基团任选地被一个或多个氘取代，直至完全氘代。The above groups are optionally substituted by one or more deuterium groups until they are fully deuterated.

在另一个方面，本发明提供了上述式(I)化合物或其药学上可接受的盐或同位素变体在制备抗体偶联药物中的用途。In another aspect, the present invention provides use of the compound of formula (I) or a pharmaceutically acceptable salt or isotopic variant thereof in the preparation of an antibody-drug conjugate.

在另一个方面，本发明提供了式(II)化合物，或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体：
In another aspect, the present invention provides a compound of formula (II), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof:

其中，in,

s＝0、1或2；s = 0, 1, or 2;

其中p＝1或2；Where p = 1 or 2;

L₁为化学键或-(CH₂)_m-(OCH₂CH₂)_n-(CH₂CH₂O)_n-(CH₂)_r-(L)_q-(CH₂)_r-(OCH₂CH₂)_n-(CH₂CH₂O)_n-(CH₂)_m-；L₁ is a chemical bond or -(CH₂ )_m -(OCH₂ CH₂ )_n -(CH₂ CH₂ O)_n -(CH₂ )_r -(L)_q -(CH₂ )_r -(OCH₂ CH₂ )_n -(CH₂ CH₂ O)_n -(CH₂ )_m -;

各个m、n、r独立地选自0、1、2、3、4、5、6、7、8、9或10；Each m, n, r is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

q＝0、1或2；q = 0, 1 or 2;

L₂选自以下氨基酸残基或2-10个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代；L₂ is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 10 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R₄ ;

其中氨基酸残基或寡肽残基的N末端与W₂相连，C末端与L₃相连；wherein the N-terminus of the amino acid residue or oligopeptide residue is connected to W₂ , and the C-terminus is connected to L₃ ;

R₄选自D、卤素、NO₂、-OR_a、-NR_bR_c、C_1-6烷基、C_1-6卤代烷基、多聚乙二醇、多聚肌氨酸、五碳糖、六碳糖、磺酸基、甲磺酰基、磷酸基、亚磷酸基、季铵盐，或选自以下基团：R₄ is selected from D, halogen, NO₂ , -OR_a , -NR_b R_c , C_1-6 alkyl, C_1-6 haloalkyl, polyethylene glycol, polysarcosine, pentose, hexose, sulfonic acid, methylsulfonyl, phosphate, phosphite, quaternary ammonium salt, or selected from the following groups:

L₃选自以下结构：
_L3 is selected from the following structures:

其中NH与L₂相连，C(O)与D相连；Among them, NH is connected to L₂ , and C(O) is connected to D;

R₅选自H、D、卤素、NO₂、-OR_a、-NR_bR_c、C_1-6烷基或C_1-6卤代烷基；R₅ is selected from H, D, halogen, NO₂ , -OR_a , -NR_b R_c , C_1-6 alkyl or C_1-6 haloalkyl;

t＝0、1、2、3或4；t = 0, 1, 2, 3 or 4;

R₆选自H、_R6 is selected from H,

D为活性化合物，选自药物、细胞毒素、检测试剂、诊断试剂或靶向载体；D is an active compound selected from drugs, cytotoxins, detection reagents, diagnostic reagents or targeting vectors;

在另一个方面，本发明提供了式(III)化合物，或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，其具有以下通式：
In another aspect, the present invention provides a compound of formula (III), or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, which has the following general formula:

其中，in,

A为靶向性分子；A is a targeting molecule;

x＝1、2、3、4、5、6、7或8；x = 1, 2, 3, 4, 5, 6, 7 or 8;

s＝0、1或2；s = 0, 1, or 2;

其中p＝1或2；Where p = 1 or 2;

q＝0、1或2；q = 0, 1 or 2;

L₃选自以下结构：
_L3 is selected from the following structures:

t＝0、1、2、3或4；t = 0, 1, 2, 3 or 4;

R₆选自H、_R6 is selected from H,

在另一个方面，本发明提供了一种药物组合物，其含有本发明所述化合物或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，以及药学上可接受的赋形剂或辅料。In another aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, and a pharmaceutically acceptable excipient or adjuvant.

在另一个方面，本发明提供了本发明所述化合物或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体在制备用于治疗疾病或病症、以及减轻所述疾病或病症的严重性的药物中的用途。In another aspect, the present invention provides the use of the compound of the present invention or its pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer in the preparation of a medicament for treating a disease or condition, and alleviating the severity of the disease or condition.

在另一个方面，本发明提供了本发明所述化合物或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或本发明所述药物组合物，其用于治疗疾病或病症、以及减轻所述疾病或病症的严重性。In another aspect, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or a pharmaceutical composition of the present invention for use in treating a disease or condition, and alleviating the severity of the disease or condition.

在另一个方面，本发明提供了一种在受试者中治疗疾病或病症、以及减轻所述疾病或病症的严重性的方法，包括向所述受试者给药本发明所述化合物或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或本发明所述药物组合物。In another aspect, the present invention provides a method for treating a disease or condition in a subject, and reducing the severity of the disease or condition, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesomorph, racemate or tautomer thereof, or a pharmaceutical composition of the present invention.

在另一方面，本申请提供了一种抗体药物偶联物的制备方法，通过将所述抗体或抗体片断铰链区的二硫链还原生成一对半胱氨酸残基，并通过将所述半胱氨酸残基中巯基与本发明化合物的连接子发生取代反应，进而将本发明化合物连接于所述抗体或抗体片段的半胱氨酸巯基上，获得抗体药物偶联物，药物抗体偶联率DAR(药物抗体偶联比率)可根据反应条件进行控制，例如常见为2～8之间。On the other hand, the present application provides a method for preparing an antibody-drug conjugate, wherein the disulfide chain in the hinge region of the antibody or antibody fragment is reduced to generate a pair of cysteine residues, and the thiol group in the cysteine residue is substituted with the linker of the compound of the present invention, and then the compound of the present invention is connected to the cysteine thiol group of the antibody or antibody fragment to obtain an antibody-drug conjugate. The drug-antibody coupling ratio DAR (drug-antibody coupling ratio) can be controlled according to the reaction conditions, for example, it is generally between 2 and 8.

在另一方面，DAR(即单个单克隆抗体分子与细胞毒性药物偶联后得到的抗体偶联药物中的药物分子与单克隆抗体分子的摩尔比的平均值)一般可以采用疏水层析(Hydrophobic-Interaction Chromatography，HIC)、反相高效液相色谱(Reverse phase HPLC,RP-HPLC)、聚丙烯酰胺-SDS凝胶电泳(SDS PAGE，electrophoresis)、液相质谱(liquid chromatograph-mass spectrometer,LC-MS)、紫外/可见光谱法(UV/Vis)等方式测定得到。On the other hand, DAR (i.e., the average molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after a single monoclonal antibody molecule is conjugated to a cytotoxic drug) can generally be measured by hydrophobic-interaction chromatography (HIC), reverse phase HPLC (RP-HPLC), polyacrylamide-SDS gel electrophoresis (SDS PAGE, electrophoresis), liquid chromatograph-mass spectrometer (LC-MS), ultraviolet/visible spectroscopy (UV/Vis), etc.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1：基于马来酰亚胺接头的ADC构建和产物的毒素脱靶反应图。Figure 1: Diagram of maleimide linker-based ADC construction and toxin off-target reactions of the product.

图2：本发明ADC在人血浆中的稳定性研究。Figure 2: Stability study of ADC of the present invention in human plasma.

图3：本发明ADC在小鼠血浆中的稳定性研究。FIG3 : Stability study of ADC of the present invention in mouse plasma.

图4：MMAE、Dxd、化合物-41和星形孢菌素(Staurosporine)的体外细胞毒性研究。FIG. 4 : In vitro cytotoxicity study of MMAE, Dxd, Compound-41 and Staurosporine.

图5：ADC-14B的体内药效研究。Figure 5: In vivo efficacy study of ADC-14B.

图6：ADC-14B与DS7300-43和IgG-14B的药效对比研究。Figure 6: Comparative study of the efficacy of ADC-14B, DS7300-43 and IgG-14B.

图7：HDC-14E(DAR4)的体内药效研究。Figure 7: In vivo efficacy study of HDC-14E (DAR4).

发明详述DETAILED DESCRIPTION OF THE INVENTION

定义definition

化学定义Chemical Definition

下面更详细地描述具体官能团和化学术语的定义。Definitions of specific functional groups and chemical terms are described in more detail below.

当列出数值范围时，既定包括每个值和在所述范围内的子范围。例如“C_1-6烷基”包括C₁、C₂、C₃、C₄、C₅、C₆、C_1-6、C_1-5、C_1-4、C_1-3、C_1-2、C_2-6、C_2-5、C_2-4、C_2-3、C_3-6、C_3-5、C_3-4、C_4-6、C_4-5和C_5-6烷基。When a numerical range is listed, each value and subrange within the stated range is intended to be included. For example, "C_1-6 alkyl" includes C₁ , C₂ , C₃ , C₄ , C₅ , C₆ , C_1-6 , C_1-5 , C_1-4 , C_1-3 , C_1-2 , C_2-6 , C_2-5 , C_2-4 , C_{2-3, C 3-6}_, C_3-5 , C_3-4 , C_4-6 , C_4-5 , and C_5-6 alkyl.

“C_1-6烷基”是指具有1至6个碳原子的直链或支链饱和烃基团。在一些实施方案中，C_1-4烷基是优选的。在一些实施方案中，C_1-2烷基是优选的。C_1-6烷基的例子包括：甲基(C₁)、乙基(C₂)、正丙基(C₃)、异丙基(C₃)、正丁基(C₄)、叔丁基(C₄)、仲丁基(C₄)、异丁基(C₄)、正戊基(C₅)、3-戊基(C₅)、戊基(C₅)、新戊基(C₅)、3-甲基-2-丁基(C₅)、叔戊基(C₅)和正己基(C₆)。术语“C_1-6烷基”还包括杂烷基，其中一或多个(例如，1、2、3或4个)碳原子被杂原子(例如，氧(O)、硫(S)、氮(N)、硼(B)、硅(Si)、磷(P))替代。烷基基团可以被一或多个取代基任选取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。常规烷基缩写包括：Me(-CH₃)、Et(-CH₂CH₃)、iPr(-CH(CH₃)₂)、nPr(-CH₂CH₂CH₃)、n-Bu(-CH₂CH₂CH₂CH₃)或i-Bu(-CH₂CH(CH₃)₂)。"C_1-6 alkyl" refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C_1-4 alkyl is preferred. In some embodiments, C_1-2 alkyl is preferred. Examples of C_1-6 alkyl include: methyl (C₁ ), ethyl (C₂ ), n-propyl (C₃ ), isopropyl (C₃ ), n-butyl (C₄ ), tert-butyl (C₄ ), sec-butyl (C₄ ), isobutyl (C₄ ), n-pentyl (C₅ ), 3-pentyl (C₅ ), pentyl (C₅ ), neopentyl (C₅ ), 3-methyl-2-butyl (C₅ ), tert-pentyl (C₅ ) and n-hexyl (C₆ ). The term "C_1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen (O), sulfur (S), nitrogen (N), boron (B), silicon (Si), phosphorus (P)). The alkyl group may be optionally substituted by one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH₃ ), Et(-CH₂ CH₃ ), iPr(-CH(CH₃ )₂ ), nPr(-CH₂ CH₂ CH₃ ), n-Bu(-CH₂ CH₂ CH₂ CH₃ ) or i-Bu(-CH₂ CH(CH₃ )₂ ).

“卤代”或“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。"Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

因此，“C_1-6卤代烷基”是指上述“C_1-6烷基”，其被一个或多个卤素基团取代。在一些实施方案中，C_1-3卤代烷基是特别优选的，更优选C_1-2卤代烷基。示例性的所述卤代烷基包括但不限于：-CF₃、-CH₂F、-CHF₂、-CHFCH₂F、-CH₂CHF₂、-CF₂CF₃、-CCl₃、-CH₂Cl、-CHCl₂、2,2,2-三氟-1,1-二甲基-乙基，等等。卤代烷基基团可以在任何可用的连接点上被取代，例如，被1至5个取代基、1至3个取代基或1个取代基取代。Therefore, "C_1-6 haloalkyl" refers to the above-mentioned "C_1-6 alkyl" substituted by one or more halogen groups. In some embodiments, C_1-3 haloalkyl is particularly preferred, more preferably C_1-2 haloalkyl. Exemplary haloalkyls include, but are not limited to: -CF₃ , -CH₂ F, -CHF₂ , -CHFCH₂ F, -CH₂ CHF₂ , -CF₂ CF₃ , -CCl₃ , -CH₂ Cl, -CHCl₂ , 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group may be substituted at any available attachment point, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

“C_2-6烯基”是指具有2至6个碳原子和一个或多个碳-碳双键(例如，1、2或3个碳-碳双键)的直链或支链烃基团。一个或多个碳-碳双键可以在内部(例如，在2-丁烯基中)或端部(例如，在1-丁烯基中)。在一些实施方案中，C_2-4烯基是特别优选的。所述烯基的实例包括但不限于：乙烯基(C₂)、1-丙烯基(C₃)、2-丙烯基(C₃)、1-丁烯基(C₄)、2-丁烯基(C₄)、丁二烯基(C₄)、戊烯基(C₅)、戊二烯基(C₅)、己烯基(C₆)，等等。不论烯基前是否修饰有“取代的”，烯基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"_C2-6 alkenyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2 or 3 carbon-carbon double bonds). One or more carbon-carbon double bonds may be internal (e.g., in 2-butenyl) or end (e.g., in 1-butenyl). In some embodiments,_C2-4 alkenyl is particularly preferred. Examples of the alkenyl include, but are not limited to, vinyl (_C2 ), 1-propenyl (_C3 ), 2-propenyl (_C3 ), 1-butenyl (_C4 ), 2-butenyl (_C4 ), butadienyl (_C4 ), pentenyl (_C5 ), pentadienyl (_C5 ), hexenyl (_C6 ), and the like. Regardless of whether the alkenyl is modified with "substituted", each of the alkenyl groups is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

“C_2-6炔基”是指具有2至6个碳原子、一个或多个碳-碳叁键(例如，1、2或3个碳-碳叁键)以及任选一个或多个碳-碳双键(例如，1、2或3个碳-碳双键)的直链或支链烃基团。在一些实施方案中，C_2-4炔基是特别优选的。在一些实施方案中，炔基不含有任何双键。一个或多个碳叁键可以在内部(例如，在2-丁炔基中)或端部(例如，在1-丁炔基中)。所述炔基的实例包括但不限于：乙炔基(C₂)、1-丙炔基(C₃)、2-丙炔基(C₃)、1-丁炔基(C₄)、2-丁炔基(C₄)、戊炔基(C₅)、己炔基(C₆)，等等。不论炔基前是否修饰有“取代的”，炔基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"C_2-6 alkynyl" refers to a straight or branched hydrocarbon group having 2 to 6 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). In some embodiments, C_2-4 alkynyl is particularly preferred. In some embodiments, the alkynyl does not contain any double bonds. The one or more carbon triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of the alkynyl include, but are not limited to, ethynyl (C₂ ), 1-propynyl (C₃ ), 2-propynyl (C₃ ), 1-butynyl (C₄ ), 2-butynyl (C₄ ), pentynyl (C₅ ), hexynyl (C₆ ), and the like. Regardless of whether the alkynyl group is preceded by "substituted", each of the alkynyl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

“C_3-10环烷基”是指具有3至10个环碳原子和零个杂原子的非芳香环烃基团。在一些实施方案中，C_3-8环烷基、C_4-6环烷基是优选的，C_3-6环烷基是特别优选的，更优选C_5-6环烷基。环烷基还包括其中上述环烷基环与一个或多个芳基或杂芳基稠合的环体系，其中连接点在环烷基环上，且在这样的情况中，碳的数目继续表示环烷基体系中的碳的数目。示例性的所述环烷基包括但不限于：环丙基(C₃)、环丙烯基(C₃)、环丁基(C₄)、环丁烯基(C₄)、环戊基(C₅)、环戊烯基(C₅)、环己基(C₆)、环己烯基(C₆)、环已二烯基(C₆)、环庚基(C₇)、环庚烯基(C₇)、环庚二烯基(C₇)、环庚三烯基(C₇)、环辛基(C₈)、环辛烯基(C₈)、二环[2.2.1]庚基(C₇)、二环[2.2.2]辛基(C₈)、环壬基(C₉)、环壬烯基(C₉)、环癸基(C₁₀)、环癸烯基(C₁₀)、八氢-1H-茚基(C₉)、十氢萘基(C₁₀)、螺[4.5]癸基(C₁₀)、冰片基、金刚烷基等等。不论环烷基前是否修饰有“取代的”，环烷基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"_C3-10 cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments,_C3-8 cycloalkyl,_C4-6 cycloalkyl are preferred,_C3-6 cycloalkyl is particularly preferred, and_C5-6 cycloalkyl is more preferred. Cycloalkyl also includes a ring system in which the above cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (_C3 ), cyclopropenyl (C3), cyclobutyl (_C4 ), cyclobutenyl (_C4 ), cyclopentyl (_C5 ), cyclopentenyl (_C5 ), cyclohexyl (_C6 ), cyclohexenyl (C6), cyclohexadienyl (_C6 ), cycloheptyl (_C7 ), cycloheptenyl (_C7 ), cycloheptadienyl (_C7 ), cycloheptatrienyl (_C7 ), cyclooctyl (_C8 ), cyclooctenyl (_C8 ), bicyclo[2.2.1]heptyl (_C7 ), bicyclo[_2.2.2 ]octyl (_C8 ), cyclononyl (C9), cyclononenyl (_C9 ), cyclodecyl (_C10 ), cyclodecenyl (_C10 ), octahydro-1H-indenyl (_C9₎ , and the_like . ), decahydronaphthyl (C₁₀ ), spiro[4.5]decyl (C₁₀ ), bornyl, adamantyl, etc. Regardless of whether the cycloalkyl group is preceded by "substituted", each of the cycloalkyl group is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined as follows.

“C_3-8亚环烷基”是指去掉“C_3-8环烷基”的另一个氢原子形成的二价基团。The "C_3-8 cycloalkylene group" means a divalent group formed by removing another hydrogen atom of the "C_3-8 cycloalkyl group".

“3-10元杂环基”是指具有环碳原子和1至4个环杂原子的3至10元非芳香环系的基团，其中，每个杂原子独立地选自氮、氧、硫、硼、磷和硅。在包含一个或多个氮原子的杂环基中，只要化合价允许，连接点可为碳或氮原子。在一些实施方案中，4-10元杂环基是优选的，其为具有环碳原子和1至4个环杂原子的4至10元非芳香环系；在一些实施方案中，5-10元杂环基是优选的，其为具有环碳原子和1至4个环杂原子的5至10元非芳香环系；在一些实施方案中，3-8元杂环基是优选的，其为具有环碳原子和1至3个环杂原子的3至8元非芳香环系；在一些实施方案中，3-6元杂环基是特别优选的，其为具有环碳原子和1至3个环杂原子的3至6元非芳香环系；更优选5-6元杂环基，其为具有环碳原子和1至3个环杂原子的5至6元非芳香环系。杂环基还包括其中上述杂环基环与一个或多个环烷基、芳基或杂芳基稠合的环体系，其中连接点在杂环基环上；且在这样的情况下，环成员的数目继续表示在杂环基环体系中环成员的数目。不论杂环基前是否修饰有“取代的”，杂环基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. In some embodiments, 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 5-10 membered heterocyclyl is preferred, which is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; in some embodiments, 3-8 membered heterocyclyl is preferred, which is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, 3-6 membered heterocyclyl is particularly preferred, which is a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably, 5-6 membered heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above heterocyclyl ring is fused to one or more cycloalkyl, aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Regardless of whether the heterocyclyl is preceded by "substituted", each of the heterocyclyl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

“3-8元亚杂环基”是指去掉“3-8元杂环基”的另一个氢原子形成的二价基团。The "3- to 8-membered heterocyclic group" means a divalent group formed by removing another hydrogen atom of the "3- to 8-membered heterocyclic group".

示例性的包含一个杂原子的3元杂环基包括但不限于：氮杂环丙烷基、氧杂环丙烷基、硫杂环丙烷基(thiorenyl)。示例性的含有一个杂原子的4元杂环基包括但不限于：氮杂环丁烷基、氧杂环丁烷基和硫杂环丁烷基。示例性的含有一个杂原子的5元杂环基包括但不限于：四氢呋喃基、二氢呋喃基、四氢噻吩基、二氢噻吩基、吡咯烷基、二氢吡咯基和吡咯基-2,5-二酮。示例性的包含两个杂原子的5元杂环基包括但不限于：二氧杂环戊烷基、氧硫杂环戊烷基(oxasulfuranyl)、二硫杂环戊烷基(disulfuranyl)和唑烷-2-酮。示例性的包含三个杂原子的5元杂环基包括但不限于：三唑啉基、二唑啉基和噻二唑啉基。示例性的包含一个杂原子的6元杂环基包括但不限于：哌啶基、四氢吡喃基、二氢吡啶基和硫杂环己烷基(thianyl)。示例性的包含两个杂原子的6元杂环基包括但不限于：哌嗪基、吗啉基、二硫杂环己烷基、二烷基。示例性的包含三个杂原子的6元杂环基包括但不限于：六氢三嗪基(triazinanyl)。示例性的含有一个杂原子的7元杂环基包括但不限于：氮杂环庚烷基、氧杂环庚烷基和硫杂环庚烷基。示例性的包含一个杂原子的8元杂环基包括但不限于：氮杂环辛烷基、氧杂环辛烷基和硫杂环辛烷基。示例性的与C₆芳基环稠合的5元杂环基(在本文中也称作5,6-双环杂环基)包括但不限于：二氢吲哚基、异二氢吲哚基、二氢苯并呋喃基、二氢苯并噻吩基、苯并唑啉酮基，等等。示例性的与C₆芳基环稠合的6元杂环基(本文还指的是6,6-双环杂环基)包括但不限于：四氢喹啉基、四氢异喹啉基，等等。Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, Examples of 6-membered heterocyclic groups that include one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Examples of 6-membered heterocyclic groups that include two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithian ... Alkyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepane and thiepanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxepane and thiecanyl. Exemplary 5-membered heterocyclic groups fused to a C₆ aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to, dihydroindole, isoindole, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzo Exemplary 6-membered heterocyclyl groups fused to a C₆ aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

“C_6-10芳基”是指具有6-10个环碳原子和零个杂原子的单环或多环的(例如，双环或三环)4n+2芳族环体系(例如，具有以环状排列共享的6或10个π电子)的基团。在一些实施方案中，芳基具有六个环碳原子(“C₆芳基”；例如，苯基)。在一些实施方案中，芳基具有十个环碳原子(“C₁₀芳基”；例如，萘基，例如，1-萘基和2-萘基)。在一些实施方案中，C_6-10芳基是特别优选的，更优选C₆芳基。芳基还包括其中上述芳基环与一个或多个环烷基或杂环基稠合的环系统，而且连接点在所述芳基环上，在这种情况下，碳原子的数目继续表示所述芳基环系统中的碳原子数目。不论芳基前是否修饰有“取代的”，芳基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"C_6-10 aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C₆ aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C₁₀ aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, C_6-10 aryl is particularly preferred, more preferably C₆ aryl. Aryl also includes a ring system in which the above-mentioned aryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Regardless of whether the aryl group is preceded by "substituted", each of the aryl groups is independently optionally substituted, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, and suitable substituents are defined below.

“C_6-10亚芳基”是指去掉“C_6-10芳基”的另一个氢原子形成的二价基团。The "C_6-10 arylene group" means a divalent group formed by removing another hydrogen atom of the "C_6-10 aryl group".

“5-10元杂芳基”是指具有环碳原子和1-4个环杂原子的5-10元单环或双环的4n+2芳族环体系(例如，具有以环状排列共享的6或10个π电子)的基团，其中每个杂原子独立地选自氮、氧和硫。在含有一个或多个氮原子的杂芳基中，只要化合价允许，连接点可以是碳或氮原子。杂芳基双环系统在一个或两个环中可以包括一个或多个杂原子。杂芳基还包括其中上述杂芳基环与一个或多个环烷基或杂环基稠合的环系统，而且连接点在所述杂芳基环上，在这种情况下，碳原子的数目继续表示所述杂芳基环系统中的碳原子数目。在一些实施方案中，5-6元杂芳基是特别优选的，其为具有环碳原子和1-4个环杂原子的5-6元单环或双环的4n+2芳族环体系。在一些实施方案中，5元杂芳基是特别优选的，其为具有环碳原子和1-4个环杂原子的5元单环或双环的4n+2芳族环体系。不论杂芳基前是否修饰有“取代的”，杂芳基的每个独立地任选被取代，例如，1至5个取代基、1至3个取代基或1个取代基，适当的取代基如下定义。"5-10 membered heteroaryl" refers to a group of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as the valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes a ring system in which the above-mentioned heteroaryl ring is fused to one or more cycloalkyl or heterocyclic groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. In some embodiments, 5-membered heteroaryl is particularly preferred, which is a 5-membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Regardless of whether the heteroaryl is preceded by "substituted", each of the heteroaryl groups is independently optionally substituted, for example, 1 to 5 substituents, 1 to 3 substituents or 1 substituent, and suitable substituents are defined as follows.

“5-10元亚杂芳基”是指去掉“5-10元杂芳基”的另一个氢原子形成的二价基团。The “5-10 membered heteroarylene group” means a divalent group formed by removing another hydrogen atom of the “5-10 membered heteroaryl group”.

示例性的含有一个杂原子的5元杂芳基包括但不限于：吡咯基、呋喃基和噻吩基。示例性的含有两个杂原子的5元杂芳基包括但不限于：咪唑基、吡唑基、噁唑基、异噁唑基、噻唑基和异噻唑基。示例性的含有三个杂原子的5元杂芳基包括但不限于：三唑基、噁二唑基和噻二唑基。示例性的含有四个杂原子的5元杂芳基包括但不限于：四唑基。示例性的含有一个杂原子的6元杂芳基包括但不限于：吡啶基。示例性的含有两个杂原子的6元杂芳基包括但不限于：哒嗪基、嘧啶基和吡嗪基。示例性的含有三个或四个杂原子的6元杂芳基分别包括但不限于：三嗪基和四嗪基。示例性的含有一个杂原子的7元杂芳基包括但不限于：氮杂环庚三烯基、氧杂环庚三烯基和硫杂环庚三烯基。示例性的5,6-双环杂芳基包括但不限于：吲哚基、异吲哚基、吲唑基、苯并三唑基、苯并噻吩基、异苯并噻吩基、苯并呋喃基、苯并异呋喃基、苯并咪唑基、苯并噁唑基、苯并异噁唑基、苯并噁二唑基、苯并噻唑基、苯并异噻唑基、苯并噻二唑基、茚嗪基和嘌呤基。示例性的6,6-双环杂芳基包括但不限于：萘啶基、喋啶基、喹啉基、异喹啉基、噌琳基、喹喔啉基、酞嗪基和喹唑啉基。Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxacycloheptatrienyl, and thiacycloheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

在本申请中，术语“任选”或“任选地”通常是指随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.

示例性的碳原子上的取代基包括但不局限于：卤素、-CN、-NO₂、-N₃、-SO₂H、-SO₃H、-OH、-OR^aa、-ON(R^bb)₂、-N(R^bb)₂、-N(R^bb)₃⁺X^-、-N(OR^cc)R^bb、-SH、-SR^aa、-SSR^cc、-C(＝O)R^aa、-CO₂H、-CHO、-C(OR^cc)₂、-CO₂R^aa、-OC(＝O)R^aa、-OCO₂R^aa、-C(＝O)N(R^bb)₂、-OC(＝O)N(R^bb)₂、-NR^bbC(＝O)R^aa、-NR^bbCO₂R^aa、-NR^bbC(＝O)N(R^bb)₂、-C(＝NR^bb)R^aa、-C(＝NR^bb)OR^aa、-OC(＝NR^bb)R^aa、-OC(＝NR^bb)OR^aa、-C(＝NR^bb)N(R^bb)₂、-OC(＝NR^bb)N(R^bb)₂、-NR^bbC(＝NR^bb)N(R^bb)₂、-C(＝O)NR^bbSO₂R^aa、-NR^bbSO₂R^aa、-SO₂N(R^bb)₂、-SO₂R^aa、-SO₂OR^aa、-OSO₂R^aa、-S(＝O)R^aa、-OS(＝O)R^aa、-Si(R^aa)₃、-OSi(R^aa)₃、-C(＝S)N(R^bb)₂、-C(＝O)SR^aa、-C(＝S)SR^aa、-SC(＝S)SR^aa、-SC(＝O)SR^aa、-OC(＝O)SR^aa、-SC(＝O)OR^aa、-SC(＝O)R^aa、-P(＝O)₂R^aa、-OP(＝O)₂R^aa、-P(＝O)(R^aa)₂、-OP(＝O)(R^aa)₂、-OP(＝O)(OR^cc)₂、-P(＝O)₂N(R^bb)₂、-OP(＝O)₂N(R^bb)₂、-P(＝O)(NR^bb)₂、-OP(＝O)(NR^bb)₂、-NR^bbP(＝O)(OR^cc)₂、-NR^bbP(＝O)(NR^bb)₂、-P(R^cc)₂、-P(R^cc)₃、-OP(R^cc)₂、-OP(R^cc)₃、-B(R^aa)₂、-B(OR^cc)₂、-BR^aa(OR^cc)、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^dd基团取代；Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2,_-N3 ,_-SO2H ,_-SO3H , -OH,_-ORaa ,^-ON (^Rbb )₂ , -N(Rbb₎₂ , -N(^Rbb )₃⁺^X- , -N(^ORcc )^Rbb , -SH,^-SRaa ,^-SSRcc , -C(=^O )^Raa ,_-CO2H , -CHO, -C(^ORcc )₂^, -CO2Raa, -OC(=O)^Raa ,_-OCO2Raa ,^-C (=O)N(^Rbb )₂ , -OC(=O)N(^Rbb )₂ , -NRbbC⁽ =_O )^Raa^,^-NRbbCO2Raa ,^-NRbb C(=O)N(R^bb )₂ , -C(=NR^bb )R^aa , -C(=NR^bb )OR^aa , -OC(=NR^bb )R^aa , -OC(=NR^bb )OR^aa , -C(=NR^bb )N(R^bb )₂ , -OC(=NR^bb )N(R^bb )₂ , -NR^bb C(=NR^bb )N(R^bb )₂ , -C(=O)NR^bb SO₂ R^aa , -NR^bb SO₂ R^aa , -SO₂ N(R^bb )₂ , -SO₂ R^aa , -SO₂ OR^aa , -OSO₂ R^aa , -S(=O)R^aa , -OS(=O)R^aa , -Si(R^aa )₃ , -OSi(R^aa )₃ , -C(=S)N(R^bb )₂ , -C(=O)SR^aa , -C(=S)SR^aa , -SC(=S)SR^aa , -SC(=O)SR^aa , -OC(=O)SR^aa , -SC(=O)OR^aa , -SC(=O)R^aa , -P(=O)₂ R^aa , -OP(＝O)₂ R^aa , -P(＝O)(R^aa )₂ , -OP(＝O)(R^aa )₂ , -OP(＝O)(OR^cc )₂ , -P(＝O)₂ N(R^bb )₂ , -OP(＝O)₂ N(R^bb )₂ , -P(＝O)(NR^bb )₂ , -OP(=O)(NR^bb )₂ , -NR^bb P(＝O)(^ORcc )₂ ,^-NRbbP (＝O)(^NRbb )₂ , -P(^Rcc )₂ , -P(^Rcc )₃ , -OP(^Rcc )2, -OP(Rcc)₃ , -B(^Raa )₂ , -B(^ORcc )₂_,^-BRaa⁽^ORcc ), alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5^Rdd groups;

或者在碳原子上的两个偕氢被基团＝O、＝S、＝NN(R^bb)₂、＝NNR^bbC(＝O)R^aa、＝NNR^bbC(＝O)OR^aa、＝NNR^bbS(＝O)₂R^aa、＝NR^bb或＝NOR^cc取代；or two geminal hydrogens on a carbon atom are replaced by groups =0, =S, =NN(^Rbb )₂ , =NNRbbC(=O)Raa, =^NNRbbC (=O^)ORaa^, =^NNRbbS (=O)_2Raa^, =^NRbb or =^NORcc^;

R^aa的每个独立地选自烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，或者两个R^aa基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^dd基团取代；each of^Raa is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl, or two^Ra groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5^Rdd groups;

R^bb的每个独立地选自：氢、-OH、-OR^aa、-N(R^cc)₂、-CN、-C(＝O)R^aa、-C(＝O)N(R^cc)₂、-CO₂R^aa、-SO₂R^aa、-C(＝NR^cc)OR^aa、-C(＝NR^cc)N(R^cc)₂、-SO₂N(R^cc)₂、-SO₂R^cc、-SO₂OR^cc、-SOR^aa、-C(＝S)N(R^cc)₂、-C(＝O)SR^cc、-C(＝S)SR^cc、-P(＝O)₂R^aa、-P(＝O)(R^aa)₂、-P(＝O)₂N(R^cc)₂、-P(＝O)(NR^cc)₂、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，或者两个R^bb基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^dd基团取代；Each of R^bb is independently selected from: hydrogen, -OH, -OR^aa , -N(R^cc )₂ , -CN, -C(=O)R^aa , -C(=O)N(R^cc )₂ , -CO₂ R^aa , -SO₂ R^aa , -C(=NR^cc )OR^aa , -C(=NR^cc )N(R^cc )₂ , -SO₂ N(R^cc )₂ , -SO₂ R^cc , -SO₂ OR^cc , -SOR^aa , -C(=S)N(R^cc )₂ , -C(=O)SR^cc , -C(=S)SR^cc , -P(=O)₂ R^aa , -P(=O)(R^aa )₂ , -P(=O)₂ N(R^cc )₂ , -P(=O)(^NRcc )₂ , alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two^Rbb groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5^Rdd groups;

R^cc的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，或者两个R^cc基团结合以形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^dd基团取代；each of R^cc is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two R^cc groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R^dd groups;

R^dd的每个独立地选自：卤素、-CN、-NO₂、-N₃、-SO₂H、-SO₃H、-OH、-OR^ee、-ON(R^ff)₂、-N(R^ff)₂,、-N(R^ff)₃⁺X^-、-N(OR^ee)R^ff、-SH、-SR^ee、-SSR^ee、-C(＝O)R^ee、-CO₂H、-CO₂R^ee、-OC(＝O)R^ee、-OCO₂R^ee、-C(＝O)N(R^ff)₂、-OC(＝O)N(R^ff)₂、-NR^ffC(＝O)R^ee、-NR^ffCO₂R^ee、-NR^ffC(＝O)N(R^ff)₂、-C(＝NR^ff)OR^ee、-OC(＝NR^ff)R^ee、-OC(＝NR^ff)OR^ee、-C(＝NR^ff)N(R^ff)₂、-OC(＝NR^ff)N(R^ff)₂、-NR^ffC(＝NR^ff)N(R^ff)₂、-NR^ffSO₂R^ee、-SO₂N(R^ff)₂、-SO₂R^ee、-SO₂OR^ee、-OSO₂R^ee、-S(＝O)R^ee、-Si(R^ee)₃、-OSi(R^ee)₃、-C(＝S)N(R^ff)₂、-C(＝O)SR^ee、-C(＝S)SR^ee、-SC(＝S)SR^ee、-P(＝O)₂R^ee、-P(＝O)(R^ee)₂、-OP(＝O)(R^ee)₂、-OP(＝O)(OR^ee)₂、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基、杂芳基，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^gg基团取代，或者两个偕R^dd取代基可结合以形成＝O或＝S；Each of R^dd is independently selected from the group consisting of halogen, -CN, -NO₂ , -N₃ , -SO₂ H, -SO₃ H, -OH, -OR^ee , -ON(R^ff )₂ , -N(R^ff )₂ , -N(R^ff )₃⁺ X^- , -N(OR^ee )R^ff , -SH, -SR^ee , -SSR^ee , -C(＝O)R^ee , -CO₂ H, -CO₂ R^ee , -OC(＝O)R^ee , -OCO₂ R^ee , -C(＝O)N(R^ff )₂ , -OC(＝O)N(R^ff )₂ , -NR^ff C(＝O)R^ee , -NR^ff CO₂ R^ee , -NR^ff C(＝O)N(R^ff )₂ , -C(=NR^ff )OR^ee , -OC(=NR^ff )R^ee , -OC(=NR^ff )OR^ee , -C(=NR^ff )N(R^ff )₂ , -OC(=NR^ff )N(R^ff )₂ , -NR^ff C(=NR^ff )N(R^ff )₂ , -NR^ff SO₂ R^ee , -SO₂ N(R^ff )₂ , -SO₂ R^ee , -SO₂ OR^ee , -OSO₂ R^ee , -S(=O)R^ee , -Si(R^ee )₃ , -OSi(R^ee )₃ , -C(=S)N(R^ff )₂ , -C(=O)SR^ee , -C(=S)SR^ee , -SC(=S)SR^ee , -P(＝O)₂ R^ee , -P(＝O)(R^ee )₂ , -OP(＝O)(R^ee )₂ , -OP(＝O)(OR^ee )₂ , alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 R^gg groups, or two geminal R^dd substituents may combine to form ＝O or ＝S;

R^ee的每个独立地选自烷基、卤代烷基、烯基、炔基、碳环基、芳基、杂环基和杂芳基，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^gg基团取代；Each of R^ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gg groups;

R^ff的每个独立地选自氢、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，或者两个R^ff基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^gg基团取代；each of^Rff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two^Rff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5^Rgg groups;

R^gg的每个独立地是：卤素、-CN、-NO₂、-N₃、-SO₂H、-SO₃H、-OH、-OC_1-6烷基、-ON(C_1-6烷基)₂、-N(C_1-6烷基)₂、-N(C_1-6烷基)₃⁺X^-、-NH(C_1-6烷基)₂⁺X^-、-NH₂(C_1-6烷基)⁺X^-、-NH₃⁺X^-、-N(OC_1-6烷基)(C_1-6烷基)、-N(OH)(C_1-6烷基)、-NH(OH)、-SH、-SC_1-6烷基、-SS(C_1-6烷基)、-C(＝O)(C_1-6烷基)、-CO₂H、-CO₂(C_1-6烷基)、-OC(＝O)(C_1-6烷基)、-OCO₂(C_1-6烷基)、-C(＝O)NH₂、-C(＝O)N(C_1-6烷基)₂、-OC(＝O)NH(C_1-6烷基)、-NHC(＝O)(C_1-6烷基)、-N(C_1-6烷基)C(＝O)(C_1-6烷基)、-NHCO₂(C_1-6烷基)、-NHC(＝O)N(C_1-6烷基)₂、-NHC(＝O)NH(C_1-6烷基)、-NHC(＝O)NH₂、-C(＝NH)O(C_1-6烷基)、-OC(＝NH)(C_1-6烷基)、-OC(＝NH)OC_1-6烷基、-C(＝NH)N(C_1-6烷基)₂、-C(＝NH)NH(C_1-6烷基)、-C(＝NH)NH₂、-OC(＝NH)N(C_1-6烷基)₂、-OC(NH)NH(C_1-6烷基)、-OC(NH)NH₂、-NHC(NH)N(C_1-6烷基)₂、-NHC(＝NH)NH₂、-NHSO₂(C_1-6烷基)、-SO₂N(C_1-6烷基)₂、-SO₂NH(C_1-6烷基)、-SO₂NH₂、-SO₂C_1-6烷基、-SO₂OC_1-6烷基、-OSO₂C_1-6烷基、-SOC_1-6烷基、-Si(C_1-6烷基)₃、-OSi(C_1-6烷基)₃、-C(＝S)N(C_1-6烷基)₂、C(＝S)NH(C_1-6烷基)、C(＝S)NH₂、-C(＝O)S(C_1-6烷基)、-C(＝S)SC_1-6烷基、-SC(＝S)SC_1-6烷基、-P(＝O)₂(C_1-6烷基)、-P(＝O)(C_1-6烷基)₂、-OP(＝O)(C_1-6烷基)₂、-OP(＝O)(OC_1-6烷基)₂、C_1-6烷基、C_1-6卤代烷基、C₂-C₆烯基、C₂-C₆炔基、C₃-C₇碳环基、C₆-C₁₀芳基、C₃-C₇杂环基、C₅-C₁₀杂芳基；或者两个偕R^gg取代基可结合形成＝O或＝S；其中，X^-为反离子。Each of R^gg is independently: halogen, -CN, -NO₂ , -N₃ , -SO₂ H, -SO₃ H, -OH, -OC_1-6 alkyl, -ON(C_1-6 alkyl)₂ , -N(C_1-6 alkyl)₂ , -N(C_1-6 alkyl)₃⁺ X^- , -NH(C_1-6 alkyl)₂⁺ X^- , -NH₂ (C_1-6 alkyl)⁺ X^- , -NH₃⁺ X^- , -N(OC_1-6 alkyl)(C_1-6 alkyl), -N(OH)(C_1-6 alkyl), -NH(OH), -SH, -SC_1-6 alkyl, -SS(C_1-6 alkyl), -C(＝O)(C_1-6 alkyl), -CO₂ H, -CO₂ (C_1-6 alkyl), -OC(＝O)(C_1-6 alkyl), -OCO₂ (C 1-6 alkyl)_-C (＝NH)₂ , -C(＝O)NH2, -C(＝O)N(C_1-6 alkyl)₂ , -OC(＝O)NH(C_1-6 alkyl), -NHC(＝O)(C_1-6 alkyl), -N(C_1-6 alkyl)C(＝O)(C_1-6 alkyl),_-NHCO2 (C_1-6 alkyl), -NHC(＝O)N(C_1-6 alkyl)₂ , -NHC(＝O)NH(C_1-6 alkyl), -NHC(＝O)_NH2 , -C(＝NH)O(C_1-6 alkyl), -OC(＝NH)(C_1-6 alkyl), -OC(＝NH)OC_1-6 alkyl, -C(＝NH)N(C_1-6 alkyl)₂ , -C(＝NH)NH(C_1-6 alkyl), -C(＝NH)_NH2 , -OC(＝NH)N(C 1-6 alkyl)_-OC (NH)NH(C_1-6 alkyl), -OC(NH)NH₂ , -NHC(NH)N(C_1-6 alkyl)₂ , -NHC(═NH)NH₂_, -NHSO₂ (C 1-6 alkyl), -SO₂ N(C_1-6 alkyl)₂ , -SO₂ NH(C_1-6 alkyl), -SO₂ NH₂ , -SO₂ C_1-6 alkyl, -SO₂ OC_1-6 alkyl, -OSO₂ C_1-6 alkyl, -SOC_1-6 alkyl, -Si(C_1-6 alkyl)₃ , -OSi(C_1-6 alkyl)₃ , -C(═S)N(C_1-6 alkyl)₂ , C(═S)NH(C_1-6 alkyl), C(═S)NH₂_, -C(═O)S(C 1-6 alkyl) -C_1-6 alkyl), -C(＝S)SC_1-6 alkyl, -SC(＝S)SC_1-6 alkyl, -P(＝O)₂ (C_1-6 alkyl), -P(＝O)(C_1-6 alkyl)₂ , -OP(＝O)(C_1-6 alkyl)₂ , -OP(＝O)(OC_1-6 alkyl)₂ , C_1-6 alkyl, C_1-6 haloalkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl, C₃ -C₇ carbocyclyl, C₆ -C₁₀ aryl, C₃ -C₇ heterocyclyl, C₅ -C₁₀ heteroaryl; or two geminal R^gg substituents may combine to form ＝O or ＝S; wherein X^- is a counter ion.

示例性的氮原子上取代基包括但不局限于：氢、-OH、-OR^aa、-N(R^cc)₂、-CN、-C(＝O)R^aa、-C(＝O)N(R^cc)₂、-CO₂R^aa、-SO₂R^aa、-C(＝NR^bb)R^aa、-C(＝NR^cc)OR^aa、-C(＝NR^cc)N(R^cc)₂、-SO₂N(R^cc)₂、-SO₂R^cc、-SO₂OR^cc、-SOR^aa、-C(＝S)N(R^cc)₂、-C(＝O)SR^cc、-C(＝S)SR^cc、-P(＝O)₂R^aa、-P(＝O)(R^aa)₂、-P(＝O)₂N(R^cc)₂、-P(＝O)(NR^cc)₂、烷基、卤代烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基，或者连接至氮原子的两个R^cc基团结合形成杂环基或杂芳基环，其中，每个烷基、烯基、炔基、碳环基、杂环基、芳基和杂芳基独立地被0、1、2、3、4或5个R^dd基团取代，且其中R^aa、R^bb、R^cc和R^dd如上所述。Exemplary substituents on the nitrogen atom include, but are not limited to, hydrogen, -OH,^-ORaa , -N(^Rcc )₂ , -CN, -C(=O)^Raa , -C(=O)_N⁽^Rcc₎₂ ,^-CO2Raa , -SO2Raa, -C(=^NRbb )Raa, -C(=^NRcc)ORaa^, -C(=^NRcc )N(^Rcc )₂ ,_-SO2N⁽^Rcc )₂^,_-SO2Rcc ,_-SO2ORcc ,^-SORaa , -C(=S)^N (^Rcc )₂ , -C(=O)^SRcc , -C(=S)^SRcc , -P(=O)_2Raa , -P(=O)(^Raa ),_-P⁽ =O)₂ N(^Rcc )₂ , -P(=O)(^NRcc )₂ , alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, or two^Rcc groups attached to the nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5^Rdd groups, and wherein^Raa ,^Rbb ,^Rcc and^Rdd are as described above.

“氘代”或“氘”指化合物或基团中的一个或多个氢被氘所取代；氘代可以是一取代、二取代、多取代或全取代。术语“一个或多个氘代的”与“一次或多次氘代”可互换使用。氘在氘代位置的氘同位素含量至少是大于天然氘同位素含量0.015％，较佳地大于30％，更佳地大于50％，更佳地大于75％，更佳地大于95％，更佳地大于99％。"Deuterated" or "deuterium" refers to a compound or group in which one or more hydrogens are replaced by deuterium; deuteration can be mono-, di-, poly- or full-substituted. The terms "one or more deuterated" and "one or more deuterated" are used interchangeably. The deuterium isotope content of deuterium at the deuterated position is at least greater than 0.015% of the natural deuterium isotope content, preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, and more preferably greater than 99%.

在本申请中，术语“化合物”通常指具有两种或两种以上不同元素的物质。例如，本申请的化合物可以是有机化合物，例如本申请的化合物可以是分子量500以下的化合物，可以是分子量1000以下的化合物，也可以是分子量1000以上的化合物，也可以是10000以上、100000以上的化合物。在本申请中，化合物还可以是指通过化学键相连的化合物，例如可以是一个或多个分子量1000以下的分子通过化学键与生物大分子相连的化合物，所述生物大分子可以是高聚糖、蛋白、核酸、多肽等。例如本申请的化合物可以包括蛋白质与一个或多个分子量1000以下的分子相连的化合物，可以是包括蛋白质与一个或多个分子量10000以下的分子相连的化合物，可以是包括蛋白质与一个或多个分子量100000以下的分子相连的化合物。In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 10000 or more than 100000. In the present application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by chemical bonds, and the biomacromolecule may be a high polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 100000.

如本文所用，术语“本申请化合物”指本申请的化合物。该术语还包括本申请化合物的各种药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体。As used herein, the term "compound of the present application" refers to the compound of the present application. The term also includes various pharmaceutically acceptable salts, prodrugs, hydrates, solvates, enantiomers, diastereomers, mesomorphs, racemates or tautomers of the compound of the present application.

生物定义Biological definition

在本申请中，术语“配体”通常指能识别和结合细胞相关的抗原或受体的大分子化合物。配体的作用可以是将药物呈递给与配体结合的细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞、受体和/或抗原结合的分子。在本申请中，配体可以表示为Ab，配体抗原通过配体上的杂原子与连接单元形成连接键，可以为抗体或其抗原结合片段，所述抗体可以选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；所述抗体可以是单克隆抗体。例如所述抗体可以是，靶向选自以下组的靶点的抗体或其抗原结合片段：In the present application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to a cell-associated antigen or receptor. The function of the ligand may be to present the drug to a cell population that binds to the ligand. These ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells, receptors and/or antigens. In the present application, the ligand may be represented by Ab, and the ligand antigen forms a connection bond with the connecting unit through the heteroatom on the ligand, and may be an antibody or an antigen-binding fragment thereof, and the antibody may be selected from a chimeric antibody, a humanized antibody, a fully human antibody or a mouse antibody; the antibody may be a monoclonal antibody. For example, the antibody may be an antibody or an antigen-binding fragment thereof that targets a target selected from the following groups:

HER2、Trop-2、Claudin-6、Claudin-9、Claudin-18.2、EGFR、c-Met、CD19、PSMA、Muc1、BCMA、PD-L1、CD33、CD30、CD22、CD79b、Nectin-4、CD19、组织因子、FRα、B7-H3、B7-H4、CDH3、CDH6、CDH17、ALPP、CD56、CD37、HER3、ROR1、MSLN、TNF-α、CD25、ENPP3、Muc1、Axl、CD20、ROR2、GPNMB、CEACAM5、CEACAM6、CD138、GC-C、LIV-1、CA6、FUT3、IGF-1R、CTLA4、RNF43、DPEP3、5T4、ITGB6、EFNA4、CD228、Notch3、CD46、CAIX、SLAMF6、ADAM9、GD3、TDGF1、SLAMF2、CLL-1、CD123、FCRL5、TIM1、sTn、ETB、Globo H、CD38、Ly6E、SLITRK6、GPR20、FGFR2，Muc16、CD5 1、SLAMF7、LAMP-1、CD74、CCR7、PTK7、SEZ6、LYFD3、TAA、PRL受体、FGFR3、KAAG1、STEAP1、Flt3、Muc1、LRRC15、CD44、CD70、EphA2、CXCR4、DDR1、DKL1、FOLR、CD45、DSG2、ALK、TRAlL、EpCAM、VEGFR2、CD47、CD49、SSEA-4、DCLK1、OacGD2、CD73、EN01、BSG、CD24、GLUT1和GPRC5D，优选HER2、HER3、EGFR、Trop-2、Claudin-6、Claudin-18.2、B7-H3、B7-H4、CDH3、CDH6、CDH17、FRα、ROR1、ALPP、CEACAM5、CEACAM6或FOLR。HER2, Trop-2, Claudin-6, Claudin-9, Claudin-18.2, EGFR, c-Met, CD19, PSMA, Muc1, BCMA, PD-L1, CD33, CD30, CD22, CD79b, Nectin-4, CD19, tissue factor, FRα, B7-H3, B7-H4, CDH3, CDH6, CDH17, ALPP, CD56, CD37, HER3, ROR1, MSLN, TNF-α, CD 25. ENPP3, Muc1, Axl, CD20, ROR2, GPNMB, CEACAM5, CEACAM6, CD138, GC-C, LIV-1, CA6, FUT3, IGF-1R, CTLA4, RNF43, D PEP3, 5T4, ITGB6, EFNA4, CD228, Notch3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, FCRL5, TIM1, sTn, ETB, Globo H, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD5 1, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, LYFD 3. TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, Muc1, LRRC15, CD44, CD70, EphA2, CXCR4, DDR1, DKL1, FOLR, CD45, DSG2, AL K, TRA1L, EpCAM, VEGFR2, CD47, CD49, SSEA-4, DCLK1, OacGD2, CD73, ENO1, BSG, CD24, GLUT1 and GPRC5D, preferably HER2, HER3, EGFR, Trop-2, Claudin-6, Claudin-18.2, B7-H3, B7-H4, CDH3, CDH6, CDH17, FRα, ROR1, ALPP, CEACAM5, CEACAM6 or FOLR.

在本申请中，术语“偶联物”通常是指本申请的化合物通过一个或多个化学反应而制备的化合物，或者通过诸如桥接(bridge)，间隔物(spacer)，或连接部分等的一个或多个连接结构彼此连接。In the present application, the term "conjugate" generally refers to a compound prepared by one or more chemical reactions of the compounds of the present application, or connected to each other through one or more connecting structures such as a bridge, a spacer, or a connecting part.

在本申请中，术语“HER2”通常是指人表皮生长因子受体2(HER2)，例如，术语“HER2”指来自任何人来源的任何天然HER2。该术语还涵盖“全长”和未加工的HER2以及源自细胞中加工的任何形式的HER2(例如成熟蛋白)。该术语还涵盖HER2的天然发生变体和同等型，例如剪接变体或等位变体。例如，Uniprot登录号P04626提供了HER2和序列的描述。In the present application, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2), for example, the term "HER2" refers to any natural HER2 from any human source. The term also encompasses "full-length" and unprocessed HER2 and any form of HER2 (e.g., mature protein) derived from processing in a cell. The term also encompasses naturally occurring variants and isoforms of HER2, such as splice variants or allelic variants. For example, Uniprot accession number P04626 provides a description of HER2 and sequences.

在本申请中，术语“B7-H3”、“CD276”通常是指一种I型跨膜蛋白，属于B7免疫共刺激和共抑制家族成员。B7-H3蛋白由染色体15q24基因编码，结构上包括涵盖胞外域、跨膜域和短胞内域在内的316个氨基酸组成。B7-H3蛋白胞内结构域很短，尚无已知的信号基序。In this application, the terms "B7-H3" and "CD276" generally refer to a type I transmembrane protein that belongs to the B7 immune co-stimulatory and co-inhibitory family. The B7-H3 protein is encoded by the chromosome 15q24 gene and consists of 316 amino acids including an extracellular domain, a transmembrane domain, and a short intracellular domain. The intracellular domain of the B7-H3 protein is very short and has no known signal motif.

在本申请中，术语“嵌合抗体(chimeric antibody)”通常是指鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，可以建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，可以根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，可以在真核系统或原核系统中表达嵌合抗体分子。In this application, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies can be established, and then the variable region gene can be cloned from the mouse hybridoma cells. The constant region gene of the human antibody can be cloned as needed, and the mouse variable region gene and the human constant region gene can be connected into a chimeric gene and inserted into an expression vector. The chimeric antibody molecule can be expressed in a eukaryotic system or a prokaryotic system.

在本申请中，术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(grafted antibody)，通常是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库。In this application, the term "humanized antibody", also known as CDR grafted antibody, generally refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. The heterologous response induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database.

在本申请中，术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区可以都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本申请所述抗体或配体可以为全人源单克隆抗体。全人抗体制备的相关技术可以为：人杂交瘤技术、EBV转化B淋巴细胞技术、嗦菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。In this application, the terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody can be both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies can be: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

在本申请中，术语“CDR”通常是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，Chothia等人和MacCallum等人提供。如本申请中使用的，CDR的Kabat定义可以应用于轻链可变结构域的CDRl、CDR2和CDR3(CDRL1、CDRL2、CDRL3或LK L2、L3)，以及重链可变结构域的CDRK、CDR2和CDR3(CDRH1、CDRH2、CDRH3或HI、H2、H3)。In this application, the term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., Chothia et al., and MacCallum et al. As used in this application, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3 or LK L2, L3) of the light chain variable domain, and CDRK, CDR2, and CDR3 (CDRH1, CDRH2, CDRH3 or HI, H2, H3) of the heavy chain variable domain.

在本申请中，术语“连接子”通常是指一端与一个基团连接而另一端与另一个基团相连的化学结构片段或键，也可以连接其他连接体后再与药物和/或配体相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过连接体连接配体。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物接头结构的化学结构片段或键作为连接体。In the present application, the term "linker" generally refers to a chemical structure fragment or bond that is connected to one group at one end and to another group at the other end, and may also be connected to other linkers before being connected to the drug and/or ligand. The directly or indirectly connected ligand may refer to the group being directly connected to the ligand through a covalent bond, or may be connected to the ligand through a linker. For example, a chemical structure fragment or bond containing an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure may be used as a linker.

在本申请中，术语“连接基团”通常是指具有与另一个基团相连的能力的基团。例如，具有连接基团的化合物，可以通过该连接基团与另一个基团的偶联反应，实现该化合物与另一个基团的连接。例如，马来酰亚胺基团可以作为连接基团。In this application, the term "linking group" generally refers to a group that has the ability to connect to another group. For example, a compound having a linking group can achieve connection with another group through a coupling reaction between the linking group and another group. For example, a maleimide group can be used as a linking group.

在本申请中，术语“药物单元”通常是指直接或间接缀合抗体或抗原结合片段以形成免疫缀合物的化学部分。例如，“药物单元”包括但不限于文中描述的抗肿瘤活性的化合物。例如药物单元包括拓扑异构酶抑制剂。In this application, the term "drug unit" generally refers to a chemical moiety that is directly or indirectly conjugated to an antibody or antigen-binding fragment to form an immunoconjugate. For example, a "drug unit" includes, but is not limited to, a compound with anti-tumor activity described herein. For example, a drug unit includes a topoisomerase inhibitor.

在本申请中，术语与某靶点“表达相关”的疾病通常是指该疾病的发生和/或进展与该靶点的表达水平有关联。例如，相对于来自组织或器官的正常细胞的表达水平，来自疾病区如患者的特定组织或器官内的细胞中某个靶点的表达水平提高，即高表达的。或者例如，相对于来自组织或器官的正常细胞的表达水平，来自疾病区如患者的特定组织或器官内的细胞中某个靶点的表达水平降低，即低表达的。或者例如，来自疾病区如患者的特定组织或器官内的细胞表达某个靶点，即阳性的。或者例如，来自疾病区如患者的特定组织或器官内的细胞不表达某个靶点，即阴性的。例如，靶点表达的特征可以由本领域己知的标准测定确定。In the present application, the term "disease associated with expression" of a target generally refers to that the occurrence and/or progression of the disease is associated with the expression level of the target. For example, relative to the expression level of normal cells from tissues or organs, the expression level of a certain target in cells from a disease area such as a specific tissue or organ of a patient is increased, i.e., highly expressed. Or, for example, relative to the expression level of normal cells from tissues or organs, the expression level of a certain target in cells from a disease area such as a specific tissue or organ of a patient is reduced, i.e., lowly expressed. Or, for example, cells from a disease area such as a specific tissue or organ of a patient express a certain target, i.e., positive. Or, for example, cells from a disease area such as a specific tissue or organ of a patient do not express a certain target, i.e., negative. For example, the characteristics of target expression can be determined by standard assays known in the art.

如本文所用，“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出所需的生物活性。抗体可以为鼠、人、人源化、嵌合的抗体或来源于其它物种。抗体为由能够识别和结合特异性抗原的免疫系统产生的蛋白质。靶抗原一般具有由多种抗体的CDRs识别的大量结合位点，也称作表位。特异性结合不同表位的各抗体具有不同的结构。因此，一种抗原可以具有一种以上相应的抗体。抗体包括全-长免疫球蛋白分子或全-长免疫球蛋白分子的免疫活性部分，即含有特异性结合所关注靶标的抗原或其部分的分子，这类靶标包括，但不限于癌细胞或产生与自身免疫性疾病相关的自身免疫抗体的细胞。本申请描述的免疫球蛋白可以具有免疫球蛋白分子的任意类型(例如IgG、IgE、IgM、IgD和IgA)、类别(例如IgGl、IgG2、IgG3、IgG4、IgAl和IgA2)或亚类。免疫球蛋白可以来源于任意的物种。然而，在一个方面中，免疫球蛋白来源于人、鼠或兔。“抗体片段”可以包含全长抗体的一部分，一般为其抗原结合区或可变区。抗体片段的实例包括：Fab、Fab\F(ab’)2和Fv片段；双抗体；线性抗体；微抗体(minibody)；Fab表达文库制备的片段；抗-独特型(抗-Id)抗体；CDR(互补决定区)；和以免疫特异性方式结合癌细胞抗原、病毒抗原或微生物抗原的上述任意的表位-结合片段；单链抗体分子；和由抗体片段形成的多特异性抗体。本申请中组成抗体药物偶联物的抗体可以保持其原有野生状态时的抗原结合能力。因此，本申请中的抗体可以，例如可以专一性地，与抗原结合。涉及的抗原包括，例如，肿瘤相关抗原(TAA)，细胞表面受体蛋白和其他细胞表面分子，细胞存活调节因子，细胞增殖调节因子，与组织生长与分化相关的分子(如己知或预知的具有功能性的)，淋巴因子，细胞因子，参与细胞循环调节的分子，参与血管生成的分子，以及与血管生成有关的分子(如己知抗体结合的抗原可以是上述分类中一个或一个子集，而其它的子集则包含其它的具有特殊性质的分子/抗原(与目标抗原相比)。应用在抗体药物偶联物中的抗体包括，但不局限于，针对细胞表面受体和肿瘤相关抗原的抗体。这样的肿瘤相关抗原是业内所熟知的，可以通过业内熟知的抗体制备方法和信息来制备。这些目标物能够特异性地表达在一种或多种癌症细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言，这样的肿瘤相关多肽在癌细胞表面可以更加过度表达。As used herein, "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric antibodies or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens generally have a large number of binding sites, also known as epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain antigens or portions thereof that specifically bind to a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins described in the present application may have any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, in one aspect, immunoglobulins are derived from humans, mice, or rabbits. "Antibody fragments" may include a portion of a full-length antibody, generally its antigen binding region or variable region. Examples of antibody fragments include: Fab, Fab\F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies; fragments prepared from Fab expression libraries; anti-idiotype (anti-Id) antibodies; CDRs (complementarity determining regions); and any of the above epitope-binding fragments that bind to cancer cell antigens, viral antigens, or microbial antigens in an immunospecific manner; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The antibodies that constitute the antibody-drug conjugates in the present application may retain their original antigen binding ability in the wild state. Therefore, the antibodies in the present application can, for example, specifically bind to antigens. The antigens involved include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as known or predicted functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as known antibodies). The antigens bound by antibodies can be one or a subset of the above categories, while other subsets contain other molecules/antigens with special properties (compared with the target antigen). The antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the industry and can be prepared by antibody preparation methods and information well known in the industry. These targets can be specifically expressed on the surface of one or more cancer cells, and rarely or not expressed on the surface of one or more non-cancerous cells. Generally, such tumor-associated polypeptides can be more overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells.

在本申请中，术语“氨基酸”是组成蛋白质的基本结构单元，是生物进行蛋白后期修饰的基础，总共有20种天然氨基酸。此外，在这些基本氨基酸的基础上，生物还会合成羟脯氨酸、羟赖氨酸等衍生出来的氨基酸类型，在萤火虫体内，甚至还会合成D-型氨基酸。天然氨基酸一般情况下是L-型的，本发明还包括D-型的氨基酸。用于本发明的氨基酸如下表所示：

In this application, the term "amino acid" is the basic structural unit of protein and the basis for later modification of proteins by organisms. There are 20 natural amino acids in total. In addition, based on these basic amino acids, organisms will also synthesize amino acid types derived from hydroxyproline, hydroxylysine, etc. In fireflies, they will even synthesize D-type amino acids. Natural amino acids are generally L-type, and the present invention also includes D-type amino acids. The amino acids used in the present invention are shown in the following table:

在本申请中，术语“多肽残基/寡肽残基”通常是指包含两个或多个氨基酸残基通过肽键连接而成的残基。例如，多肽残基中的两个或多个氨基酸可以是任选取代的。例如，本申请的多肽残基可以选自以下组：缬氨酸-瓜氨酸(Val-Cit)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-赖氨酸(Val-Lys)、苯丙氨酸-赖氨酸(Phe-Lys)、赖氨酸-赖氨酸(Lys-Lys)、丙氨酸-赖氨酸(Ala-Lys)、苯丙氨酸-瓜氨酸(Phe-Cit)、亮氨酸-瓜氨酸(Leu-Cit)、异亮氨酸-瓜氨酸(Ile-Cit)、苯丙氨酸-丙氨酸(Phe-Ala)、赖氨酸-缬氨酸-瓜氨酸(Lys-Val-Cit)、赖氨酸-缬氨酸-丙氨酸(Lys-Val-Ala)、缬氨酸-赖氨酸-甘氨酸(Val-Lys-Gly)、甘氨酸-缬氨酸-赖氨酸(Gly-Val-Lys)、甘氨酸-缬氨酸-丙氨酸(Gly-Val-Ala)、谷氨酸-缬氨酸-丙氨酸(Glu-Val-Ala)、谷氨酸-缬氨酸-瓜氨酸(Glu-Val-Cit)、谷氨酰胺-缬氨酸-丙氨酸(Gln-Val-Ala)、谷氨酰胺-缬氨酸-瓜氨酸(Gln-Val-Cit)、丙氨酸-丙氨酸-丙氨酸(Ala-Ala-Ala)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、苯丙氨酸-苯丙氨酸-赖氨酸(Phe-Phe-Lys)、甘氨酸-苯丙氨酸-赖氨酸(Gly-Phe-Lys)、亮氨酸-丙氨酸-亮氨酸(Leu-Ala-Leu)、异亮氨酸-丙氨酸-亮氨酸(Ile-Ala-Leu)、缬氨酸-丙氨酸-缬氨酸(Val-Ala-Val)、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)、(Ala-Leu-Ala-Leu)和(Gly-Phe-Leu-Gly)，优选选自以下组：苯丙氨酸-赖氨酸(Phe-Lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-瓜氨酸(Val-Cit)、谷氨酸-缬氨酸-丙氨酸(Glu-Val-Ala)、谷氨酸-缬氨酸-瓜氨酸(Glu-Val-Cit)、谷氨酰胺-缬氨酸-丙氨酸(Gln-Val-Ala)、谷氨酰胺-缬氨酸-瓜氨酸(Gln-Val-Cit)、缬氨酸-赖氨酸(Val-Lys)、丙氨酸-丙氨酸-丙氨酸(Ala-Ala-Ala)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)，优选选自以下组：缬氨酸-瓜氨酸(Val-Cit)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-赖氨酸(Val-Lys)、苯丙氨酸-赖氨酸(Phe-Lys)、赖氨酸-缬氨酸-瓜氨酸(Lys-Val-Cit)、赖氨酸-缬氨酸-丙氨酸(Lys-Val-Ala)、谷氨酰胺-缬氨酸-丙氨酸(Gln-Val-Ala)、谷氨酰胺-缬氨酸-瓜氨酸(Gln-Val-Cit)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)，优选选自以下组：缬氨酸-瓜氨酸(Val-Cit)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-赖氨酸(Val-Lys)、赖氨酸-缬氨酸-瓜氨酸(Lys-Val-Cit)、赖氨酸-缬氨酸-丙氨酸(Lys-Val-Ala)和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。In the present application, the term "polypeptide residue/oligopeptide residue" generally refers to a residue comprising two or more amino acid residues connected by peptide bonds. For example, two or more amino acids in a polypeptide residue may be optionally substituted. For example, the polypeptide residues of the present application can be selected from the following groups: valine-citrulline (Val-Cit), valine-alanine (Val-Ala), valine-lysine (Val-Lys), phenylalanine-lysine (Phe-Lys), lysine-lysine (Lys-Lys), alanine-lysine (Ala-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), phenylalanine-alanine (Phe-Ala), lysine-valine-citrulline (Lys-Val-Cit), lysine-valine-alanine (Lys-Val-Ala), valine-lysine-glycine (Val-Lys-Gly), glycine-valine-lysine (Gly-Val-Lys), glycine-valine-alanine (Gly-Val-Ala), glutamic acid- Valine-Alanine (Glu-Val-Ala), Glutamic acid-Valine-Citrulline (Glu-Val-Cit), Glutamine-Valine-Alanine (Gln-Val-Ala), Glutamine-Valine-Citrulline (Gln-Val-Cit), Alanine-Alanine-Alanine (Ala-Ala-Ala), Alanine-Alanine-Asparagine (Ala-Ala-Asn), Phenylalanine-Phenylalanine-Lysine (Phe-Phe-Lys), Glycine-Phenylalanine-Lysine (Gly-Phe-Lys), Leucine-Alanine-Leucine (Leu-Ala-Leu), Isoleucine-Alanine-Leucine (Ile-Ala-Leu), Valine-Alanine-Valine (Val-Ala-Val), Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), (Ala-Leu-Al α-Leu) and (Gly-Phe-Leu-Gly), preferably selected from the following group: phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-alanine (Glu-Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), glutamine-valine-alanine (Gln-Val-Ala), glutamine-valine-citrulline (Gln-Val-Cit), valine-lysine (Val-Lys), alanine-alanine-alanine (Ala-Ala-Ala), alanine-alanine-asparagine (Ala-Ala-Asn) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), preferably selected from the following group: valine-citrulline (Val-Cit), valine-alanine ( Preferably, the amino acid sequence of the present invention is selected from the group consisting of valine-citrulline (Val-Ala), valine-lysine (Val-Lys), phenylalanine-lysine (Phe-Lys), lysine-valine-citrulline (Lys-Val-Cit), lysine-valine-alanine (Lys-Val-Ala), glutamine-valine-alanine (Gln-Val-Ala), glutamine-valine-citrulline (Gln-Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and is preferably selected from the group consisting of valine-citrulline (Val-Cit), valine-alanine (Val-Ala), valine-lysine (Val-Lys), lysine-valine-citrulline (Lys-Val-Cit), lysine-valine-alanine (Lys-Val-Ala) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

在本申请中，术语“多聚乙二醇基/聚乙二醇基”通常是指包含一个或多个乙二醇残基连接而成的残基。例如，聚乙二醇基可以包含-(CH₂CH₂O)_p-，其中p为至少为1的数。例如本申请中的聚乙二醇基可以是任选取代的。In the present application, the term "polyethylene glycol group" generally refers to a residue comprising one or more ethylene glycol residues connected together. For example, the polyethylene glycol group may comprise -(CH₂ CH₂ O)_p -, wherein p is a number at least 1. For example, the polyethylene glycol group in the present application may be optionally substituted.

在本申请中，术语“多聚肌氨酸残基/聚肌氨酸残基”通常是指包含一个或多个肌氨酸残基连接而成的残基。例如，聚肌氨酸残基可以包含-(COCH₂N(CH₃))_q-，其中q为至少为1的数。In the present application, the term "polysarcosine residue" generally refers to a residue comprising one or more sarcosine residues linked together. For example, the polysarcosine residue may comprise -(COCH₂ N(CH₃ ))_q -, wherein q is a number of at least 1.

在本申请中，术语“五碳糖”是指分子中所含碳原子的数目为五个的单糖，又称为戊糖。戊糖在生物界分布很广，在生命活动中具有重要作用。主要有D-木糖、L-阿拉伯糖、D-核糖及其衍生物D-2-脱氧核糖。作为糖代谢中间产物的戊酮糖有D-核酮糖和D-木酮糖。In this application, the term "pentose" refers to a monosaccharide with five carbon atoms in the molecule, also known as pentose. Pentose is widely distributed in the biological world and plays an important role in life activities. It mainly includes D-xylose, L-arabinose, D-ribose and its derivative D-2-deoxyribose. Ketopentose as an intermediate product of sugar metabolism includes D-ribulose and D-xylulose.

在本申请中，术语“六碳糖”是指含有6个碳原子的单糖，又称为六碳糖。己糖在自然界分布最广，数量也最多，与机体的营养代谢也最密切。在1位含有醛基的6碳糖称为己醛糖，在2位含有酮基的己糖称为己酮糖。己糖六个碳原子中有五个碳原子连上了羟基(-OH)，余下的一个碳原子是属于一个醛基(-COH)或是酮基(-CO-)的。重要的己醛糖有D-葡萄糖、D-半乳糖和D-甘露糖；己酮糖则有D-果糖。In the present application, the term "hexose" refers to a monosaccharide containing 6 carbon atoms, also known as a hexose. Hexoses are the most widely distributed in nature, have the largest number, and are most closely related to the body's nutritional metabolism. The 6-carbon sugar containing an aldehyde group at position 1 is called an aldohexose, and the hexose containing a ketone group at position 2 is called a ketohexose. Five of the six carbon atoms in a hexose are connected to a hydroxyl group (-OH), and the remaining carbon atom belongs to an aldehyde group (-COH) or a ketone group (-CO-). Important aldohexoses include D-glucose, D-galactose, and D-mannose; and ketohexoses include D-fructose.

在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。对于本申请来说，血管生成是肿瘤特征的一部分。肿瘤可能是良性的，也可能是恶件的。术语“肿瘤”一般用于指良性或恶性的肿瘤，而术语“癌”一般用于指恶性肿瘤，可以是转移癌，也可以是非转移癌。可用本申请的方法治疗的肿瘤选自以下组：乳腺癌、卵巢癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性淋巴细胞性白血病、间变性大细胞淋巴瘤、多发性骨髓瘤、前列腺癌、恶性黑色素瘤、鳞状细胞癌、胶质母细胞瘤、肾细胞癌、胃肠道肿瘤、胰腺癌、前列腺癌、直结肠、胃癌、神经胶质瘤和间皮瘤。用于研究时，可通过本领域技术人员熟知的方法从易于获得的资源中将这些组织分离出来。In this application, the term "tumor" generally refers to any new pathological tissue proliferation. For the present application, angiogenesis is part of the characteristics of tumors. Tumors may be benign or malignant. The term "tumor" is generally used to refer to benign or malignant tumors, while the term "cancer" is generally used to refer to malignant tumors, which may be metastatic or non-metastatic. Tumors that can be treated by the method of the present application are selected from the following groups: breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, rectum, gastric cancer, glioma and mesothelioma. When used for research, these tissues can be separated from readily available resources by methods well known to those skilled in the art.

其他定义Other definitions

在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。术语“以上”、“以下”通常是指包含本数的情况。In the present application, the term "comprising" generally refers to including the features explicitly specified, but not excluding other elements. The terms "above" and "below" generally refer to the case where the number is inclusive.

在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

本文所用的术语“药学上可接受的盐”表示本发明化合物的酸加成盐或碱加成盐，它们在可靠的医学判断范围内适用于与患者组织接触，不会产生不恰当的毒性、刺激作用、变态反应等，与合理的益处/风险比相称，就它们的预期应用而言是有效的，包括(可能的话)本发明化合物的两性离子形式。As used herein, the term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt of the compounds of the invention which are suitable, within the scope of sound medical judgment, for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including, where possible, zwitterionic forms of the compounds of the invention.

药学上可接受的盐可以是从无机酸制备的硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、硝酸盐、硼酸盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物等。代表性盐包括：氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、硝酸盐、硼酸盐和磷酸盐等。盐也可以是从有机酸制备的，例如脂肪族一元与二元羧酸、苯基取代的烷酸、羟基烷酸、烷二酸、芳香族酸、脂肪族与芳香族磺酸等。代表性盐包括乙酸盐、丙酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、辛酸盐、异丁酸盐、草酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、扁桃酸盐、苯甲酸盐、氯苯甲酸盆、甲基苯甲酸盐、二硝基苯甲酸盐、萘甲酸盐、苯磺酸盐、甲苯磺酸盐、苯乙酸盐、柠檬酸盐、乳酸盐、马来酸盐、酒石酸盐、甲磺酸盐、葡庚糖酸盐、乳糖酸盐、月桂基磺酸盐和羟乙磺酸盐等。药学上可接受的盐可以包括基于碱金属与碱土金属的阳离子，例如钠、锂、钾、钙、镁等，以及无毒的铵、季铵和胺阳离子，包括但不限于铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、乙胺等。还涵盖氨基酸的盐，例如精氨酸盐、葡糖酸盐、半乳糖醛酸盐等(例如参见Berge S.M.et al.，"Pharmaceutical Salts,”J.Pharm.Sci.，1977；66:1-19，引入此作为参考)。Pharmaceutically acceptable salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, borates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, etc. prepared from inorganic acids. Representative salts include hydrobromides, hydrochlorides, sulfates, bisulfates, nitrates, borates, and phosphates, etc. Salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, valerate, oleate, palmitate, stearate, laurate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, glucoheptonate, lactobionate, laurylsulfonate, and isethionate, etc. Pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also covered are salts of amino acids, such as argininate, gluconate, galacturonate, and the like (see, e.g., Berge S.M. et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

本发明还包括同位素标记的化合物(同位素变体)，它们等同于本申请所述的那些通式或具体化合物，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如²H、³H、¹³C、¹¹C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F和³⁶Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如³H和¹⁴C)的那些可用于药物和/或底物组织分布测定。氚、即³H和碳-14、即¹⁴C同位素是特别优选的，因为它们容易制备和检测。另外，被较重的同位素例如氘(即²H)取代，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或减少剂量需求，因而在有些情况下是优选的。同位素标记的本发明化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。The present invention also includes isotope-labeled compounds (isotope variants), which are equivalent to those general formulas or specific compounds described in the present application, but one or more atoms are replaced by atoms whose atomic mass or mass number is different from the atomic mass or mass number commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as² H,³ H,¹³ C,¹¹ C,¹⁴ C,¹⁵ N,¹⁸ O,¹⁷ O,³¹ P,³² P,³⁵ S,¹⁸ F and³⁶ Cl, respectively. Compounds of the present invention containing the above-mentioned isotopes and/or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs all belong to the scope of the present invention. Certain isotope-labeled compounds of the present invention, such as those that introduce radioactive isotopes (such as³ H and¹⁴ C), can be used for drug and/or substrate tissue distribution assays. Tritium, i.e.,³ H, and carbon-14, i.e.,¹⁴ C isotopes are particularly preferred because they are easy to prepare and detect. In addition, substitution with heavier isotopes, such as deuterium (i.e.,² H), may provide therapeutic benefits due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and is therefore preferred in some cases. Isotopically labeled compounds of the invention and prodrugs thereof can generally be prepared by replacing non-isotopically labeled reagents with readily available isotopically labeled reagents when performing the processes disclosed in the following schemes and/or the Examples and Preparations.

本发明化合物包括一个或多个不对称中心，且因此可以存在多种立体异构体形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。The compounds of the present invention include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and/or diastereoisomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

在本申请中，术语“药物组合物”通常是指含有一种或多种本申请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。药物组合物可以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。常规的药物组合物的制备可以见本领域常用技术。In the present application, the term "pharmaceutical composition" generally refers to 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 pharmaceutical composition can promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. The preparation of conventional pharmaceutical compositions can be found in the common techniques in the art.

在本申请中，术语“药学上可接受的载体”通常是指给予治疗剂，例如抗体或多肽、基因和其它治疗剂的载体。该术语指本身不诱导对接受组合物的个体有害的抗体产生并且可以给予而不产生过度毒性的任何药物载体。例如，药学上可接受的载体(carrier)可以与基因工程中用于包含基因的核酸载体(vector)相区分。合适的载体可以是大的、代谢缓慢的大分子，例如蛋白质、多糖、聚乳酸、聚乙醇酸、多聚氨基酸、氨基酸共聚物、脂质聚集物和灭活的病毒颗粒。本领域技术人员熟知这些载体。治疗组合物中药学上可接受的载体可包括液体，例如水、盐水、甘油和乙醇。这些载体中也可存在辅助物质，例如润湿剂或乳化剂、pH缓冲物质等。In the present application, the term "pharmaceutically acceptable carrier" generally refers to a carrier for administering therapeutic agents, such as antibodies or polypeptides, genes and other therapeutic agents. The term refers to any drug carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity. For example, a pharmaceutically acceptable carrier can be distinguished from a nucleic acid vector used to contain a gene in genetic engineering. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates and inactivated viral particles. Those skilled in the art are familiar with these carriers. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.

在本申请中，术语“抗肿瘤活性的化合物”通常是指具有使肿瘤细胞的增殖速率、存活力或转移活性的降低的能力的化合物。例如，抗肿瘤活性可以由治疗期间出现的异常细胞的生长速率减少或肿瘤尺寸稳定或缩减显示的，或与在无治疗情况下对照相比因治疗所致的存活期更长显示的。抗肿瘤活性可使用公认的体外或体内肿瘤模型评估，例如异种移植模型。In the present application, the term "compound with anti-tumor activity" generally refers to a compound that has the ability to reduce the proliferation rate, viability or metastatic activity of tumor cells. For example, the anti-tumor activity can be shown by a reduction in the growth rate of abnormal cells that occur during treatment or a stabilization or reduction in tumor size, or a longer survival period due to treatment compared to a control without treatment. The anti-tumor activity can be evaluated using recognized in vitro or in vivo tumor models, such as xenograft models.

在本发明的部分实施方案中，偶联物中的生物活性分子为具体有抗肿瘤活性的化合物，具体例如：放射性同位素，例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212或Lu的放射性同位素；金属配合物，例如金属铂配合物(如奥沙利铂)或金属金配合物；糖肽类抗生素，例如博来霉素或平阳霉素；拓扑异构酶抑制剂；干扰DNA合成的药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨或奈拉滨等；作用于结构蛋白的药物，例如微管蛋白抑制剂(诸如长春花生物碱类、长春新碱、长春碱、紫杉醇类、美登素类、澳瑞他汀类、Tubulysin B或艾瑞布林等)；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂等；蛋白酶体抑制剂；表观遗传相关靶点抑制剂；肿瘤新生血管生成抑制剂；细胞周期蛋白抑制剂。In some embodiments of the present invention, the biologically active molecule in the conjugate is a compound with specific anti-tumor activity, such as: radioactive isotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 or radioactive isotopes of Lu; metal complexes, such as metal platinum complexes (such as oxaliplatin) or metal gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; topoisomerase inhibitors; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, glycosides, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nelarabine; drugs acting on structural proteins, such as microtubule inhibitors (such as vinca alkaloids, vincristine, vinblastine, paclitaxel, maytansine, auristatins, Tubulysin B or eribulin); tumor signaling pathway inhibitors, such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors; epigenetic-related target inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors.

在本申请中，术语“拓扑异构酶抑制剂”通常是指包括拓扑异构酶I抑制剂和拓扑异构酶II抑制剂的化合物或其衍生物。拓扑异构酶I抑制剂的实例包括但不限于喜树碱及其类似物；拓扑异构酶II抑制剂(诸如放线菌素D、阿霉素、多柔比星、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素或依托泊苷等)。拓扑异构酶(topoisomerase)可以是指通过切断DNA的一条或两条链中的磷酸二酯键，然后重新缠绕和封口来更正DNA连环数的酶。In the present application, the term "topoisomerase inhibitor" generally refers to compounds including topoisomerase I inhibitors and topoisomerase II inhibitors or their derivatives. Examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs; topoisomerase II inhibitors (such as actinomycin D, adriamycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide, etc.). Topoisomerase can refer to an enzyme that corrects the number of DNA loops by cutting the phosphodiester bonds in one or both strands of DNA and then rewinding and sealing.

在本申请中，术语“喜树碱类似物”通常是指与喜树碱结构类似或衍生自喜树碱的化合物。例如，喜树碱的结构可以在CAS登录号7689-03-4中记载。例如，喜树碱类似物可以是指伊沙替康(Exatecan，CAS登录号171335-80-1)或贝洛替康(Belotecan，CAS登录号256411-32-2)。术语“非喜树碱类拓扑异构酶I抑制剂”通常是指吲哚咔唑类、茚并异喹啉酮类、苯并菲啶类以及二苯并萘啶酮类具有拓扑异构酶I抑制活性的杂环分子，主要指Genz-644282(CAS登录号529488_28-6)。In the present application, the term "camptothecin analogue" generally refers to a compound that is structurally similar to or derived from camptothecin. For example, the structure of camptothecin can be recorded in CAS accession number 7689-03-4. For example, a camptothecin analogue can refer to Exatecan (CAS accession number 171335-80-1) or Belotecan (CAS accession number 256411-32-2). The term "non-camptothecin topoisomerase I inhibitor" generally refers to heterocyclic molecules with topoisomerase I inhibitory activity of indolecarbazoles, indenoisoquinolinones, benzophenanthridines and dibenzonaphthyridones, mainly referring to Genz-644282 (CAS accession number 529488_28-6).

在本申请中，术语“有效量”通常是指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。In this application, the term "effective amount" generally refers to the amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation, which a clinician is able to determine.

术语“患者”包括哺乳动物和非哺乳动物。哺乳动物的实例包括但不限于哺乳动物纲的任何成员：人、非人灵长类如黑猩猩及其它猿类和猴类；农场动物如牛、马、绵羊、山羊、猪；家养动物如兔、狗和猫；实验室动物，包括啮齿类动物如大鼠、小鼠和豚鼠等，且包含未出生(in utero)的哺乳动物。非哺乳动物的实例包括但不限于鸟、鱼等。The term "patient" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs, etc., and include in utero mammals. Examples of non-mammals include, but are not limited to, birds, fish, etc.

术语“患者”包括确诊患者，但所述“患者”并不需要对医院、诊所或研究设备具有任何特殊的身份(如作为确诊的病人、研究参与者等)。The term "patient" includes confirmed patients, but the "patient" does not need to have any special identity with respect to the hospital, clinic or research facility (such as being a confirmed patient, research participant, etc.).

在本申请中，术语“疏水层析”通常是指一种基于的是物质疏水差异的分析技术。In this application, the term "hydrophobic chromatography" generally refers to an analytical technique based on differences in the hydrophobicity of substances.

在本申请中，术语“液相质谱”通常是指一种鉴定物质组分的分析方法。例如液相质谱可以通过液相色谱-质谱联用分析待测物质的分子量。In this application, the term "liquid phase mass spectrometry" generally refers to an analytical method for identifying the components of a substance. For example, liquid phase mass spectrometry can analyze the molecular weight of a substance to be tested by liquid chromatography-mass spectrometry.

具体实施方案Specific implementation plan

在一个方面，本发明提供了式(I)化合物，或其药学上可接受的盐或同位素变体：
In one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or isotopic variant thereof:

其中，in,

其中p＝1或2；Where p = 1 or 2;

q＝0、1或2；q = 0, 1 or 2;

s＝0、1或2；s = 0, 1, or 2;

其中，in,

s＝0、1或2；s = 0, 1, or 2;

其中p＝1或2；Where p = 1 or 2;

q＝0、1或2；q = 0, 1 or 2;

L₃选自以下结构：
_L3 is selected from the following structures:

t＝0、1、2、3或4；t = 0, 1, 2, 3 or 4;

R₆选自H、_R6 is selected from H,

其中，in,

A为靶向性分子；A is a targeting molecule;

x＝1、2、3、4、5、6、7或8；x = 1, 2, 3, 4, 5, 6, 7 or 8;

s＝0、1或2；s = 0, 1, or 2;

其中p＝1或2；Where p = 1 or 2;

q＝0、1或2；q = 0, 1 or 2;

L₃选自以下结构：
_L3 is selected from the following structures:

t＝0、1、2、3或4；t = 0, 1, 2, 3 or 4;

R₆选自H、_R6 is selected from H,

AA

在一个实施方式中，A为靶向性分子；在一个具体实施方式中，A选自蛋白、抗体、抗体片段、融合蛋白、多肽、酶和小分子；在一个具体实施方式中，A为蛋白；在另一个具体实施方式中，A为抗体，优选为单克隆抗体，例如单特异性单克隆抗体和双特异性单克隆抗体；在另一个具体实施方式中，A为靶向肿瘤相关抗原的IgG型抗体和HCAb型抗体；在一个具体实施方式中，A为抗体片段；在一个具体实施方式中，A为融合蛋白；在一个具体实施方式中，A为多肽；在一个具体实施方式中，A为酶；在一个具体实施方式中，A为小分子。In one embodiment, A is a targeting molecule; in a specific embodiment, A is selected from a protein, an antibody, an antibody fragment, a fusion protein, a polypeptide, an enzyme and a small molecule; in one embodiment, A is a protein; in another embodiment, A is an antibody, preferably a monoclonal antibody, such as a monospecific monoclonal antibody and a bispecific monoclonal antibody; in another embodiment, A is an IgG antibody and an HCAb antibody targeting a tumor-associated antigen; in one embodiment, A is an antibody fragment; in one embodiment, A is a fusion protein; in one embodiment, A is a polypeptide; in one embodiment, A is an enzyme; in one embodiment, A is a small molecule.

xx

在一个实施方式中，x＝1；在另一个实施方式中，x＝2；在另一个实施方式中，x＝3；在另一个实施方式中，x＝4；在另一个实施方式中，x＝5；在另一个实施方式中，x＝6；在另一个实施方式中，x＝7；在另一个实施方式中，x＝8；在另一个实施方式中，x＝2、3、4、5、6、7或8；在另一个实施方式中，x＝2、4、6或8；在另一个实施方式中，x＝4、6或8。In one embodiment, x=1; in another embodiment, x=2; in another embodiment, x=3; in another embodiment, x=4; in another embodiment, x=5; in another embodiment, x=6; in another embodiment, x=7; in another embodiment, x=8; in another embodiment, x=2, 3, 4, 5, 6, 7 or 8; in another embodiment, x=2, 4, 6 or 8; in another embodiment, x=4, 6 or 8.

R₃R₃

在一个实施方式中，R₃为H；在另一个实施方式中，R₃为D；在另一个实施方式中，R₃为卤素；在另一个实施方式中，R₃为C_1-6烷基；在另一个实施方式中，R₃为C_1-6卤代烷基。In one embodiment, R₃ is H; in another embodiment, R₃ is D; in another embodiment, R₃ is halogen; in another embodiment, R₃ is C_1-6 alkyl; in another embodiment, R₃ is C_1-6 haloalkyl.

ss

在一个实施方式中，s＝0；在另一个实施方式中，s＝1；在另一个实施方式中，s＝2。In one embodiment, s=0; in another embodiment, s=1; in another embodiment, s=2.

W₁W₁

在一个实施方式中，W₁为-O-；在另一个实施方式中，W₁为-S-；在另一个实施方式中，W₁为-NR_b-；在另一个实施方式中，W₁为-C(O)O-；在另一个实施方式中，W₁为-C(O)NR_b-；在另一个实施方式中，W₁为-O-C(O)-；在另一个实施方式中，W₁为-NR_b-C(O)-；在另一个实施方式中，W₁为-S(O)_pO-；在另一个实施方式中，W₁为-O-S(O)_p-。In one embodiment, W₁ is -O-; in another embodiment, W₁ is -S-; in another embodiment, W₁ is -NR_b -; in another embodiment, W₁ is -C(O)O-; in another embodiment, W₁ is -C(O)NR_b -; in another embodiment, W₁ is -OC(O)-; in another embodiment, W₁ is -NR_b -C(O)-; in another embodiment, W₁ is -S(O)_p O-; in another embodiment, W₁ is -OS(O)_p -.

在一个具体实施方式中，p＝1；在另一个具体实施方式中，p＝2。In one embodiment, p=1; in another embodiment, p=2.

L₁L₁

在一个实施方式中，L₁为化学键；在另一个实施方式中，L₁为-(CH₂)_m1-(OCH₂CH₂)_n1-(CH₂CH₂O)_n2-(CH₂)_r1-(L)_q-(CH₂)_r2-(OCH₂CH₂)_n3-(CH₂CH₂O)_n4-(CH₂)_m2-。In one embodiment,_L1 is a chemical bond; in another embodiment,_L1 is_- (_CH2 )_m1- (_OCH2CH2 )_n1- (_CH2CH2O )_n2- (_CH2 )_r1- (L)_q- (_CH2 )_r2- (_OCH2CH2₎_n3-₍ CH2CH2O)_n4-₍_CH2₎_m2- .

在另一个实施方式中，L₁为-(CH₂)_m1-(CH₂CH₂O)_n1-(CH₂)_r1-L-(CH₂)_r2-(CH₂CH₂O)_n2-(CH₂)_m2-；在另一个实施方式中，L₁为-(CH₂)_m3-(OCH₂CH₂)_n3-(CH₂)_r3-L-(CH₂)_r4-(OCH₂CH₂)_n4-(CH₂)_m4-。In another embodiment, L1_is -(_CH2 )_m1- (CH2CH2O)_n1- (_CH2 )_r1 -L-(_CH2₎_r2- (_CH2CH2O )_n2- (_CH2₎_m2- ; in another embodiment,_L1 is -(_CH2 )_m3- (_OCH2CH2 )_n3-₍_CH2 )_r3 -L-(_CH2 )_r4- (_OCH2CH2₎_n4- (_CH2₎_m4- .

在另一个实施方式中，L₁为-(CH₂CH₂O)_n5-(CH₂)_r5-L-(CH₂)_m5-；在另一个实施方式中，L₁为-(OCH₂CH₂)_n6-(CH₂)_r6-L-(CH₂)_m6-；在另一个实施方式中，L₁为-(CH₂)_m7-L-(CH₂CH₂O)_n7-(CH₂)_r7-；在另一个实施方式中，L₁为-(CH₂)_m8-L-(OCH₂CH₂)_n8-(CH₂)_r8-。In another embodiment, L₁ is -(CH₂ CH₂ O)_n5 -(CH₂ )_r5 -L-(CH₂ )_m5 -; in another embodiment, L₁ is -(OCH₂ CH₂ )_n6 -(CH₂ )_r6 -L-(CH₂ )_m6 -; in another embodiment, L₁ is -(CH₂ )_m7 -L-(CH₂ CH₂ O)_n7 -(CH₂ )_r7 -; in another embodiment, L₁ is -(CH₂ )_m8 -L-(OCH₂ CH₂ )_n8 -(CH₂ )_r8 -.

在另一个实施方式中，L₁为-(CH₂)_m9-(CH₂CH₂O)_n9-(CH₂)_r9-；在另一个实施方式中，L₁为-(CH₂)_m10-(OCH₂CH₂)_n10-(CH₂)_r10-。In another embodiment,_L1 is -(_CH2 )_m9- (_CH2CH2O )_n9- (_CH2 )_r9- ; in_another embodiment,_L1 is -(_CH2 )_m10-₍_OCH2CH2 )_n10- (_CH2 )_r10- .

在另一个实施方式中，L₁为-(CH₂CH₂O)_n11-(CH₂)_m11-；在另一个实施方式中，L₁为-(CH₂)_m12-(CH₂CH₂O)_n12-；在另一个实施方式中，L₁为-(OCH₂CH₂)_n13-(CH₂)_m13-；在另一个实施方式中，L₁为-(CH₂)_m14-(OCH₂CH₂)_n14-。In another embodiment, L₁ is -(CH₂ CH₂ O)_n11 -(CH₂ )_m11 -; in another embodiment, L₁ is -(CH₂ )_m12 -(CH₂ CH₂ O)_n12 -; in another embodiment, L₁ is -(OCH₂ CH₂ )_n13 -(CH₂ )_m13 -; in another embodiment, L₁ is -(CH₂ )_m14 -(OCH₂ CH₂ )_n14 -.

在另一个实施方式中，L₁为-(CH₂)_m15-O-(CH₂)_m16-；在另一个实施方式中，L₁为-(CH₂)_m17-O-、-(CH₂)_m18-NH-；在另一个实施方式中，L₁为-O-(CH₂)_m19-；在另一个实施方式中，L₁为-NR_b-(CH₂)_m20-；在另一个实施方式中，L₁为-(CH₂)_m21-；在另一个实施方式中，L₁为-L-；在另一个实施方式中，L₁为-(CH₂)_m22-L-；在另一个实施方式中，L₁为-L-(CH₂)_m23-。In another embodiment, L₁ is -(CH₂ )_m15 -O-(CH₂ )_m16 -; in another embodiment, L₁ is -(CH₂ )_m17 -O-, -(CH₂ )_m18 -NH-; in another embodiment, L₁ is -O-(CH₂ )_m19 -; in another embodiment, L₁ is -NR_b -(CH₂ )_m20 -; in another embodiment, L₁ is -(CH₂ )_m21 -; in another embodiment, L₁ is -L-; in another embodiment, L₁ is -(CH₂ )_m22 -L-; in another embodiment, L₁ is -L-(CH₂ )_m23 -.

在另一个实施方式中，L₁为-(CH₂CH₂O)_n11-(CH₂)_m11-，优选-(CH₂CH₂O)_n11-；在另一个实施方式中，L₁为-(CH₂)_m21-。In another embodiment, L₁ is -(CH₂ CH₂ O)_n11 -(CH₂ )_m11 -, preferably -(CH₂ CH₂ O)_n11 -; in another embodiment, L₁ is -(CH₂ )_m21 -.

在一个具体实施方式中，-L-为-O-；在另一个具体实施方式中，-L-为-NR_b-；在另一个具体实施方式中，-L-为-C(O)NR_b-；在另一个具体实施方式中，-L-为-C(O)O-；在另一个具体实施方式中，-L-为-NR_b-C(O)-；在另一个具体实施方式中，-L-为-O-C(O)-；在另一个具体实施方式中，-L-为-C_3-8亚环烷基-；在另一个具体实施方式中，-L-为-3-8元亚杂环基-；在另一个具体实施方式中，-L-为-C_6-10亚芳基-；在另一个具体实施方式中，-L-为-5-10元亚杂芳基。In one embodiment, -L- is -O-; in another embodiment, -L- is -NR_b -; in another embodiment, -L- is -C(O)NR_b -; in another embodiment, -L- is -C(O)O-; in another embodiment, -L- is -NR_b -C(O)-; in another embodiment, -L- is -OC(O)-; in another embodiment, -L- is -C_3-8 cycloalkylene-; in another embodiment, -L- is -3-8 membered heterocyclylene-; in another embodiment, -L- is -C_6-10 arylene-; in another embodiment, -L- is -5-10 membered heteroarylene.

在一个具体实施方式中，m＝0；在另一个具体实施方式中，m＝1；在另一个具体实施方式中，m＝2；在另一个具体实施方式中，m＝3；在另一个具体实施方式中，m＝4；在另一个具体实施方式中，m＝5；在另一个具体实施方式中，m＝6；在另一个具体实施方式中，m＝7；在另一个具体实施方式中，m＝8；在另一个具体实施方式中，m＝9；在另一个具体实施方式中，m＝10。In one embodiment, m=0; in another embodiment, m=1; in another embodiment, m=2; in another embodiment, m=3; in another embodiment, m=4; in another embodiment, m=5; in another embodiment, m=6; in another embodiment, m=7; in another embodiment, m=8; in another embodiment, m=9; in another embodiment, m=10.

在一个具体实施方式中，n＝0；在另一个具体实施方式中，n＝1；在另一个具体实施方式中，n＝2；在另一个具体实施方式中，n＝3；在另一个具体实施方式中，n＝4；在另一个具体实施方式中，n＝5；在另一个具体实施方式中，n＝6；在另一个具体实施方式中，n＝7；在另一个具体实施方式中，n＝8；在另一个具体实施方式中，n＝9；在另一个具体实施方式中，n＝10。In one embodiment, n=0; in another embodiment, n=1; in another embodiment, n=2; in another embodiment, n=3; in another embodiment, n=4; in another embodiment, n=5; in another embodiment, n=6; in another embodiment, n=7; in another embodiment, n=8; in another embodiment, n=9; in another embodiment, n=10.

在一个具体实施方式中，r＝0；在另一个具体实施方式中，r＝1；在另一个具体实施方式中，r＝2；在另一个具体实施方式中，r＝3；在另一个具体实施方式中，r＝4；在另一个具体实施方式中，r＝5；在另一个具体实施方式中，r＝6；在另一个具体实施方式中，r＝7；在另一个具体实施方式中，r＝8；在另一个具体实施方式中，r＝9；在另一个具体实施方式中，r＝10。In one embodiment, r=0; in another embodiment, r=1; in another embodiment, r=2; in another embodiment, r=3; in another embodiment, r=4; in another embodiment, r=5; in another embodiment, r=6; in another embodiment, r=7; in another embodiment, r=8; in another embodiment, r=9; in another embodiment, r=10.

在一个具体实施方式中，q＝0；在另一个具体实施方式中，q＝1；在另一个具体实施方式中，q＝2。In one embodiment, q=0; in another embodiment, q=1; in another embodiment, q=2.

在另一个具体实施方式中，其中m1、m2、m3、m4、m5、m6、m7、m8、m9、m10、m11、m12、m13、m14、m15、m16、m17、m18、m19、m20、m21、m22、m23各自独立地选自0、1、2、3、4、5、6、7、8、9或10；n1、n2、n3、n4、n5、n6、n7、n8、n9、n10、n11、n12、n13、n14各自独立地选自0、1、2、3、4、5、6、7、8、9或10；r1、r2、r3、r4、r5、r6、r7、r8、r9、r10各自独立地选自0、1、2、3、4、5、6、7、8、9或10。In another specific embodiment, wherein m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, m12, m13, m14, m15, m16, m17, m18, m19, m20, m21, m22, m23 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n1, n2, n3, n4, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; r1, r2, r3, r4, r5, r6, r7, r8, r9, r10 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

W₂W₂

在一个实施方式中，W₂为化学键；在另一个实施方式中，W₂为-O-；在另一个实施方式中，W₂为-S-；在另一个实施方式中，W₂为-NR_b-；在另一个实施方式中，W₂为-C(O)-。In one embodiment, W₂ is a chemical bond; in another embodiment, W₂ is -O-; in another embodiment, W₂ is -S-; in another embodiment, W₂ is -NR_b -; in another embodiment, W₂ is -C(O)-.

L₂L₂

在一个实施方式中，L₂选自以下氨基酸残基或2-10个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代；在另一个实施方式中，L₂选自以下氨基酸残基或2-8个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代；在另一个实施方式中，L₂选自以下氨基酸残基或2-6个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代；在另一个实施方式中，L₂选自以下氨基酸残基或2-5个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代；在另一个实施方式中，L₂选自以下氨基酸残基或2-4个以下氨基酸构成的寡肽残基，其中所述氨基酸选自半胱氨酸、苯丙氨酸、异亮氨酸、亮氨酸、色氨酸、缬氨酸、甲硫氨酸、酪氨酸、丙氨酸、苏氨酸、组氨酸、丝氨酸、谷氨酰胺、精氨酸、赖氨酸、天冬酰胺、谷氨酸、脯氨酸、瓜氨酸、天冬氨酸和甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In one embodiment,_L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-10 amino acids, wherein the amino acids are selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6_R4 ; in another embodiment,_L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-8 amino acids, wherein the amino acids are selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6_R4 ; in another embodiment, L In another embodiment,_L is selected from the following amino acid residues or an oligopeptide residue consisting of 2-6 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R₄ ; in another embodiment,_L is selected from the following amino acid residues or an oligopeptide residue consisting of 2-5 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6 R₄ ; in another embodiment, L_R4 is selected from the following amino acid residues or oligopeptide residues consisting of 2 to 4 amino acids, wherein the amino acid is selected from cysteine, phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6_R4 .

在一个具体实施方式中，L₂选自以下氨基酸残基或2-5个以下氨基酸构成的寡肽残基，其中所述氨基酸选自缬氨酸、瓜氨酸、丙氨酸、甘氨酸、苯丙氨酸、赖氨酸、精氨酸、天冬氨酸、谷氨酸和丝氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In a specific embodiment,_L2 is selected from the following amino acid residues or oligopeptide residues consisting of 2-5 amino acids, wherein the amino acid is selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acid is optionally substituted by 1, 2, 3, 4, 5 or 6_R4 .

在另一个具体实施方式中，L₂选自以下氨基酸构成的二肽、三肽或四肽残基，其中所述氨基酸选自缬氨酸、瓜氨酸、丙氨酸、甘氨酸、苯丙氨酸、赖氨酸、精氨酸、天冬氨酸、谷氨酸和丝氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In another specific embodiment,_L2 is selected from the dipeptide, tripeptide or tetrapeptide residue consisting of the following amino acids, wherein the amino acids are selected from valine, citrulline, alanine, glycine, phenylalanine, lysine, arginine, aspartic acid, glutamic acid and serine, and the amino acids are optionally substituted by 1, 2, 3, 4, 5 or 6_R4 .

在另一个具体实施方式中，L₂选自缬氨酸-瓜氨酸、缬氨酸-丙氨酸、缬氨酸-赖氨酸、苯丙氨酸-赖氨酸、赖氨酸-赖氨酸、丙氨酸-赖氨酸、苯丙氨酸-瓜氨酸、亮氨酸-瓜氨酸、异亮氨酸-瓜氨酸、苯丙氨酸-丙氨酸、赖氨酸-缬氨酸-瓜氨酸、赖氨酸-缬氨酸-丙氨酸、缬氨酸-赖氨酸-甘氨酸、甘氨酸-缬氨酸-赖氨酸、甘氨酸-缬氨酸-丙氨酸、谷氨酰胺-缬氨酸-丙氨酸、谷氨酰胺-缬氨酸-瓜氨酸、谷氨酸-缬氨酸-丙氨酸、谷氨酸-缬氨酸-瓜氨酸、丙氨酸-丙氨酸-丙氨酸、丙氨酸-丙氨酸-天冬酰胺、苯丙氨酸-苯丙氨酸-赖氨酸、甘氨酸-苯丙氨酸-赖氨酸、亮氨酸-丙氨酸-亮氨酸、异亮氨酸-丙氨酸-亮氨酸、缬氨酸-丙氨酸-缬氨酸、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸、丙氨酸-亮氨酸-丙氨酸-亮氨酸和甘氨酸-苯丙氨酸-亮氨酸-甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In another specific embodiment,_L2 is selected from valine-citrulline, valine-alanine, valine-lysine, phenylalanine-lysine, lysine-lysine, alanine-lysine, phenylalanine-citrulline, leucine-citrulline, isoleucine-citrulline, phenylalanine-alanine, lysine-valine-citrulline, lysine-valine-alanine, valine-lysine-glycine, glycine-valine-lysine, glycine-valine-alanine, glutamine-valine-alanine, glutamine-valine-citrulline, glutamate-valine R4-, glutamic acid-valine-citrulline, alanine-alanine-alanine, alanine-alanine-asparagine, phenylalanine-phenylalanine-lysine, glycine-phenylalanine-lysine, leucine-alanine-leucine, isoleucine-alanine-leucine, valine-alanine-valine, glycine-glycine-phenylalanine-glycine, alanine-leucine-alanine-leucine and glycine-phenylalanine-leucine-glycine, and the amino acids are optionally substituted with 1, 2, 3, 4, 5 or 6_R4 .

在另一个具体实施方式中，L₂选自谷氨酰胺-缬氨酸-丙氨酸、谷氨酰胺-缬氨酸-瓜氨酸、缬氨酸-瓜氨酸、缬氨酸-丙氨酸、缬氨酸-赖氨酸、赖氨酸-缬氨酸-瓜氨酸、赖氨酸-缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In another specific embodiment,_L2 is selected from glutamine-valine-alanine, glutamine-valine-citrulline, valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine, and the amino acid is optionally substituted with 1, 2, 3, 4, 5 or 6_R4 .

在另一个具体实施方式中，L₂选自缬氨酸-瓜氨酸、缬氨酸-丙氨酸、缬氨酸-赖氨酸、赖氨酸-缬氨酸-瓜氨酸、赖氨酸-缬氨酸-丙氨酸和甘氨酸-甘氨酸-苯丙氨酸-甘氨酸，并且所述氨基酸任选地被1、2、3、4、5或6个R₄取代。In another specific embodiment,_L2 is selected from valine-citrulline, valine-alanine, valine-lysine, lysine-valine-citrulline, lysine-valine-alanine and glycine-glycine-phenylalanine-glycine, and the amino acid is optionally substituted with 1, 2, 3, 4, 5 or 6_R4 .

R₄R₄

在一个实施方式中，R₄为D；在另一个实施方式中，R₄为卤素；在另一个实施方式中，R₄为NO₂；在另一个实施方式中，R₄为-OR_a；在另一个实施方式中，R₄为-NR_bR_c；在另一个实施方式中，R₄为C_1-6烷基；在另一个实施方式中，R₄为C_1-6卤代烷基；在另一个实施方式中，R₄为多聚乙二醇；在另一个实施方式中，R₄为多聚肌氨酸；在另一个实施方式中，R₄为五碳糖；在另一个实施方式中，R₄为六碳糖；在另一个实施方式中，R₄为磺酸基；在另一个实施方式中，R₄为甲磺酰基；在另一个实施方式中，R₄为磷酸基；在另一个实施方式中，R₄为亚磷酸基；在另一个实施方式中，R₄为季铵盐；在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为在另一个实施方式中，R₄为In one embodiment, R₄ is D; in another embodiment, R₄ is halogen; in another embodiment, R₄ is NO₂ ; in another embodiment, R₄ is -OR_a ; in another embodiment, R₄ is -NR_b R_c ; in another embodiment, R₄ is C_1-6 alkyl; in another embodiment, R₄ is C_1-6 haloalkyl; in another embodiment, R₄ is polyethylene glycol; in another embodiment, R₄ is polysarcosine; in another embodiment, R₄ is a pentose; in another embodiment, R₄ is a hexose; in another embodiment, R₄ is a sulfonic acid group; in another embodiment, R₄ is a mesyl group; in another embodiment, R₄ is a phosphate group; in another embodiment, R₄ is a phosphite group; in another embodiment, R₄ is a quaternary ammonium salt; in another embodiment, R₄ is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is In another embodiment,_R4 is

L₃L₃

在一个实施方式中，L₃为在另一个实施方式中，L₃为在另一个实施方式中，L₃为In one embodiment,_L3 is In another embodiment,_L3 is In another embodiment,_L3 is

R₅R₅

在一个实施方式中，R₅为H；在另一个实施方式中，R₅为D；在另一个实施方式中，R₅为卤素；在另一个实施方式中，R₅为NO₂；在另一个实施方式中，R₅为-OR_a；在另一个实施方式中，R₅为-NR_bR_c；在另一个实施方式中，R₅为C_1-6烷基；在另一个实施方式中，R₅为C_1-6卤代烷基。In one embodiment, R₅ is H; in another embodiment, R₅ is D; in another embodiment, R₅ is halogen; in another embodiment, R₅ is NO₂ ; in another embodiment, R₅ is -OR_a ; in another embodiment, R₅ is -NR_b R_c ; in another embodiment, R₅ is C_1-6 alkyl; in another embodiment, R₅ is C_1-6 haloalkyl.

tt

在一个实施方式中，t＝0；在另一个实施方式中，t＝1；在另一个实施方式中，t＝2；在另一个实施方式中，t＝3；在另一个实施方式中，t＝4。In one embodiment, t=0; in another embodiment, t=1; in another embodiment, t=2; in another embodiment, t=3; in another embodiment, t=4.

R₆R₆

在一个实施方式中，R₆为H；在另一个实施方式中，R₆为在另一个实施方式中，R₆为In one embodiment, R₆ is H; in another embodiment, R₆ is In another embodiment,_R6 is

DD

在一个实施方式中，D为活性化合物；在另一个实施方式中，D为药物；在另一个实施方式中，D为细胞毒素；在另一个实施方式中，D为检测试剂；在另一个实施方式中，D为诊断试剂；在另一个实施方式中，D为靶向载体。In one embodiment, D is an active compound; in another embodiment, D is a drug; in another embodiment, D is a cytotoxin; in another embodiment, D is a detection reagent; in another embodiment, D is a diagnostic reagent; in another embodiment, D is a targeting vector.

具体地，本发明涉及选自以下的连接子：
In particular, the present invention relates to a linker selected from the group consisting of:

具体地，本发明涉及选自下表的连接子-有效荷载：

In particular, the present invention relates to a linker-payload selected from the following table:

具体地，本发明涉及选自下表的ADC：

In particular, the present invention relates to an ADC selected from the following table:

其中，in,

表示单克隆抗体，优选为抗HER2和B7-H3的IgG1和HCAb型单克隆抗体。 It represents a monoclonal antibody, preferably an IgG1 and HCAb type monoclonal antibody against HER2 and B7-H3.

治疗方法Treatment

本申请所述的靶向性分子可以是蛋白类激素、凝集素、生长因子、抗体或其他能与细胞、受体和/或抗原结合的分子。例如，本申请的靶向性分子可以是抗HER2抗体、抗B7-H3的抗体、包括IgG型抗体和HCAb型抗体或其抗原结合片段。The targeting molecule described in the present application can be a protein hormone, a lectin, a growth factor, an antibody or other molecule that can bind to a cell, a receptor and/or an antigen. For example, the targeting molecule of the present application can be an anti-HER2 antibody, an anti-B7-H3 antibody, including an IgG antibody and an HCAb antibody or an antigen-binding fragment thereof.

由于本申请提供的抗体药物偶联物可以靶向瞄准特殊的细胞群体，与细胞表面特异性蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内。因此，本申请的抗体药物偶联物可以用于治疗疾病。本申请的抗体药物偶联物可以以治疗有效量，通过合适的途径给予受试者(例如人)。需要治疗的受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。Because the antibody drug conjugate provided by the present application can target a special cell population, bind to a cell surface specific protein (antigen), and thereby release the drug into the cell in an active form through conjugate endocytosis or drug infiltration. Therefore, the antibody drug conjugate of the present application can be used to treat diseases. The antibody drug conjugate of the present application can be administered to a subject (e.g., a human) in a therapeutically effective amount through a suitable route. The subject in need of treatment can be a patient who is at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such a patient can be identified by a routine physical examination.

当用本申请的抗体药物偶联物治疗时，可以通过本领域常规的方法进行递送。例如，它可以通过使用脂质体，水凝胶，环糊精，生物可降解的纳米胶囊，或生物粘附性微球被引入到细胞中。或者，所述核酸或载体可在本地通过直接注射或通过使用输注泵递送。其它方法可以包括通过使用缀合物和生物可降解的聚合物的使用各种运输和载体系统。When using the antibody drug conjugate of the present application for treatment, it can be delivered by methods conventional in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump. Other methods can include various transport and carrier systems by using conjugates and biodegradable polymers.

如本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用具体化合物的活性、患者的年龄、患者的体重、患者的健康状况、患者的行为、患者的饮食、给药时间、给药方式、排泄的速率、药物的组合等；另外，最佳的治疗方式如治疗的模式、本申请所述化合物或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或其混合物形式的日用量或可药用的盐的种类可以根据传统的治疗方案来验证。As is well known to those skilled in the art, the dosage of a drug depends on a variety of factors, including but not limited to the following factors: the activity of the specific compound used, the age of the patient, the weight of the patient, the health status of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal treatment method such as the mode of treatment, the daily dosage of the compound described in the present application or its pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesomorph, racemate or tautomer, or a mixture thereof, or the type of pharmaceutically acceptable salt can be verified according to traditional treatment regimens.

本申请化合物可以单独给药，或者可以与其他药学上可接受的治疗剂联合给药。使用药物组合物时，可以是将安全有效量的本申请化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量可以为药学上认为的有效给药剂量，具体剂量还可以考虑给药途径、病人健康状况等因素。The compounds of the present application can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents. When using a pharmaceutical composition, a safe and effective amount of the compounds of the present application can be applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration can be a pharmaceutically effective dosage, and the specific dosage can also take into account factors such as the route of administration and the patient's health status.

本申请提供一种本申请的化合物，或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或本申请的药物组合物在制备可以用于治疗和/或预防肿瘤的药物中的用途。例如，所述肿瘤可以选自与HER2、B7-H3等靶点表达相关的肿瘤。例如，与所述HER2、B7-H3等靶点表达相关的肿瘤包含所述靶点高表达的肿瘤和/或所述靶点阳性的肿瘤。例如，所述肿瘤包含实体肿瘤和/或血液肿瘤。例如，所述肿瘤包括乳腺癌、卵巢癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性淋巴细胞性白血病、间变性大细胞淋巴瘤、多发性骨髓瘤、前列腺癌、肺癌、恶性黑色素瘤、鳞状细胞癌、胶质母细胞瘤、肾细胞癌、胃肠道肿瘤、胰腺癌、前列腺癌、直结肠、胃癌、神经胶质瘤或者间皮瘤。The present application provides a compound of the present application, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or the use of the pharmaceutical composition of the present application in the preparation of a drug that can be used to treat and/or prevent tumors. For example, the tumor can be selected from tumors related to the expression of targets such as HER2 and B7-H3. For example, tumors related to the expression of targets such as HER2 and B7-H3 include tumors with high expression of the target and/or tumors with positive target. For example, the tumor includes solid tumors and/or blood tumors. For example, the tumor includes breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, rectum, gastric cancer, glioma or mesothelioma.

本申请提供一种化合物，或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或包含本申请的药物组合物，其可以用于治疗和/或预防肿瘤。例如，所述肿瘤可以选自与以下组靶点表达相关的肿瘤：HER2、B7-H3等。例如，所述与所述靶点表达相关的肿瘤包含所述靶点高表达的肿瘤和/或所述靶点阳性的肿瘤。例如，所述肿瘤包含实体肿瘤和/或血液肿瘤。例如，所述肿瘤选自以下组：乳腺癌、卵巢癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性淋巴细胞性白血病、间变性大细胞淋巴瘤、多发性骨髓瘤、前列腺癌、肺癌、恶性黑色素瘤、鳞状细胞癌、胶质母细胞瘤、肾细胞癌、胃肠道肿瘤、胰腺癌、前列腺癌、直结肠、胃癌、神经胶质瘤和间皮瘤。The present application provides a compound, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or a pharmaceutical composition comprising the present application, which can be used to treat and/or prevent tumors. For example, the tumor can be selected from tumors associated with the expression of the following groups of targets: HER2, B7-H3, etc. For example, the tumor associated with the expression of the target includes a tumor with high expression of the target and/or a tumor positive for the target. For example, the tumor includes a solid tumor and/or a blood tumor. For example, the tumor is selected from the following group: breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, rectum, gastric cancer, glioma and mesothelioma.

本申请提供一种预防和/或治疗肿瘤的方法，可以包括向受试者施用本申请的化合物，或其药学上可接受的盐、前药、水合物、溶剂合物、对映异构体、非对映异构体、内消旋体、外消旋体或互变异构体，或本申请的药物组合物。例如，所述肿瘤可以选自与以下组靶点表达相关的肿瘤：HER2、B7-H3等。例如，所述与所述靶点表达相关的肿瘤包含所述靶点高表达的肿瘤和/或所述靶点阳性的肿瘤。例如，所述肿瘤包含实体肿瘤和/或血液肿瘤。例如，所述肿瘤选自以下组：乳腺癌、卵巢癌、非霍奇金淋巴瘤、霍奇金淋巴瘤、急性淋巴细胞性白血病、间变性大细胞淋巴瘤、多发性骨髓瘤、前列腺癌、肺癌、恶性黑色素瘤、鳞状细胞癌、胶质母细胞瘤、肾细胞癌、胃肠道肿瘤、胰腺癌、前列腺癌、直结肠、胃癌、神经胶质瘤和间皮瘤。The present application provides a method for preventing and/or treating tumors, which may include administering to a subject a compound of the present application, or a pharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomer, diastereomer, mesoform, racemate or tautomer thereof, or a pharmaceutical composition of the present application. For example, the tumor may be selected from tumors associated with the expression of the following groups of targets: HER2, B7-H3, etc. For example, the tumor associated with the expression of the target comprises a tumor with high expression of the target and/or a tumor positive for the target. For example, the tumor comprises a solid tumor and/or a blood tumor. For example, the tumor is selected from the following groups: breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, rectum, gastric cancer, glioma and mesothelioma.

药物组合物Pharmaceutical composition

本申请所述的药物组合物除活性化合物外，可以含有一种或多种辅料，所述辅料可以选自以下组的成分：填充剂(稀释剂)、粘合剂、润湿剂、崩解剂和赋形剂等。根据给药方法的不同，组合物可以含有0.1至99重量％的活性化合物。The pharmaceutical composition described in the present application may contain one or more excipients in addition to the active compound, and the excipients may be selected from the following groups of ingredients: fillers (diluents), binders, wetting agents, disintegrants and excipients, etc. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

含活性成分的药物组合物可以是适用于口服的形式，例如片剂、糖锭剂、锭剂、水或油混悬液、可分散粉末或颗粒、乳液、硬或软胶囊，或糖浆剂。可以按照本领域任何己知制备药用组合物的方法制备口服组合物，所述组合物可以含有粘合剂、填充剂、润滑剂、崩解剂或药学上可接受的润湿剂等，所述组合物还可以含有一种或多种选自以下组的成分：甜味剂、矫味剂、着色剂和防腐剂。The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, lozenges, pastilles, water or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups. The oral composition may be prepared according to any method known in the art for preparing a pharmaceutical composition, and the composition may contain a binder, a filler, a lubricant, a disintegrant or a pharmaceutically acceptable wetting agent, etc. The composition may also contain one or more ingredients selected from the following group: sweeteners, flavoring agents, coloring agents and preservatives.

水悬浮液可以含有活性物质和用于混合的适宜制备的水悬浮液的赋形剂。水混悬液也可以含有一种或多种防腐剂，例如一种或多种着色剂、一种或多种矫味剂和一种或多种甜味剂。油混悬液可以通过使活性成分悬浮于植物油中配制而成。油悬浮液可以含有增稠剂。还可以加入上述的甜味剂和矫味剂。Aqueous suspensions may contain active substances and excipients for mixing suitable aqueous suspensions. Aqueous suspensions may also contain one or more preservatives, such as one or more coloring agents, one or more flavoring agents, and one or more sweeteners. Oil suspensions may be prepared by suspending the active ingredient in a vegetable oil. Oil suspensions may contain thickeners. The above-mentioned sweeteners and flavoring agents may also be added.

药物组合物还可以是用于制备水混悬液的可分散粉末和颗粒提供活性成分，通过加入水混合分散剂、湿润剂、悬浮剂或防腐剂中的一种或多种。也可加入其他赋形剂例如甜味剂、矫味剂和着色剂。通过加入抗氧化剂例如抗坏血酸保存这些组合物。本申请的药物组合物也可以是水包油乳剂的形式。The pharmaceutical composition can also be a dispersible powder and granules for preparing an aqueous suspension to provide the active ingredient, by adding water to mix one or more of a dispersant, a wetting agent, a suspending agent or a preservative. Other excipients such as sweeteners, flavoring agents and coloring agents can also be added. These compositions are preserved by adding antioxidants such as ascorbic acid. The pharmaceutical composition of the present application can also be in the form of an oil-in-water emulsion.

药物组合物可以是无菌注射水溶液形式。可以使用的可接受的溶媒或溶剂有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后可以将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，可以按可保持本申请化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。例如，所述装置可以是静脉注射泵。The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable solvents or solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution may then be added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion may be injected into the patient's bloodstream by local mass injection. Alternatively, the solution and microemulsion may be administered in a manner that maintains a constant circulating concentration of the compound of the present application. To maintain this constant concentration, a continuous intravenous drug delivery device may be used. For example, the device may be an intravenous injection pump.

药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按己知技术，用上述本申请所述适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在肠胃外可接受的无毒稀释剂或溶剂中制备的无菌注射溶液或混悬液。或者，可方便地用无菌固定油作为溶剂或悬浮介质。The pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents described in the present application as described above. Sterile injection preparations can also be sterile injection solutions or suspensions prepared in parenteral acceptable non-toxic diluents or solvents. Alternatively, sterile fixed oils can be conveniently used as solvents or suspension media.

可按用于直肠给药的栓剂形式给予本申请化合物。可通过将药物与在普通温度下为固体但在直肠中为液体，因而在直肠中会溶化而释放药物的适宜的无刺激性赋形剂混合来制备这些药物组合物。此类物质包括可可脂、甘油明胶、氢化植物油、各种分子量的聚乙二醇和聚乙二醇的脂肪酸酯的混合物。The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and which will dissolve in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and mixtures of fatty acid esters of polyethylene glycol.

本申请的药物组合物可以含有安全有效量的本申请的抗体药物偶联物以及药学上可接受的载体。这类载体可以包括(但并不限于)盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本申请的药物组合物可以被制成溶液剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物可以在无菌条件下制造。活性成分的给药量可以是治疗有效量。The pharmaceutical composition of the present application may contain a safe and effective amount of the antibody drug conjugate of the present application and a pharmaceutically acceptable carrier. Such carriers may include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the mode of administration, and the pharmaceutical composition of the present application may be prepared in the form of a solution, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition may be manufactured under aseptic conditions. The dosage of the active ingredient may be a therapeutically effective amount.

本申请所述的抗体药物偶联物的有效量可以随给药的模式和待治疗的疾病的严重程度等而变化。有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素可以包括但不限于：所述的双功能抗体偶联物的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本申请的抗体药物偶联物每天以合适的剂量给予，可以得到令人满意的效果。例如，由治疗状况的迫切要求，可以每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the antibody drug conjugate described in the present application may vary with the mode of administration and the severity of the disease to be treated. The selection of the effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors may include, but are not limited to: pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the antibody drug conjugate of the present application is administered at an appropriate dose every day, a satisfactory effect can be obtained. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

实施例Example

以下由特定的具体实施例说明本申请的具体实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的化合物、制备方法和用途等，而不用于限制本申请发明的范围。The specific embodiments of the present invention are described below by specific specific examples. People familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Without being limited by any theory, the following examples are only for illustrating the compounds, preparation methods and uses of the present invention, and are not used to limit the scope of the present invention.

本文中用到的缩略语的定义如下：The definitions of the abbreviations used in this document are as follows:

ADC(antibody-drug conjugate)：抗体偶联药物；ADC (antibody-drug conjugate): antibody-drug conjugate;

Ala(Alanine)：丙氨酸；Ala(Alanine): alanine;

Boc(t-Butyloxy carbonyl)：叔丁氧羰基；Boc (t-Butyloxy carbonyl): tert-Butyloxycarbonyl;

Cit(Citrulline)：瓜氨酸；Cit(Citrulline): Citrulline;

CD276(Cluster of Differentiation 276)：B7-H3；CD276 (Cluster of Differentiation 276): B7-H3;

DAR(Drug to antibody ratio)：抗体药物摩尔比；DAR (Drug to antibody ratio): antibody-drug molar ratio;

DCC(Dicyclohexylcarbodiimide)：二环己基碳二亚胺；DCC (Dicyclohexylcarbodiimide): Dicyclohexylcarbodiimide;

DCM(Dichloromethane)：二氯甲烷；DCM (Dichloromethane): dichloromethane;

DIPEA(N,N-Diisopropylethylamine)：N,N-二异丙基乙胺；DIPEA(N,N-Diisopropylethylamine): N,N-diisopropylethylamine;

DMAC(Dimethylacetamide)：N,N-二甲基乙酰胺；DMAC (Dimethylacetamide): N,N-dimethylacetamide;

DMF(N,N-Dimethylformamide)：N,N-二甲基甲酰胺；DMF (N,N-Dimethylformamide): N,N-dimethylformamide;

DMSO(Dimethyl Sulphoxide)：二甲基亚砜；DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide;

DX-8951f(Exatecan Mesylate)：依喜替康甲磺酸盐；DX-8951f (Exatecan Mesylate): Exatecan mesylate;

EA(ethyl acetate)：乙酸乙酯；EA (ethyl acetate): ethyl acetate;

EEDQ(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline)：N-乙氧酰基-2-乙氧基-1,2-二氢喹啉；EEDQ (N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline): N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline;

Fmoc(Fluorenylmethyloxycarbonyl)：芴甲氧羰基；Fmoc (Fluorenylmethyloxycarbonyl): fluorenylmethyloxycarbonyl;

HATU：N,N,N′,N′-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲HATU: N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate

HER2(Human epidermal growth factor receptor 2)：人表皮生长因子受体2；HER2 (Human epidermal growth factor receptor 2): human epidermal growth factor receptor 2;

HIC(Hydrophobic Interaction Chromatography)：疏水作用色谱；HIC (Hydrophobic Interaction Chromatography): Hydrophobic interaction chromatography;

His(Histidine)：组氨酸；His(Histidine): histidine;

HOBt(N-Hydroxybenzotrizole)：1-羟基苯并三唑；HOBt (N-Hydroxybenzotrizole): 1-hydroxybenzotriazole;

m-CPBA(m-Chloroperbenzoic Acid)：间氯过氧苯甲酸；m-CPBA (m-Chloroperbenzoic Acid): m-chloroperbenzoic acid;

mM(Millimoles)：毫摩尔；mM (Millimoles): millimole;

Me(methyl)：甲基Me(methyl): methyl

mAB(Monoclonal Antibody)：单克隆抗体；mAB (Monoclonal Antibody): monoclonal antibody;

MMAE(Monomethyl auristatin E)：单甲基奥里斯他汀E；MMAE (Monomethyl auristatin E): Monomethyl auristatin E;

MMAF(Monomethyl auristatin F)：单甲基奥里斯他汀F；MMAF (Monomethyl auristatin F): Monomethyl auristatin F;

NHS(N-Hydroxysuccinimide)：N-羟基琥珀酰亚胺；NHS(N-Hydroxysuccinimide): N-hydroxysuccinimide;

NAC(N-Acetyl-L-cysteine)：N-乙酰-L-半胱氨酸；NAC(N-Acetyl-L-cysteine): N-acetyl-L-cysteine;

NPC(Bis(4-nitrophenyl)carbonate)：二(对硝基苯基)碳酸酯NPC(Bis(4-nitrophenyl)carbonate): Bis(4-nitrophenyl)carbonate

PAB(p-Aminobenzyl alcohol)：对氨基苄醇；PAB (p-Aminobenzyl alcohol): p-aminobenzyl alcohol;

Su/SuOH(Succinimide)：琥珀酰亚胺；Su/SuOH(Succinimide): succinimide;

TCEP(Tris(2-carboxyethyl)phosphine)：三(2-羧乙基)膦；TCEP (Tris(2-carboxyethyl)phosphine): tris(2-carboxyethyl)phosphine;

TFA(Trifluoroacetic acid)：三氟乙酸；TFA (Trifluoroacetic acid): trifluoroacetic acid;

THF(Tetrahydrofuran)：四氢呋喃；THF (Tetrahydrofuran): Tetrahydrofuran;

VA(Valine-Alanine，Val-Ala)：缬氨酸-丙氨酸二肽；VA (Valine-Alanine, Val-Ala): Valine-Alanine dipeptide;

Val(Valine)：缬氨酸；Val(Valine): valine;

VC(Valine-Citrulline，Val-Cit)：缬氨酸-瓜氨酸二肽；VC (Valine-Citrulline, Val-Cit): Valine-Citrulline dipeptide;

DCU：二环己基脲；DCU: dicyclohexylurea;

EDCI：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

TEA：三乙胺；TEA: triethylamine;

Lys(Lysine)：赖氨酸；Lys(Lysine): lysine;

Gly(Glycine)：甘氨酸；Gly(Glycine): glycine;

Thr(Threonine)：苏氨酸；Thr(Threonine): threonine;

Leu(Leucine)：亮氨酸；Leu(Leucine): Leucine;

Ile(Isoleucine)：异亮氨酸；Ile(Isoleucine): Isoleucine;

Asn(Asparagine)：天冬氨酸；Asn(Asparagine): Aspartic acid;

Phe(Phenylalanine)苯丙氨酸。Phe (Phenylalanine) Phenylalanine.

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 the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

实施例1：化合物-1的制备
Example 1: Preparation of Compound-1

具体合成操作如下所述。The specific synthesis operation is as follows.

步骤1-1：M1的制备Step 1-1: Preparation of M1

将Fmoc-Ala-NHS(25g，57.28mM)溶于DME/THF混合溶液中(2:1，300mL)，全部溶解后加入瓜氨酸(Cit；13g，74.46mM)的NaHCO₃溶液，室温下搅拌反应20小时。LC/MS监测化合物Fmoc-Ala-NHS反应完全，M1为主产物。反应完成后，在搅拌状态下将反应液倒入15％的柠檬酸水溶液中，析出果冻状不溶物。过滤，将滤饼充分抽干，进一步烘干后采用乙醚打浆两次，得到产物M1为白色固体粉末，27.88g，收率约为98％。¹H NMR(400MHz,DMSO-d6):δ0.89(dd,6H),1.42(m,2H),1.58(m,1H),1.72(m,1H),1.98(m,1H),2.96(q,2H),3.94(t,1H),4.16(q,1H),4.25(m,3H),5.43(s,2H),5.98(t,1H),7.33(t,2H),7.43(q,3H),7.76(t,2H),7.89(d,2H),8.21(d,1H),12.61(s,1H).MS(EI)m/z:497.52[M+H]⁺；495.62[M-H]^-.Fmoc-Ala-NHS (25g, 57.28mM) was dissolved in a DME/THF mixed solution (2:1, 300mL). After all the solution was dissolved, a NaHCO₃ solution of citrulline (Cit; 13g, 74.46mM) was added, and the reaction was stirred at room temperature for 20 hours. LC/MS monitored that the compound Fmoc-Ala-NHS reacted completely, and M1 was the main product. After the reaction was completed, the reaction solution was poured into a 15% citric acid aqueous solution under stirring, and a jelly-like insoluble substance was precipitated. Filter, fully drain the filter cake, further dry it, and use ether to beat it twice to obtain the product M1 as a white solid powder, 27.88g, with a yield of about 98%.^1H NMR (400MHz, DMSO-d6): δ0.89(dd,6H),1.42(m,2H),1.58(m,1H),1.72(m,1H),1.98(m,1H),2.96(q,2H),3.94(t,1H),4.16(q,1H),4.25( m,3H),5.43(s,2H),5.98(t,1H),7.33(t,2H),7.43(q,3H),7.76(t,2H),7.89(d,2H),8.21(d,1H),12.61(s,1H).MS(EI)m/z:497.52[M+H]⁺ ;495.62[MH]^- .

步骤1-2：M2的制备Step 1-2: Preparation of M2

将化合物M1(15g，30.2mM；1eq)溶于DCM/MeOH混合溶液中(2:1，450mL)，全部溶解后加入对氨基苄醇(PAB；7.44g，60.4mM)和EEDQ(14.94g，60.4mM)；室温下避光搅拌反应超过36小时。反应完成后，减压浓缩去除溶剂，所得残渣采用乙醚打浆3次，得到产物M2为白色固体粉末，17g，收率约为95％。¹H NMR(400MHz,DMSO-d6):δ0.86(dd,6H),1.41(m,2H),1.57(m,1H),1.69(m,1H),1.98(m,1H),3.00(m,2H),3.92(q,1H),4.23(q,2H),4.30(q,1H),4.41(q,3H),5.12(t,1H),5.42(s,2H),5.98(t,1H),7.23(d,2H),7.32(m,2H),7.41(m,2H),7.46(d,1H),7.54(d,2H),7.74(t,2H),7.89(d,2H),8.12(d,1H),9.99(s,1H).MS(EI)m/z:602.67[M+H]⁺；624.48[M+Na]⁺.Compound M1 (15 g, 30.2 mM; 1 eq) was dissolved in a DCM/MeOH mixed solution (2:1, 450 mL). After all the solution was dissolved, p-aminobenzyl alcohol (PAB; 7.44 g, 60.4 mM) and EEDQ (14.94 g, 60.4 mM) were added. The mixture was stirred at room temperature in the dark for more than³⁶ hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was slurried with ether for 3 times to obtain product M2 as a white solid powder, 17 g, with a yield of about 95%. NMR (400MHz, DMSO-d6): δ0.86(dd,6H),1.41(m,2H),1.57(m,1H),1.69(m,1H),1.98( m,1H),3.00(m,2H),3.92(q,1H),4.23(q,2H),4.30(q,1H),4.41(q,3H),5.12(t,1H) ,5.42(s,2H),5.98(t,1H),7.23(d,2H),7.32(m,2H),7.41(m,2H),7.46(d,1H),7.54 (d,2H),7.74(t,2H),7.89(d,2H),8.12(d,1H),9.99(s,1H).MS(EI)m/z:602.67[M+H]⁺ ;624.48[M+Na]⁺ .

步骤1-3：M3的制备Steps 1-3: Preparation of M3

将化合物M2(1.5g，2.49mM)溶解于DMF中(40mL)，全部溶解后加入对硝基苯酚碳酸酯(NPC；1.52g，4.99mM)和DIPEA(0.48g，3.74mM)。室温下搅拌反应过夜，待应完成后，减压浓缩去除溶剂，所得残渣采用乙醚打浆3次。产物M3为白色固体粉末1.2g，收率约为68％。所得物料不经进一步纯化而直接进行下一步反应。MS(EI)m/z:767.05[M+H]⁺；789.26[M+Na]⁺.Compound M2 (1.5 g, 2.49 mM) was dissolved in DMF (40 mL). After all the solution was dissolved, p-nitrophenol carbonate (NPC; 1.52 g, 4.99 mM) and DIPEA (0.48 g, 3.74 mM) were added. The reaction was stirred overnight at room temperature. After completion, the solvent was removed by concentration under reduced pressure. The residue was slurried with ether three times. The product M3 was 1.2 g of white solid powder with a yield of about 68%. The obtained material was directly used for the next step reaction without further purification. MS (EI) m/z: 767.05 [M + H]⁺ ; 789.26 [M + Na]⁺ .

步骤1-4：M4的制备Steps 1-4: Preparation of M4

将化合物M3(128mg，0.167mM)溶解于DMF中(40mL)，全部溶解后加入MMAE(100mg，0.139mM)、HOBt(18.82mg，0.139)和DIPEA(36mg，0.279mM)。室温下搅拌反应过夜，待应完成后，减压浓缩去除溶剂，所得残渣采用乙酸乙酯(EA)打浆3次。过滤后得白色固体粉末产物M4共150mg，收率约为80％。MS(EI)m/z:673.80[M+2H]²⁺.Compound M3 (128 mg, 0.167 mM) was dissolved in DMF (40 mL). After all the solution was dissolved, MMAE (100 mg, 0.139 mM), HOBt (18.82 mg, 0.139) and DIPEA (36 mg, 0.279 mM) were added. The reaction was stirred overnight at room temperature. After completion, the solvent was removed by concentration under reduced pressure. The residue was slurried 3 times with ethyl acetate (EA). After filtration, a total of 150 mg of white solid powder product M4 was obtained with a yield of about 80%. MS (EI) m/z: 673.80 [M + 2H]^{2 +} .

步骤1-5：M5的制备Steps 1-5: Preparation of M5

将化合物M4(135mg，0.1mM)溶解于DMF中(20mL)，加入哌啶(1mL)，室温下搅拌反应2小时，LC/MS检测原料消失。减压浓缩去除溶剂，所得残渣采用乙醚打浆并过滤。滤饼采用柱层析进一步纯化得产物M5为淡黄色固体粉末，83mg，收率约为74％。MS(EI)m/z:1123.68[M+H]⁺；MS(EI)m/z:562.41[M+2H]²⁺.Compound M4 (135 mg, 0.1 mM) was dissolved in DMF (20 mL), piperidine (1 mL) was added, and the reaction was stirred at room temperature for 2 hours. The raw material disappeared after LC/MS detection. The solvent was removed by concentration under reduced pressure, and the obtained residue was slurried with ether and filtered. The filter cake was further purified by column chromatography to obtain product M5 as a light yellow solid powder, 83 mg, with a yield of about 74%. MS (EI) m/z: 1123.68 [M + H]⁺ ; MS (EI) m/z: 562.41 [M + 2H]²⁺ .

步骤1-6：M6的制备Steps 1-6: Preparation of M6

将5-氯-4硝基-2吡啶甲酸甲酯(5g，23mM)、硫脲(5g，2.8eq)溶解于环丁砜(30mL)中，加热至130度搅拌反应2～4小时。待反应完成后，将反应液静置缓慢降温，并直接采用柱层析纯化得物M6为橙黄色固体粉末，3.68g，收率约为77％。¹H NMR(400MHz,DMSO-d₆)δ8.26(s,2H),7.95(d,J＝8.3Hz,1H),7.68(d,J＝8.3Hz,1H),3.86(s,3H).MS(EI)m/z:209.29[M+H]⁺.Dissolve 5-chloro-4-nitro-2-picolinic acid methyl ester (5g, 23mM) and thiourea (5g, 2.8eq) in sulfolane (30mL), heat to 130 degrees and stir to react for 2 to 4 hours. After the reaction is completed, the reaction solution is allowed to stand and slowly cool down, and directly purified by column chromatography to obtain M6 as an orange-yellow solid powder, 3.68g, with a yield of about 77%.¹ H NMR (400MHz, DMSO-d₆ )δ8.26(s,2H),7.95(d,J＝8.3Hz,1H),7.68(d,J＝8.3Hz,1H),3.86(s,3H).MS(EI)m/z:209.29[M+H]⁺ .

步骤1-7：M7的制备Steps 1-7: Preparation of M7

将M6(3.77g，18mM)分散于乙腈中，室温搅拌下加入溴化铜(8.4g，36mM)，全部溶解后并分批滴入亚硝酸叔丁酯(3.71g，36mM)。加毕，室温搅拌反应约5小时。待反应完成后，减压浓缩去除乙腈，残渣加水复溶，并采用乙酸乙酯萃取2次，有机相采用水和饱和NaCl溶液分别洗涤2次，再经Na₂SO₄干燥后，浓缩得黄色固体残渣。粗产品进一步经硅胶柱层析纯化得产物M7为白色固体粉末，4.03g，收率约为82％。¹H NMR(400MHz,DMSO-d₆)δ8.57(d,J＝8.5Hz,1H),8.24(d,J＝8.5Hz,1H),3.94(s,3H).MS(EI)m/z:272.74/274.84[M+H]⁺.M6 (3.77 g, 18 mM) was dispersed in acetonitrile, and copper bromide (8.4 g, 36 mM) was added under stirring at room temperature. After all the solution was dissolved, tert-butyl nitrite (3.71 g, 36 mM) was added dropwise in batches. After the addition was completed, the reaction was stirred at room temperature for about 5 hours. After the reaction was completed, the acetonitrile was removed by concentration under reduced pressure, the residue was redissolved in water, and extracted twice with ethyl acetate. The organic phase was washed twice with water and saturated NaCl solution, respectively, and then dried over Na₂ SO₄ , and concentrated to obtain a yellow solid residue. The crude product was further purified by silica gel column chromatography to obtain the product M7 as a white solid powder, 4.03 g, with a yield of about 82%.¹ H NMR (400MHz, DMSO-d₆ ) δ8.57 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 3.94 (s, 3H). MS (EI) m/z: 272.74/274.84 [M + H]⁺ .

步骤1-8：M8的制备Step 1-8: Preparation of M8

将M7(2.05g，7.47mM)溶解于四氢呋喃(THF，100mL)中，室温搅拌下加入LiOH一水化合物(313mg，7.47mM)，再加入水约20mL，反应约10分钟。LC/MS监测化合物M7反应完全，化合物M8为主产物。减压浓缩去除溶剂，残渣加水复溶，采用稀盐酸调pH到3～4，悬浊液静置15分钟后过滤，滤饼采用蒸馏水洗涤2～3次，充分干燥得产物M8为淡黄色固体粉末，1.8g，收率约为93％。¹H NMR(400MHz,DMSO-d₆)δ13.61(s,1H),8.55(d,J＝8.4Hz,1H),8.23(d,J＝8.5Hz,1H).MS(EI)m/z:258.83/260.73[M+H]⁺.M7 (2.05 g, 7.47 mM) was dissolved in tetrahydrofuran (THF, 100 mL), and LiOH monohydrate (313 mg, 7.47 mM) was added under stirring at room temperature, and then about 20 mL of water was added, and the reaction was carried out for about 10 minutes. LC/MS monitored that the reaction of compound M7 was complete, and compound M8 was the main product. The solvent was removed by concentration under reduced pressure, and the residue was redissolved in water, and the pH was adjusted to 3-4 with dilute hydrochloric acid. The suspension was allowed to stand for 15 minutes and then filtered. The filter cake was washed with distilled water 2-3 times and fully dried to obtain the product M8 as a light yellow solid powder, 1.8 g, with a yield of about 93%.¹ H NMR (400MHz, DMSO-d₆ ) δ13.61 (s, 1H), 8.55 (d, J = 8.4Hz, 1H), 8.23 (d, J = 8.5Hz, 1H). MS (EI) m/z: 258.83/260.73 [M+H]⁺ .

步骤1-9：M9的制备Step 1-9: Preparation of M9

将M8(1.80g，6.95mM)溶解于四氢呋喃和MeOH的混合溶剂(1:1，80mL)中，室温搅拌下加入甲硫醇钠的水溶液(20％，0.974g，13.9mM)，加毕在室温下搅拌反应约10分钟。LC/MS监测化合物M8反应完全，化合物M9为主产物。减压浓缩去除溶剂，残渣加水复溶，先采用稀的NaOH溶液调pH～10，超声使残渣充分溶解后，再采用稀盐酸调pH到3～4，悬浊液静置15分钟后过滤，滤饼采用蒸馏水洗涤2～3次，充分干燥得产物M9为淡黄色固体粉末，1.5g，收率约为95％。¹H NMR(400MHz,DMSO-d₆)δ13.26(s,1H),8.31(d,J＝8.4Hz,1H),8.16(d,J＝8.4Hz,1H),2.85(s,3H).MS(EI)m/z:226.90[M+H]⁺；MS(EI)m/z:224.78[M-H]^-.M8 (1.80 g, 6.95 mM) was dissolved in a mixed solvent of tetrahydrofuran and MeOH (1:1, 80 mL), and an aqueous solution of sodium thiomethoxide (20%, 0.974 g, 13.9 mM) was added under stirring at room temperature. After addition, the mixture was stirred at room temperature for about 10 minutes. LC/MS monitored that the reaction of compound M8 was complete, and compound M9 was the main product. The solvent was removed by concentration under reduced pressure, and the residue was redissolved in water. The pH was first adjusted to 10 with a dilute NaOH solution, and the residue was fully dissolved by ultrasound. Then, the pH was adjusted to 3-4 with dilute hydrochloric acid. The suspension was allowed to stand for 15 minutes and then filtered. The filter cake was washed 2-3 times with distilled water and fully dried to obtain the product M9 as a light yellow solid powder, 1.5 g, with a yield of about 95%.¹ H NMR (400MHz, DMSO-d₆ ) δ13.26 (s, 1H), 8.31 (d, J = 8.4Hz, 1H), 8.16 (d, J = 8.4Hz, 1H), 2.85 (s, 3H). MS (EI) m/z: 226.90 [M + H]⁺ ; MS (EI) m/z: 224.78 [MH]^- .

步骤1-10：M10的制备Steps 1-10: Preparation of M10

将M9(1.3g，5.74mM)溶解于DMF中(30mL)，全部溶解后分别加入NHS(1.32g，11.49mM)，DCC(1.78g，8.62mM)和DIPEA(cat.,0.2mL)。室温搅拌下反应过夜。LC/MS监测化合物M9反应完全，化合物M10为主产物。过滤去除不溶物DCU，减压浓缩去除溶剂，所得残渣直接采用粗硅胶拌样，硅胶柱层析纯化，得产物M10为类白色固体粉末，1.78g，收率约为96％。¹H NMR(400MHz,DMSO-d₆)δ8.43(d,J＝8.5Hz,1H),8.33(d,J＝8.5Hz,1H),2.93(s,4H),2.88(s,3H).MS(EI)m/z:323.98[M+H]⁺.M9 (1.3 g, 5.74 mM) was dissolved in DMF (30 mL). After all the solution was dissolved, NHS (1.32 g, 11.49 mM), DCC (1.78 g, 8.62 mM) and DIPEA (cat., 0.2 mL) were added respectively. The mixture was stirred at room temperature overnight. LC/MS monitored that the reaction of compound M9 was complete, and compound M10 was the main product. The insoluble matter DCU was removed by filtration, and the solvent was removed by concentration under reduced pressure. The residue was directly mixed with crude silica gel and purified by silica gel column chromatography to obtain product M10 as an off-white solid powder, 1.78 g, with a yield of about 96%.¹ H NMR (400MHz, DMSO-d₆ ) δ8.43 (d, J = 8.5 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H), 2.93 (s, 4H), 2.88 (s, 3H). MS (EI) m/z: 323.98 [M + H]⁺ .

步骤1-11：M11的制备Steps 1-11: Preparation of M11

将M10(1.0g，3.1mM)溶解于DMF中(30mL)，全部溶解后分别加入6-氨基己酸(0.61g，4.65mM)和催化量的DIPEA。室温搅拌下反应过夜，待反应完成后，减压浓缩去除溶剂得淡黄色固体残渣，经硅胶柱色谱层析纯化得产物M11为白色固体粉末，916mg，收率约为87％。¹H NMR(500MHz,DMSO-d6):δ12.01(br,1H),8.16(d,J＝7.7Hz,1H),8.06(d,J＝7.5Hz,1H),8.01(t,1H),3.35(q,J＝5.4Hz,2H),2.77(s,3H),2.26(t,J＝8.9Hz,2H),1.62-1.56(m,2H),1.59-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m/z:340.06[M+H]⁺；MS(EI)m/z:338.08[M-H]^-.M10 (1.0 g, 3.1 mM) was dissolved in DMF (30 mL), and 6-aminocaproic acid (0.61 g, 4.65 mM) and a catalytic amount of DIPEA were added after all the solution was dissolved. The mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a light yellow solid residue, which was purified by silica gel column chromatography to obtain the product M11 as a white solid powder, 916 mg, with a yield of about 87%.¹ H NMR (500MHz, DMSO-d6): δ12.01(br,1H),8.16(d,J＝7.7Hz,1H),8.06(d,J＝7.5Hz,1H),8.01(t,1H),3.35(q,J＝5.4Hz,2H ),2.77(s,3H),2.26(t,J＝8.9Hz,2H),1.62-1.56(m,2H),1.59-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m/z:340.06[M+H]⁺ ;MS(EI)m/z:338.08[MH]^- .

步骤1-12：M12的制备Steps 1-12: Preparation of M12

将M11(500mg，1.47mM)溶解于DCM中(30mL)，全部溶解后加入间氯过氧苯甲酸(m-CPBA，85％；1.0g，5.89mM)。室温搅拌下反应过夜。LC/MS监测化合物M11反应完全，化合物M12为主产物。减压浓缩去除溶剂，所得残渣采用粗硅胶拌样，经硅胶柱层析纯化得产物M12为白色固体粉末，509mg，收率约为93％。¹H NMR(500MHz,DMSO-d6)δ8.21(d,J＝7.5Hz,1H),8.09(d,J＝7.5Hz,1H),8.01(t,1H),3.48(s,3H),3.35(q,J＝5.4Hz,2H),2.26(t,J＝8.9Hz,2H),1.61-1.55(m,2H),1.58-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m/z:372.08[M+H]⁺；MS(EI)m/z:370.05[M-H]^-.Dissolve M11 (500 mg, 1.47 mM) in DCM (30 mL), add m-chloroperbenzoic acid (m-CPBA, 85%; 1.0 g, 5.89 mM) after all dissolved. Stir at room temperature and react overnight. LC/MS monitored that compound M11 reacted completely, and compound M12 was the main product. Concentrate under reduced pressure to remove the solvent, and the residue was mixed with crude silica gel and purified by silica gel column chromatography to obtain product M12 as a white solid powder, 509 mg, with a yield of about 93%.¹ H NMR (500MHz, DMSO-d6) δ8.21(d,J＝7.5Hz,1H),8.09(d,J＝7.5Hz,1H),8.01(t,1H),3.48(s,3H),3.35(q,J＝5.4H z,2H),2.26(t,J＝8.9Hz,2H),1.61-1.55(m,2H),1.58-1.51(m,2H),1.42-1.32(m,2H).MS(EI)m/z:372.08[M+H]⁺ ;MS(EI)m/z:370.05[MH]^- .

步骤1-13：化合物-1的制备Step 1-13: Preparation of Compound-1

将M5(50mg，0.0445mM)和M12(20mg，0.0534mM)溶解于DMF(10mL)中，搅拌使全部溶解后，将反应体系转移至冰浴中进行降温。之后，向反应溶液中加入HATU(25.4mg，0.668mM)和催化量的DIPEA。加毕在冰浴中搅拌反应30分钟后，逐渐恢复至室温，并在室温下搅拌反应过夜。待反应完成后，减压浓缩去除溶剂，所得残渣直接采用粗硅胶拌样，经硅胶柱层析纯化得化合物-1为白色固体，进一步冻干变为蓬松状白色固体36.8mg，收率约为56％。MS(EI)m/z:738.38[M+2H]²⁺；MS(ESI)m/z:1476.7643[M+H]⁺.M5 (50 mg, 0.0445 mM) and M12 (20 mg, 0.0534 mM) were dissolved in DMF (10 mL), stirred until completely dissolved, and then the reaction system was transferred to an ice bath for cooling. After that, HATU (25.4 mg, 0.668 mM) and a catalytic amount of DIPEA were added to the reaction solution. After the addition was completed, the reaction was stirred in an ice bath for 30 minutes, then gradually returned to room temperature, and stirred at room temperature overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the resulting residue was directly mixed with crude silica gel and purified by silica gel column chromatography to obtain compound-1 as a white solid, which was further freeze-dried to become a fluffy white solid of 36.8 mg, with a yield of about 56%. MS (EI) m/z: 738.38 [M + 2H]^{2 +} ; MS (ESI) m/z: 1476.7643 [M + H]⁺ .

实施例2：化合物-2的制备
Example 2: Preparation of Compound-2

步骤2-1：M13的制备Step 2-1: Preparation of M13

按照M4的合成方法，采用Dx-8951甲磺酸盐替换MMAE与M3反应，制备得到M13，淡黄色固体粉末，收率约为83％。MS(EI)m/z:1063.45[M+H]⁺.According to the synthesis method of M4, Dx-8951 mesylate was used to replace MMAE and react with M3 to prepare M13, a light yellow solid powder with a yield of about 83%. MS (EI) m/z: 1063.45 [M + H]⁺ .

步骤2-2：M14的制备Step 2-2: Preparation of M14

按照M5的合成方法，采用M13替换M4在哌啶溶液中脱保护基，制备得到M14，淡黄色固体粉末，收率约为70％。MS(EI)m/z:841.41[M+H]⁺.According to the synthesis method of M5, M13 was used to replace M4 and the protecting group was removed in a piperidine solution to prepare M14, a light yellow solid powder, with a yield of about 70%. MS (EI) m/z: 841.41 [M+H]⁺ .

步骤2-3：化合物-2的制备Step 2-3: Preparation of Compound-2

按照化合物-1的合成方法，采用M14替换M5与M12反应，制备得到化合物-2，淡黄色固体粉末，收率约为56％。MS(EI)m/z:597.68[M+2H]²⁺；MS(ESI)m/z:1194.4175[M+H]⁺.According to the synthesis method of compound-1, M14 was used to replace M5 and react with M12 to prepare compound-2, a light yellow solid powder, with a yield of about 56%. MS (EI) m/z: 597.68 [M + 2H]^{2 +} ; MS (ESI) m/z: 1194.4175 [M + H]⁺ .

实施例3：化合物-3的制备
Example 3: Preparation of Compound-3

步骤3-1：M15的制备Step 3-1: Preparation of M15

采用1-氨基-3,6,9,12-四氧杂十五烷-15-酸替换6-氨基己酸与M10反应，按照M11的合成方法制备得到M15，浅黄色粘稠油状物，收率约为92％。¹H NMR(500MHz,Chloroform-d)δ12.04(br,1H),8.26(t,1H),8.16(d,J＝7.7Hz,1H),8.06(d,J＝7.5Hz,1H),3.69-3.59(m,15H),3.59-3.53(m,2H),3.40(dt,J＝5.6,4.2Hz,2H),2.77(s,3H),2.48(t,J＝6.4Hz,2H).MS(EI)m/z:474.07[M+H]⁺.1-Amino-3,6,9,12-tetraoxapentadecan-15-acid was used to replace 6-aminohexanoic acid and react with M10. M15 was prepared according to the synthetic method of M11. The light yellow viscous oil had a yield of about 92%.¹ H NMR (500MHz, Chloroform-d) δ12.04 (br, 1H), 8.26 (t, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 3.69-3.59 (m, 15H), 3.59-3.53 (m, 2H), 3.40 (dt, J = 5.6, 4.2 Hz, 2H), 2.77 (s, 3H), 2.48 (t, J = 6.4 Hz, 2H). MS (EI) m/z: 474.07 [M+H]⁺ .

步骤3-2：M16的制备Step 3-2: Preparation of M16

按照M12的合成方法，采用M15替换M11与m-CPBA反应，制备得到M16，浅黄色粘稠油状物，收率约为80％。¹H NMR(500MHz,Chloroform-d)δ12.04(br,1H),8.26(t,1H),8.16(d,J＝7.7Hz,1H),8.06(d,J＝7.5Hz,1H),3.69-3.59(m,15H),3.59-3.53(m,2H),3.40(dt,J＝5.6,4.2Hz,2H),2.77(s,3H),2.48(t,J＝6.4Hz,2H).MS(EI)m/z:505.98[M+H]⁺；528.10[M+Na]⁺.According to the synthesis method of M12, M15 was used to replace M11 and react with m-CPBA to prepare M16, a light yellow viscous oil, with a yield of about 80%.¹ H NMR (500MHz, Chloroform-d) δ12.04 (br, 1H), 8.26 (t, 1H), 8.16 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 3.69-3.59 (m, 15H), 3.59-3.53 (m, 2H), 3.40 (dt, J = 5.6, 4.2 Hz, 2H), 2.77 (s, 3H), 2.48 (t, J = 6.4 Hz, 2H). MS (EI) m/z: 505.98 [M+H]⁺ ; 528.10 [M+Na]⁺ .

步骤3-3：化合物-3的制备Step 3-3: Preparation of Compound-3

按照化合物-1的合成方法，采用M16替换M12与M5反应，制备得到化合物-3，白色固体粉末，收率约为63％。MS(EI)m/z:805.92[M+2H]²⁺；MS(ESI)m/z:1610.8215[M+H]⁺.According to the synthesis method of compound-1, M16 was used to replace M12 and react with M5 to prepare compound-3, a white solid powder, with a yield of about 63%. MS (EI) m/z: 805.92 [M + 2H]^{2 +} ; MS (ESI) m/z: 1610.8215 [M + H]⁺ .

实施例4：化合物-4的制备
Example 4: Preparation of Compound-4

步骤4-1：化合物-4的制备Step 4-1: Preparation of Compound-4

按照化合物-2的合成方法，采用M16替换M12与M14反应，制备得到化合物-4，淡黄色固体粉末，收率约为55％。MS(EI)m/z:664.71[M+2H]²⁺；MS(ESI)m/z:1328.4768[M+H]⁺.According to the synthesis method of compound-2, M16 was used to replace M12 and react with M14 to prepare compound-4, a light yellow solid powder, with a yield of about 55%. MS (EI) m/z: 664.71 [M + 2H]^{2 +} ; MS (ESI) m/z: 1328.4768 [M + H]⁺ .

实施例5：化合物-5的制备
Example 5: Preparation of Compound-5

步骤5-1：M17的制备Step 5-1: Preparation of M17

按照M1的合成方法，采用L-丙氨酸替换L-瓜氨酸与Fmoc-Ala-NHS反应，制备得到化合物M17，白色固体粉末，收率约为83％。¹H NMR(400MHz,DMSO-d6):δ0.88(dd,6H),1.27(d,3H),1.96(m,1H),3.89(q,1H),4.22(m,4H),7.33(t,2H),7.43(m,3H),7.74(t 2H),7.89(d,2H),8.25(d,1H),12.48(s,1H).MS(EI)m/z:411.23[M+H]⁺；455.05[M+Na]⁺.According to the synthesis method of M1, L-alanine was used to replace L-citrulline and react with Fmoc-Ala-NHS to prepare compound M17, a white solid powder, with a yield of about 83%.¹ H NMR (400MHz, DMSO-d6): δ0.88 (dd, 6H), 1.27 (d, 3H), 1.96 (m, 1H), 3.89 (q, 1H), 4.22 (m, 4H), 7.33 (t, 2H), 7.43 (m, 3H), 7.74 (t 2H), 7.89 (d, 2H), 8.25 (d, 1H), 12.48 (s, 1H). MS (EI) m/z: 411.23 [M+H]⁺ ; 455.05 [M+Na]⁺ .

步骤5-2：M18的制备Step 5-2: Preparation of M18

按照M2的合成方法，采用M17替换M1采用EEDQ与PAB发生缩合反应，制备得到化合物M18，白色固体粉末，收率约为87％。¹H NMR(400MHz,DMSO-d6):δ0.88(dd,6H),1.30(d,3H),2.00(m,1H),3.91(t,1H),4.22(q,2H),4.30(t,1H),4.40(br,1H),4.42(d,2H),5.13(t,1H),7.24(t,2H),7.34(t,2H),7.41(t,2H),7.52(q,3H),7.75(t,2H),7.89(d,2H),8.22(d,1H),9.96(s,1H).MS(EI)m/z:516.29[M+H]⁺；538.20[M+Na]⁺.According to the synthesis method of M2, M17 was used to replace M1, and EEDQ and PAB were used for condensation reaction to prepare compound M18 as a white solid powder with a yield of about 87%.¹ H NMR (400MHz, DMSO-d6): δ0.88(dd,6H),1.30(d,3H),2.00(m,1H),3.91(t,1H),4.22(q,2H),4.30(t,1H),4.40(br,1H),4.42(d,2H),5.13 (t,1H),7.24(t,2H),7.34(t,2H),7.41(t,2H),7.52(q,3H),7.75(t,2H),7.89(d,2H),8.22(d,1H),9.96(s,1H).MS(EI)m/z:516.29[M+H]⁺ ;538.20[M+Na]⁺ .

步骤5-3：M19的制备Step 5-3: Preparation of M19

按照M3的合成方法，采用M18替换M2与对硝基苯酚碳酸酯进行反应，制备得到化合物M19，白色固体粉末，收率约为92％。所得物料不经进一步纯化而直接进行下一步反应。MS(EI)m/z:681.24[M+H]⁺；703.20[M+Na]⁺.According to the synthesis method of M3, M18 was used to replace M2 and react with p-nitrophenol carbonate to prepare compound M19, a white solid powder with a yield of about 92%. The obtained material was directly used for the next reaction without further purification. MS (EI) m/z: 681.24 [M+H]⁺ ; 703.20 [M+Na]⁺ .

步骤5-4：M20的制备Step 5-4: Preparation of M20

按照M4的合成方法，采用M19替换M3与MMAE进行反应，制备得到化合物M20，淡黄色固体粉末，收率约为67％。MS(EI)m/z:630.34[M+2H]²⁺；MS(ESI)m/z:1259.7341[M+H]⁺.According to the synthesis method of M4, M19 was used to replace M3 and react with MMAE to prepare compound M20, a light yellow solid powder, with a yield of about 67%. MS (EI) m/z: 630.34 [M + 2H]^{2 +} ; MS (ESI) m/z: 1259.7341 [M + H]⁺ .

步骤5-5：M21的制备Step 5-5: Preparation of M21

按照M5的合成方法，采用M20替换M4在哌啶溶解中脱去Fmoc保护基，制备得到化合物M21，淡黄色固体粉末，收率约为55％。产物有一定的吸湿性，产物不久置而直接进行下一步反应。MS(EI)m/z:1123.68[M+H]⁺.According to the synthesis method of M5, M20 was used to replace M4 to remove the Fmoc protecting group in piperidine solution to prepare compound M21, a light yellow solid powder, with a yield of about 55%. The product has a certain hygroscopicity, and the product was not left for a long time and directly carried out the next reaction. MS (EI) m/z: 1123.68 [M + H]⁺ .

步骤5-6：化合物-5的制备Step 5-6: Preparation of Compound-5

按照化合物-1的合成方法，采用M21替换M5与化合物M12进行反应，制备得到化合物-5，类白色固体粉末，收率约为53％。MS(EI)m/z:695.86[M+2H]²⁺；MS(ESI)m/z:1390.7162[M+H]⁺.According to the synthesis method of compound-1, M21 was used to replace M5 and react with compound M12 to prepare compound-5, an off-white solid powder, with a yield of about 53%. MS (EI) m/z: 695.86 [M + 2H]^{2 +} ; MS (ESI) m/z: 1390.7162 [M + H]⁺ .

实施例6：化合物-6的制备
Example 6: Preparation of Compound-6

步骤6-1：化合物-6的制备Step 6-1: Preparation of Compound-6

按照化合物-5的合成方法，采用M16替换M12与M21与化合物进行反应，制备得到化合物-6，类白色固体粉末，收率约为61％。MS(EI)m/z:762.93[M+2H]²⁺；MS(ESI)m/z:1524.7745[M+H]⁺.According to the synthesis method of compound-5, M16 was used to replace M12 and M21 to react with the compound to prepare compound-6, an off-white solid powder with a yield of about 61%. MS (EI) m/z: 762.93 [M + 2H]^{2 +} ; MS (ESI) m/z: 1524.7745 [M + H]⁺ .

实施例7：化合物-7的制备
Example 7: Preparation of Compound-7

步骤7-1：M22的制备Step 7-1: Preparation of M22

按照M13的合成方法，采用M19替换M3与Dx-8951f进行缩合反应，制备得到M22，淡黄色固体粉末，收率约为85％。MS(EI)m/z:977.40[M+H]⁺.According to the synthesis method of M13, M19 was used to replace M3 and condensed with Dx-8951f to prepare M22, a light yellow solid powder, with a yield of about 85%. MS (EI) m/z: 977.40 [M + H]⁺ .

步骤7-2：M23的制备Step 7-2: Preparation of M23

按照M14的合成方法，采用M22替换M13在哌啶溶液中脱去Fmoc保护基，制备得到M23，淡黄色固体粉末，收率约为75％。MS(EI)m/z:755.36[M+H]⁺.According to the synthesis method of M14, M22 was used to replace M13 to remove the Fmoc protecting group in a piperidine solution to prepare M23, a light yellow solid powder, with a yield of about 75%. MS (EI) m/z: 755.36 [M + H]⁺ .

步骤7-3：化合物-7的制备Step 7-3: Preparation of Compound-7

按照化合物-2的合成方法，采用M23替换M14与M12在HATU的作用下发生缩合反应，制备得到化合物-7，淡黄色固体粉末，收率约为63％。MS(EI)m/z:1108.33[M+H]⁺；MS(ESI)m/z:1130.32[M+Na]⁺.According to the synthesis method of compound-2, M23 was used to replace M14 and react with M12 under the action of HATU to prepare compound-7, a light yellow solid powder, with a yield of about 63%. MS (EI) m/z: 1108.33 [M + H]⁺ ; MS (ESI) m/z: 1130.32 [M + Na]⁺ .

实施例8：化合物-8的制备
Example 8: Preparation of Compound-8

步骤8-1：化合物-8的制备Step 8-1: Preparation of Compound-8

按照化合物-7的合成方法，采用M16替换M12与M23在HATU的作用下发生缩合反应，制备得到化合物-8，淡黄色固体粉末，收率约为56％。MS(EI)m/z:621.74[M+2H]²⁺；MS(ESI)m/z:1242.4294[M+H]⁺.According to the synthesis method of compound-7, M16 was used to replace M12 and M23 to undergo condensation reaction under the action of HATU to prepare compound-8, a light yellow solid powder, with a yield of about 56%. MS (EI) m/z: 621.74 [M + 2H]^{2 +} ; MS (ESI) m/z: 1242.4294 [M + H]⁺ .

实施例9：化合物-9的制备
Example 9: Preparation of Compound-9

步骤9-1：M24的制备Step 9-1: Preparation of M24

将苄氧羰基保护的二甘氨酸(10g，37.56mM)溶解于乙腈(100mL)中，在搅拌状态下加入NHS(4.75g，41.31mM)和EDCI(8.64g，45.07mM)。在氮气保护下，混合物搅拌反应过夜。待反应完成后，减压浓缩去除溶剂得无色油状物残渣。将油状物复溶与乙酸乙酯中，采用蒸馏水洗涤并分液，重复3次。最终得到的有机相经饱和食盐水洗涤3次后，浓缩反应液至100mL左右。在室温下放置24小时，析出白色不溶物，过滤并采用EA洗涤滤饼，滤饼充分干燥后得产物M24为白色固体粉末，13g，收率约为96％。M24为活性酯中间体，不经久置而直接进行下一步反应。MS(EI)m/z:364.18[M+H]⁺；MS(EI)m/z:386.10[M+Na]⁺.The benzyloxycarbonyl protected diglycine (10g, 37.56mM) was dissolved in acetonitrile (100mL), and NHS (4.75g, 41.31mM) and EDCI (8.64g, 45.07mM) were added under stirring. Under nitrogen protection, the mixture was stirred and reacted overnight. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a colorless oily residue. The oil was redissolved in ethyl acetate, washed with distilled water and separated, and repeated 3 times. The organic phase finally obtained was washed 3 times with saturated brine, and the reaction solution was concentrated to about 100mL. It was placed at room temperature for 24 hours, and white insoluble matter was precipitated. The filter cake was filtered and washed with EA. After the filter cake was fully dried, the product M24 was a white solid powder, 13g, and the yield was about 96%. M24 is an active ester intermediate, and the next step of the reaction is directly carried out without long-term placement. MS(EI)m/z:364.18[M+H]⁺ ; MS(EI)m/z:386.10[M+Na]⁺ .

步骤9-2：M25的制备Step 9-2: Preparation of M25

将苯丙氨酸(4.55g，27.52mM)溶解于乙腈和水(1:1；100mL)的混合溶液中，加入三乙胺(TEA，2.78g，27.52mM)并在室温下搅拌5分钟使固体完全溶解。加入M24(10g，27.52mM)，并在室温下继续搅拌反应3小时。待反应完成后，向反应液中加入水(50mL)，并在搅拌状态下滴加盐酸调pH～2。减压浓缩去除大部分的乙腈，剩余液体在室温下放置过夜析出白色固体。过滤并采用乙醚洗涤滤饼，充分干燥后得产物M25为白色固体粉末，13.2g，收率约为84％。¹H NMR(400MHz,DMSO-d6)δ7.77(d,J＝9.1Hz,1H),7.48(t,J＝6.0Hz,1H),7.38-7.18(m,10H),5.89(t,J＝6.6Hz,1H),5.10(d,J＝0.8Hz,2H),4.45(dt,J＝9.2,7.0Hz,1H),3.86(d,J＝6.6Hz,2H),3.84-3.71(m,2H),3.12-3.06(m,2H).MS(EI)m/z:414.19[M+H]⁺；MS(EI)m/z:412.13[M-H]^-.Phenylalanine (4.55 g, 27.52 mM) was dissolved in a mixed solution of acetonitrile and water (1:1; 100 mL), triethylamine (TEA, 2.78 g, 27.52 mM) was added and stirred at room temperature for 5 minutes to completely dissolve the solid. M24 (10 g, 27.52 mM) was added and the reaction was continued to stir at room temperature for 3 hours. After the reaction was completed, water (50 mL) was added to the reaction solution, and hydrochloric acid was added dropwise under stirring to adjust the pH to 2. Most of the acetonitrile was removed by vacuum concentration, and the remaining liquid was left at room temperature overnight to precipitate a white solid. Filter and wash the filter cake with ether, and after sufficient drying, the product M25 was obtained as a white solid powder, 13.2 g, with a yield of about 84%.^1H NMR (400MHz, DMSO-d6) δ7.77(d,J＝9.1Hz,1H),7.48(t,J＝6.0Hz,1H),7.38-7.18(m,10H),5.89(t,J＝6.6Hz,1H),5.10(d,J＝0 .8Hz,2H),4.45(dt,J＝9.2,7.0Hz,1H),3.86(d,J＝6.6Hz,2H),3.84-3.71(m,2H),3.12-3.06(m,2H).MS(EI)m/z:414.19[M+H]⁺ ;MS(EI)m/z:412.13[MH]^- .

步骤9-3：M26的制备Step 9-3: Preparation of M26

将Fmoc保护的二甘氨酸(12.49g，35.24mM)分散于四氢呋喃(200mL)和乙酸(50mL)的混合溶液中，加热至40℃在搅拌状态下固体全部溶解。加入四乙酸铅(25g，56.39mM)后开始升温回流2小时。待反应完成后，停止加热自然放凉，搅拌状态下，向混合物中加入20％的柠檬酸钠溶液约100mL析出淡黄色沉淀，过滤去除不溶物后，滤液采用20％的柠檬酸钠溶液洗涤2～3次，将分液所得的有机相浓缩至60mL左右，并向浓缩后得溶液中加入蒸馏水约100mL，室温下静置过夜后，过滤，滤饼采用蒸馏水洗涤3次后，充分抽干得到M26为白色固体粉末，13.2g，收率约为99％。Fmoc-protected diglycine (12.49 g, 35.24 mM) was dispersed in a mixed solution of tetrahydrofuran (200 mL) and acetic acid (50 mL), heated to 40 ° C, and all solids were dissolved under stirring. After adding lead tetraacetate (25 g, 56.39 mM), the temperature was raised and refluxed for 2 hours. After the reaction was completed, the heating was stopped and cooled naturally. Under stirring, about 100 mL of 20% sodium citrate solution was added to the mixture to precipitate a light yellow precipitate. After filtering to remove insoluble matter, the filtrate was washed 2 to 3 times with 20% sodium citrate solution, and the organic phase obtained by separation was concentrated to about 60 mL, and about 100 mL of distilled water was added to the concentrated solution. After standing overnight at room temperature, it was filtered, and the filter cake was washed 3 times with distilled water, and then fully drained to obtain M26 as a white solid powder, 13.2 g, with a yield of about 99%.

¹H NMR(400MHz,Chloroform-d)δ7.82(dd,J＝8.2,1.3Hz,2H),7.62(dd,J＝7.6,1.4Hz,2H),7.53-7.46(m,3H),7.41(td,J＝7.8,1.5Hz,2H),5.81(t,J＝6.6Hz,1H),5.44-5.39(m,1H),5.04(d,J＝3.5Hz,2H),4.42(d,J＝4.8Hz,2H),3.84(d,J＝6.6Hz,2H),2.19(s,3H).MS(EI)m/z:369.21[M+H]⁺.¹ H NMR(400MHz,Chloroform-d)δ7.82(dd,J＝8.2,1.3Hz,2H),7.62(dd,J＝7.6,1.4Hz,2H),7.53-7.46(m,3H),7.41(td,J＝7.8,1.5Hz,2H),5.81( t,J＝6.6Hz,1H),5.44-5.39(m,1H),5.04(d,J＝3.5Hz,2H),4.42(d,J＝4.8Hz,2H),3.84(d,J＝6.6Hz,2H),2.19(s,3H).MS(EI)m/z:369.21[M+H]⁺ .

步骤9-4：M27的制备Step 9-4: Preparation of M27

将M26(12.2g，33.12mM)溶解于乙二醇二甲醚(DME；200mL)中，并向反应液混合物中加入乙醇酸苄酯(11g，66.23mM)。待固体全部溶解后，将反应液置于冰浴中进行降温。向反应液中滴加10M的NaOH(10eq，1.32g)溶液，并在低温下搅拌反应1小时。向反应液中滴入冰醋酸约1.3mL，混合物在低温下继续反应1小时。待反应完成后，向反应体系中加入水150mL，并在低温下继续搅拌2.5小时。过滤，滤饼采用水和乙二醇二甲醚的混合溶液(1:1)洗涤3次，充分干燥得M27为白色固体粉末，13.94g，收率约为89％。所得固体产物直接用于下一步反应。MS(EI)m/z:475.22[M+H]⁺.M26 (12.2 g, 33.12 mM) was dissolved in ethylene glycol dimethyl ether (DME; 200 mL), and benzyl glycolate (11 g, 66.23 mM) was added to the reaction mixture. After the solid was completely dissolved, the reaction solution was placed in an ice bath for cooling. A 10 M NaOH (10 eq, 1.32 g) solution was added dropwise to the reaction solution, and the reaction was stirred at low temperature for 1 hour. About 1.3 mL of glacial acetic acid was added dropwise to the reaction solution, and the mixture continued to react at low temperature for 1 hour. After the reaction was completed, 150 mL of water was added to the reaction system, and stirring was continued at low temperature for 2.5 hours. After filtration, the filter cake was washed 3 times with a mixed solution of water and ethylene glycol dimethyl ether (1:1), and fully dried to obtain M27 as a white solid powder, 13.94 g, with a yield of about 89%. The obtained solid product was directly used for the next step reaction. MS (EI) m/z: 475.22 [M + H]⁺ .

步骤9-5：M28的制备Step 9-5: Preparation of M28

将M27(13g，23.4mM)溶解于乙腈(400mL)中，向反应液混合物中加入DBU(4.59g，30.14mM)，混合物在常温下继续搅拌反应4小时。待反应完成后，减压浓缩去除溶剂得粘稠油状物残渣，进一步经硅胶柱层析纯化得到物，收率>100％，疑似包裹有未除尽的溶剂。所得游离胺产物M28稳定性差直接用于下一步反应。MS(EI)m/z:253.23[M+H]⁺.M27 (13 g, 23.4 mM) was dissolved in acetonitrile (400 mL), DBU (4.59 g, 30.14 mM) was added to the reaction mixture, and the mixture was stirred and reacted for 4 hours at room temperature. After the reaction was completed, the solvent was removed by concentration under reduced pressure to obtain a viscous oily residue, which was further purified by silica gel column chromatography to obtain a product with a yield of >100%, which was suspected to be encapsulated with unremoved solvent. The obtained free amine product M28 had poor stability and was directly used in the next step. MS (EI) m/z: 253.23 [M + H]⁺ .

步骤9-6：M29的制备Step 9-6: Preparation of M29

将M25(9.0g，22mM)和M28(6.3g，25mM)溶解于乙腈(200mL)中，加入催化量的DIPEA(0.5eq)之后，将反应体系置于冰浴中降温至0℃左右，分批加入EDCI(5.75g，30mM)，在低温下继续反应1小时后，转移至室温继续反应2小时。待反应完成后，减压浓缩去除溶剂得到黄色油状物残渣，采用乙酸乙酯复溶，采用水洗涤并分液3次后，浓缩有机溶剂至100mL左右。室温下搅拌过夜，过滤析出的糊状不溶物，充分抽干并充分烘干，得到M29为淡黄色固体粉末，13.4g，收率约为94％。MS(EI)m/z:648.33[M+H]⁺；MS(EI)m/z:670.26[M+Na]⁺.M25 (9.0 g, 22 mM) and M28 (6.3 g, 25 mM) were dissolved in acetonitrile (200 mL), and after adding a catalytic amount of DIPEA (0.5 eq), the reaction system was placed in an ice bath and cooled to about 0 ° C. EDCI (5.75 g, 30 mM) was added in batches. After continuing to react at low temperature for 1 hour, the mixture was transferred to room temperature and continued to react for 2 hours. After the reaction was completed, the solvent was removed by vacuum concentration to obtain a yellow oily residue, which was redissolved in ethyl acetate, washed with water and separated 3 times, and the organic solvent was concentrated to about 100 mL. Stir overnight at room temperature, filter the precipitated paste insolubles, fully drain and fully dry to obtain M29 as a light yellow solid powder, 13.4 g, with a yield of about 94%. MS (EI) m/z: 648.33 [M + H]⁺ ; MS (EI) m/z: 670.26 [M + Na]⁺ .

步骤9-7：M30的制备Step 9-7: Preparation of M30

将M29(1.0g，1.54mM)溶解于甲醇(50mL)中，室温搅拌下加入LiOH一水化合物(77.7mg，1.85mM)和催化量的水。室温下反应过夜，减压浓缩去除溶剂，得到的固体残渣采用粗硅胶拌样，硅胶柱层析纯化得到产物M30为白色固体粉末，300mg，收率约为35％。MS(EI)m/z:580.31[M+Na]⁺；MS(EI)m/z:556.24[M-H]^-.M29 (1.0 g, 1.54 mM) was dissolved in methanol (50 mL), and LiOH monohydrate (77.7 mg, 1.85 mM) and a catalytic amount of water were added under stirring at room temperature. The reaction was allowed to proceed overnight at room temperature, and the solvent was removed by concentration under reduced pressure. The obtained solid residue was mixed with crude silica gel and purified by silica gel column chromatography to obtain the product M30 as a white solid powder, 300 mg, with a yield of about 35%. MS (EI) m/z: 580.31 [M+Na]⁺ ; MS (EI) m/z: 556.24 [MH]^- .

步骤9-8：M31的制备Step 9-8: Preparation of M31

将M30(100mg，0.179mM)溶于DMF(20mL)中，待固体全部溶解后，依次加入Dx-8951f甲磺酸盐(64mg，0.1196mM)，HATU(136.39mg，0.359mM)和DIPEA(92.73mg，0.7174mM)。室温下搅拌反应4小时后，减压浓缩去除溶剂，所得油状物残渣经硅胶柱层析纯化得到产物M31，210mg，收率>100％，疑似包裹少量硅胶。所得到的粗产物不经进一步纯化而直接进行下一步。MS(EI)m/z:975.41[M+H]⁺.M30 (100 mg, 0.179 mM) was dissolved in DMF (20 mL). After the solid was completely dissolved, Dx-8951f mesylate (64 mg, 0.1196 mM), HATU (136.39 mg, 0.359 mM) and DIPEA (92.73 mg, 0.7174 mM) were added in sequence. After stirring and reacting at room temperature for 4 hours, the solvent was removed by concentration under reduced pressure. The obtained oily residue was purified by silica gel column chromatography to obtain product M31, 210 mg, with a yield of >100%, suspected to be wrapped with a small amount of silica gel. The obtained crude product was directly carried out to the next step without further purification. MS (EI) m/z: 975.41 [M + H]⁺ .

步骤9-9：M32的制备Step 9-9: Preparation of M32

按照M14的合成方法，采用M31替换M13在哌啶溶液中脱去Fmoc保护基，制备得到M32，淡黄色固体粉末，收率约为57％。所得到的氨基产物稳定性较差，不经进一步纯化而直接投入下一步反应。MS(EI)m/z:841.45[M+H]⁺.According to the synthesis method of M14, M31 was used to replace M13 to remove the Fmoc protecting group in a piperidine solution to prepare M32, a light yellow solid powder, with a yield of about 57%. The obtained amino product had poor stability and was directly used in the next step without further purification. MS (EI) m/z: 841.45 [M + H]⁺ .

步骤9-10：化合物-9的制备Step 9-10: Preparation of Compound-9

按照化合物-2的合成方法，采用M32替换M14与M12在HATU的作用下发生缩合反应，制备得到化合物-9，淡黄色固体粉末，收率约为45％。MS(EI)m/z:597.72[M+2H]²⁺；MS(ESI)m/z:1194.3832[M+H]⁺.According to the synthesis method of compound-2, M32 was used to replace M14 and condensed with M12 under the action of HATU to prepare compound-9, a light yellow solid powder, with a yield of about 45%. MS (EI) m/z: 597.72 [M + 2H]^{2 +} ; MS (ESI) m/z: 1194.3832 [M + H]⁺ .

实施例10：化合物-10的制备
Example 10: Preparation of Compound-10

步骤10-1：化合物-10的制备Step 10-1: Preparation of Compound-10

按照化合物-9的合成方法，采用M16替换M12与M32在HATU的作用下发生缩合反应，制备得到化合物-10，淡黄色固体粉末，收率约为47％。MS(EI)m/z:664.69[M+2H]²⁺；MS(ESI)m/z:1328.4436[M+H]⁺.According to the synthesis method of compound-9, M16 was used to replace M12 and condensed with M32 under the action of HATU to prepare compound-10, a light yellow solid powder, with a yield of about 47%. MS (EI) m/z: 664.69 [M + 2H]^{2 +} ; MS (ESI) m/z: 1328.4436 [M + H]⁺ .

实施例11：化合物-11的制备
Example 11: Preparation of Compound-11

步骤11-1：M33的制备Step 11-1: Preparation of M33

按照化合物M31的合成方法，采用MMAE替换Dx-8951f甲磺酸盐与M30在HATU的作用下发生缩合反应，制备得到中间体产物M33，白色固体粉末，收率约为78％。MS(EI)m/z:629.33[M+2H]²⁺；MS(ESI)m/z:1257.7135[M+H]⁺.According to the synthesis method of compound M31, MMAE was used to replace the mesylate of Dx-8951f and react with M30 under the action of HATU to obtain the intermediate product M33, a white solid powder, with a yield of about 78%. MS (EI) m/z: 629.33 [M + 2H]^{2 +} ; MS (ESI) m/z: 1257.7135 [M + H]⁺ .

步骤11-2：M34的制备Step 11-2: Preparation of M34

将化合物M33(100mg，0.08mM)溶解于甲醇(10mL)中，加入催化量的Pd/C(～15mg)，在氢气氛围下搅拌反应。室温下反应3小时后，去除氢气球，过滤去除不溶物后，减压浓缩得到淡黄色玻璃状残渣。进一步经硅胶柱层析纯化得到化合物M34，白色固体粉末，61mg，收率约为67％。MS(ESI)m/z:1123.68[M+H]⁺.Compound M33 (100 mg, 0.08 mM) was dissolved in methanol (10 mL), and a catalytic amount of Pd/C (~15 mg) was added. The mixture was stirred under a hydrogen atmosphere. After reacting at room temperature for 3 hours, the hydrogen balloon was removed, the insoluble matter was filtered out, and the mixture was concentrated under reduced pressure to obtain a light yellow glassy residue. Compound M34 was further purified by silica gel column chromatography to obtain a white solid powder, 61 mg, with a yield of about 67%. MS (ESI) m/z: 1123.68 [M+H]⁺ .

步骤11-3：化合物-11的制备Step 11-3: Preparation of Compound-11

按照化合物-1的合成方法，采用M34替换M5与M12在HATU的作用下发生缩合反应，制备得到化合物-11，白色固体粉末，收率约为46％。MS(EI)m/z:738.79[M+2H]²⁺；MS(ESI)m/z:1476.7281[M+H]⁺.According to the synthesis method of compound-1, M34 was used to replace M5 and M12 to undergo condensation reaction under the action of HATU to prepare compound-11, a white solid powder, with a yield of about 46%. MS (EI) m/z: 738.79 [M + 2H]^{2 +} ; MS (ESI) m/z: 1476.7281 [M + H]⁺ .

实施例12：化合物-12的制备
Example 12: Preparation of Compound-12

步骤12-1：化合物-12的制备Step 12-1: Preparation of Compound-12

按照化合物-11的合成方法，采用M16替换M12与M34在HATU的作用下发生缩合反应，制备得到化合物-12，白色固体粉末，收率约为53％。MS(EI)m/z:805.95[M+2H]²⁺；MS(ESI)m/z:1610.7848[M+H]⁺.According to the synthesis method of compound-11, M16 was used to replace M12 and M34 to undergo condensation reaction under the action of HATU to prepare compound-12, a white solid powder, with a yield of about 53%. MS (EI) m/z: 805.95 [M + 2H]^{2 +} ; MS (ESI) m/z: 1610.7848 [M + H]⁺ .

实施例13：化合物-13的制备
Example 13: Preparation of Compound-13

步骤13-1：M35的制备Step 13-1: Preparation of M35

将M2(1.0g，1.66mM)溶解于DMF(15mL)中，待固体全部溶解后，加入哌啶(1mL)，并在室温下搅拌反应2小时。待反应结束后，减压浓缩去除溶剂，所得油状物残渣经硅胶柱层数纯化得产物为无色粘稠液体，进一步减压干燥并放置，得到M35为白色固体，530mg，收率约为84％。¹H NMR(400MHz,DMSO-d6):δ0.86(dd,6H),1.38(m,2H),1.64(m,2H),1.94(m,1H),2.95(m,2H),3.05(d,1H),4.43(s,2H),4.47(s,1H),5.13(br,1H),5.44(d,2H),6.01(br,1H),7.23(d,2H),7.54(d,2H),8.17(br,1H),10.07(s,1H).MS(EI)m/z:380.32[M+H]⁺；MS(EI)m/z:402.31[M+Na]⁺.M2 (1.0 g, 1.66 mM) was dissolved in DMF (15 mL). After the solid was completely dissolved, piperidine (1 mL) was added and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The obtained oily residue was purified by silica gel column to obtain a colorless viscous liquid. It was further dried under reduced pressure and allowed to stand to obtain M35 as a white solid, 530 mg, with a yield of about 84%.^1H NMR (400MHz, DMSO-d6): δ0.86(dd,6H),1.38(m,2H),1.64(m,2H),1.94(m,1H),2.95(m,2H),3.05(d,1H),4.43(s,2H),4.47(s, 1H),5.13(br,1H),5.44(d,2H),6.01(br,1H),7.23(d,2H),7.54(d,2H),8.17(br,1H),10.07(s,1H).MS(EI)m/z:380.32[M+H]⁺ ;MS(EI)m/z:402.31[M+Na]⁺ .

步骤13-2：M36的制备Step 13-2: Preparation of M36

将2,5,8,11-四氧十四烷-14酸(5.0g，21.16mM)溶解于二氯甲烷(50mL)中，并依次加入NHS(3.65mg，31.74mM)和EDCI(4.78g，25.4mM)。室温下搅拌反应过夜。待反应完成后，减压浓缩去除溶剂，所得无色油状物残渣经进一步的柱层析纯化得到M36，无色粘稠油状物，8.0g，收率>100％，油状物包裹少量未能蒸干的溶剂。所得油状物不经进一步纯化直接尽快用于下一步反应。MS(EI)m/z:334.42[M+H]⁺.2,5,8,11-Tetrahydrotetradecane-14 acid (5.0 g, 21.16 mM) was dissolved in dichloromethane (50 mL), and NHS (3.65 mg, 31.74 mM) and EDCI (4.78 g, 25.4 mM) were added in sequence. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the colorless oily residue was further purified by column chromatography to obtain M36, a colorless viscous oil, 8.0 g, with a yield of >100%, and the oil contained a small amount of solvent that was not evaporated. The obtained oil was used directly in the next step as soon as possible without further purification. MS (EI) m/z: 334.42 [M + H]⁺ .

步骤13-3：M37的制备Step 13-3: Preparation of M37

将Fmoc-²N-Boc-⁶N-L-Lys(10.0g，21.3mM)分散于DCM中(100mL)，不全溶，加入TFA(10mL)后反应物全部溶解并呈浅黄色；室温下搅拌反应3小时后，减压浓缩去除溶剂，加入乙酸乙酯复溶后重新蒸干，重复3次带去残留的TFA，继续用隔膜泵充分抽干，得粘稠油状物17g。收率>100％，仍包含少量未除尽溶剂。所得无色油状物M37直接用于下一步。MS(EI)m/z:369.21[M+H]⁺.Fmoc^-2N -Boc^-6NL -Lys (10.0g, 21.3mM) was dispersed in DCM (100mL), but was not completely dissolved. After adding TFA (10mL), the reactant was completely dissolved and light yellow. After stirring and reacting at room temperature for 3 hours, the solvent was removed by concentration under reduced pressure, and ethyl acetate was added for redissolution and then evaporated to dryness. This was repeated 3 times to remove the residual TFA, and the diaphragm pump was used to fully pump out to obtain 17g of viscous oil. The yield was >100%, and a small amount of solvent was still contained. The obtained colorless oil M37 was used directly in the next step. MS (EI) m/z: 369.21 [M+H]⁺ .

步骤13-4：M38的制备Step 13-4: Preparation of M38

将化合物M37(粗品，～15mM)和M36(粗品，～15mM)分散于DCM中(100mL)，加入DIPEA(1.94g，15mM)在室温下搅拌反应过夜。减压浓缩，隔膜泵充分抽干得油状物粗品。进一步经硅胶柱层析纯化得到产物M38为无色粘稠油状物，13g，收率>100％，仍包含少量未除尽溶剂。所得无色油状物M38直接用于下一步反应。MS(EI)m/z:587.32[M+H]⁺.Compounds M37 (crude, ~15 mM) and M36 (crude, ~15 mM) were dispersed in DCM (100 mL), and DIPEA (1.94 g, 15 mM) was added and stirred at room temperature for overnight reaction. The mixture was concentrated under reduced pressure and fully pumped dry by a diaphragm pump to obtain a crude oil. The product M38 was further purified by silica gel column chromatography as a colorless viscous oil, 13 g, with a yield of >100%, and still contained a small amount of unremoved solvent. The obtained colorless oil M38 was directly used for the next step reaction. MS (EI) m/z: 587.32 [M+H]⁺ .

步骤13-5：M39的制备Step 13-5: Preparation of M39

将化合物M38(～2.0g，3.4mM)和M35(1.0g，2.6mM)溶解于DMF(20mL)，室温下搅拌待全部溶解后，依次加入HATU(1.50g，3.93mM)和DIPEA(0.508g，3.93mM)。室温下搅拌反应过夜，减压浓缩去除溶剂得玻璃状物残渣。向残渣中加入乙酸乙酯超声分散并打浆，抽滤糊状物，滤饼采用乙酸乙酯洗涤(重复3次)，所得滤饼充分干燥后得M39为淡黄色固体粉末，2.5g，收率约95％。MS(EI)m/z:948.56[M+H]⁺；MS(EI)m/z:983.17[M+Cl]^-.Compounds M38 (~2.0 g, 3.4 mM) and M35 (1.0 g, 2.6 mM) were dissolved in DMF (20 mL). After stirring at room temperature until all the mixture was dissolved, HATU (1.50 g, 3.93 mM) and DIPEA (0.508 g, 3.93 mM) were added in sequence. The mixture was stirred at room temperature overnight and the solvent was removed by concentration under reduced pressure to obtain a glassy residue. Ethyl acetate was added to the residue for ultrasonic dispersion and slurrying. The paste was filtered and the filter cake was washed with ethyl acetate (repeated 3 times). After the filter cake was fully dried, M39 was obtained as a light yellow solid powder, 2.5 g, with a yield of about 95%. MS (EI) m/z: 948.56 [M + H]⁺ ; MS (EI) m/z: 983.17 [M + Cl]^- .

步骤13-6：M40的制备Step 13-6: Preparation of M40

按照化合物M3的合成方法，采用M39替换M2与对硝基苯酚碳酸酯反应，制备得到中间体产物M40，白色固体粉末，收率约为74％。MS(EI)m/z:1113.69[M+H]⁺；MS(ESI)m/z:1135.49[M+Na]⁺.According to the synthesis method of compound M3, M39 was used to replace M2 and react with p-nitrophenol carbonate to prepare the intermediate product M40, a white solid powder, with a yield of about 74%. MS (EI) m/z: 1113.69 [M + H]⁺ ; MS (ESI) m/z: 1135.49 [M + Na]⁺ .

步骤13-7：M41的制备Step 13-7: Preparation of M41

按照化合物M13的合成方法，采用M40替换M3与Dx-8951f甲磺酸盐反应，制备得到中间体产物M41，淡黄色固体粉末，收率约为57％。MS(EI)m/z:705.27[M+2H]²⁺；MS(ESI)m/z:1409.6475[M+H]⁺.According to the synthesis method of compound M13, M40 was used to replace M3 and react with Dx-8951f mesylate to prepare the intermediate product M41, a light yellow solid powder, with a yield of about 57%. MS (EI) m/z: 705.27 [M + 2H]^{2 +} ; MS (ESI) m/z: 1409.6475 [M + H]⁺ .

步骤13-8：M42的制备Step 13-8: Preparation of M42

按照化合物M14的合成方法，采用M41替换M13在哌啶溶液中脱除Fmoc保护基，制备得到中间体产物M42，土灰色固体粉末，收率约为68％。MS(EI)m/z:594.37[M+2H]²⁺；MS(ESI)m/z:1187.5790[M+H]⁺.According to the synthesis method of compound M14, M41 was used to replace M13 to remove the Fmoc protecting group in a piperidine solution to prepare the intermediate product M42, a gray solid powder, with a yield of about 68%. MS (EI) m/z: 594.37 [M + 2H]^{2 +} ; MS (ESI) m/z: 1187.5790 [M + H]⁺ .

步骤13-9：化合物-13的制备Step 13-9: Preparation of Compound-13

按照化合物-2的合成方法，采用M42替换M14与M12在HATU的作用下发生缩合反应，制备得到化合物-13，白色固体粉末，收率约为59％。MS(EI)m/z:770.86[M+2H]²⁺；MS(ESI)m/z:1540.6295[M+H]⁺.According to the synthesis method of compound-2, M42 was used to replace M14 and condensed with M12 under the action of HATU to prepare compound-13, a white solid powder, with a yield of about 59%. MS (EI) m/z: 770.86 [M + 2H]^{2 +} ; MS (ESI) m/z: 1540.6295 [M + H]⁺ .

实施例14：化合物-14的制备
Example 14: Preparation of Compound-14

步骤14-1：化合物-14的制备Step 14-1: Preparation of Compound-14

按照化合物-13的合成方法，采用M16替换M12与M42在HATU的作用下发生缩合反应，制备得到化合物-14，白色固体粉末，收率约为43％。MS(EI)m/z:838.13[M+2H]²⁺；MS(ESI)m/z:1674.6877[M+H]⁺.According to the synthesis method of compound-13, M16 was used to replace M12 and M42 to undergo condensation reaction under the action of HATU to prepare compound-14, a white solid powder, with a yield of about 43%. MS (EI) m/z: 838.13 [M + 2H]^{2 +} ; MS (ESI) m/z: 1674.6877 [M + H]⁺ .

实施例15：化合物-15的制备
Example 15: Preparation of Compound-15

步骤15-1：M43的制备Step 15-1: Preparation of M43

按照化合物M4的合成方法，采用M40替换M3与MMAE进行反应，制备得到M43，白色固体粉末，收率约为76％。MS(EI)m/z:846.57[M+2H]²⁺；MS(ESI)m/z:1691.9921[M+H]⁺.According to the synthesis method of compound M4, M40 was used to replace M3 and react with MMAE to prepare M43, a white solid powder, with a yield of about 76%. MS (EI) m/z: 846.57 [M + 2H]^{2 +} ; MS (ESI) m/z: 1691.9921 [M + H]⁺ .

步骤15-2：M44的制备Step 15-2: Preparation of M44

按照化合物M5的合成方法，采用M43替换M4在哌啶溶液中脱去Fmoc保护基，制备得到M44，玻璃状固体粉末，收率约为61％。MS(EI)m/z:735.54[M+2H]²⁺；MS(ESI)m/z:1469.9233[M+H]⁺.According to the synthesis method of compound M5, M43 was used to replace M4 to remove the Fmoc protecting group in a piperidine solution to prepare M44, a glassy solid powder with a yield of about 61%. MS (EI) m/z: 735.54 [M + 2H]^{2 +} ; MS (ESI) m/z: 1469.9233 [M + H]⁺ .

步骤15-3：化合物-15的制备Step 15-3: Preparation of Compound-15

按照化合物-1的合成方法，采用M44替换M5与M12在HATU的作用下发生缩合反应，制备得到化合物-15，白色固体粉末，收率约为48％。MS(EI)m/z:911.95[M+2H]²⁺；MS(ESI)m/z:1822.9741[M+H]⁺.According to the synthesis method of compound-1, M44 was used to replace M5 and M12 to undergo condensation reaction under the action of HATU to prepare compound-15, a white solid powder, with a yield of about 48%. MS (EI) m/z: 911.95 [M + 2H]^{2 +} ; MS (ESI) m/z: 1822.9741 [M + H]⁺ .

实施例16：化合物-16的制备
Example 16: Preparation of Compound-16

步骤16-1：化合物-16的制备Step 16-1: Preparation of Compound-16

按照化合物-15的合成方法，采用M16替换M12与M44在HATU的作用下发生缩合反应，制备得到化合物-16，白色固体粉末，收率约为59％。MS(EI)m/z:979.23[M+2H]²⁺；MS(ESI)m/z:1957.0324[M+H]⁺.According to the synthesis method of compound-15, M16 was used to replace M12 and M44 to undergo condensation reaction under the action of HATU to prepare compound-16, a white solid powder, with a yield of about 59%. MS (EI) m/z: 979.23 [M + 2H]^{2 +} ; MS (ESI) m/z: 1957.0324 [M + H]⁺ .

实施例17：化合物-17的制备
Example 17: Preparation of Compound-17

步骤17-1：M45的制备Step 17-1: Preparation of M45

按照M35的合成方法，采用M18替换M2在哌啶溶液中脱去Fmoc保护基，制备得到M45，无色粘稠油状物，收率约为75％。M45在室温下久置不稳定，应尽快用于下一步反应。¹H NMR(400MHz,DMSO-d6):δ0.85(dd,6H),1.29(d,3H),1.92(m,1H),2.80(d,1H),3.00(d,1H),4.43(s,1H),4.48(t,1H),5.13(s,1H),7.24(d,2H),7.53(d,2H),8.18(s,1H),10.0(s,1H).MS(EI)m/z:294.23[M+H]⁺；MS(EI)m/z:316.22[M+Na]⁺.According to the synthesis method of M35, M18 was used to replace M2 to remove the Fmoc protecting group in a piperidine solution to prepare M45, a colorless viscous oil with a yield of about 75%. M45 is unstable at room temperature for a long time and should be used in the next reaction as soon as possible.^1H NMR (400MHz, DMSO-d6): δ0.85(dd,6H),1.29(d,3H),1.92(m,1H),2.80(d,1H),3.00(d,1H),4.43(s,1H) ,4.48(t,1H),5.13(s,1H),7.24(d,2H),7.53(d,2H),8.18(s,1H),10.0(s,1H).MS(EI)m/z:294.23[M+H]⁺ ;MS(EI)m/z:316.22[M+Na]⁺ .

步骤17-2：M46的制备Step 17-2: Preparation of M46

按照M39的合成方法，采用M45替换M35与M38在HATU的作用下发生酰化反应，制备得到M46，淡黄色固体粉末，收率约为68％。MS(EI)m/z:862.48[M+H]⁺；MS(EI)m/z:884.46[M+Na]⁺.According to the synthesis method of M39, M45 was used to replace M35 and acylated with M38 under the action of HATU to prepare M46, a light yellow solid powder, with a yield of about 68%. MS (EI) m/z: 862.48 [M + H]⁺ ; MS (EI) m/z: 884.46 [M + Na]⁺ .

步骤17-3：M47的制备Step 17-3: Preparation of M47

按照M40的合成方法，采用M46替换M39与对硝基苯酚碳酸酯发生反应，制备得到M47，淡黄色固体粉末，收率约为85％。MS(EI)m/z:1027.47[M+H]⁺；MS(EI)m/z:1049.45[M+Na]⁺.According to the synthesis method of M40, M46 was used to replace M39 and react with p-nitrophenol carbonate to prepare M47, a light yellow solid powder, with a yield of about 85%. MS (EI) m/z: 1027.47 [M + H]⁺ ; MS (EI) m/z: 1049.45 [M + Na]⁺ .

步骤17-4：M48的制备Step 17-4: Preparation of M48

按照M41的合成方法，采用M47替换M40与Dx-8951f甲磺酸盐发生反应，制备得到M48，灰白色固体粉末，收率约为44％。MS(EI)m/z:662.32[M+2H]²⁺；MS(ESI)m/z:1323.5974[M+H]⁺.According to the synthesis method of M41, M47 was used to replace M40 and react with Dx-8951f mesylate to prepare M48, an off-white solid powder with a yield of about 44%. MS (EI) m/z: 662.32 [M + 2H]^{2 +} ; MS (ESI) m/z: 1323.5974 [M + H]⁺ .

步骤17-5：M49的制备Step 17-5: Preparation of M49

按照M42的合成方法，采用M48替换M41在哌啶的DMF溶液中脱除Fmoc保护基，制备得到M49，土灰色固体粉末，收率约为56％。MS(EI)m/z:1101.55[M+H]⁺；MS(EI)m/z:1123.54[M+Na]⁺.According to the synthesis method of M42, M48 was used to replace M41 to remove the Fmoc protecting group in a piperidine DMF solution to prepare M49, a gray solid powder with a yield of about 56%. MS (EI) m/z: 1101.55 [M + H]⁺ ; MS (EI) m/z: 1123.54 [M + Na]⁺ .

步骤17-6：化合物-17的制备Step 17-6: Preparation of Compound-17

按照化合物-13的合成方法，采用M49替换M42与M12在HATU的作用下发生酰化反应，制备得到化合物-17，灰白色固体粉末，收率约为49％。MS(EI)m/z:727.72[M+2H]²⁺；MS(ESI)m/z:1454.5831[M+H]⁺.According to the synthesis method of compound-13, M49 was used to replace M42 and acylated with M12 under the action of HATU to prepare compound-17, a grayish white solid powder, with a yield of about 49%. MS (EI) m/z: 727.72 [M + 2H]^{2 +} ; MS (ESI) m/z: 1454.5831 [M + H]⁺ .

实施例18：化合物-18的制备
Example 18: Preparation of Compound-18

步骤18-1：化合物-18的制备Step 18-1: Preparation of Compound-18

按照化合物-17的合成方法，采用M16替换M12与M49在HATU的作用下发生酰化反应，制备得到化合物-18，灰白色固体粉末，收率约为50％。MS(EI)m/z:794.76[M+2H]²⁺；MS(ESI)m/z:1588.6387[M+H]⁺.According to the synthesis method of compound-17, M16 was used to replace M12 and acylated with M49 under the action of HATU to prepare compound-18, a grayish white solid powder, with a yield of about 50%. MS (EI) m/z: 794.76 [M + 2H]^{2 +} ; MS (ESI) m/z: 1588.6387 [M + H]⁺ .

实施例19：化合物-19的制备
Example 19: Preparation of Compound-19

步骤19-1：M50的制备Step 19-1: Preparation of M50

按照M48的合成方法，采用MMAE替换Dx-8951f甲磺酸盐与M47发生酰化反应，制备得到M50，白色固体粉末，收率约为61％。MS(EI)m/z:803.49[M+2H]²⁺.According to the synthesis method of M48, MMAE was used to replace Dx-8951f mesylate and react with M47 to produce M50, a white solid powder, with a yield of about 61%. MS (EI) m/z: 803.49 [M + 2H]²⁺ .

步骤19-2：M51的制备Step 19-2: Preparation of M51

按照M5的合成方法，采用M50替换M4在哌啶的DMF溶液中脱除Fmoc保护基，制备得到M51，白色蜡状固体，收率约为68％。MS(EI)m/z:692.43[M+2H]²⁺；MS(ESI)m/z:1383.8751[M+H]⁺.According to the synthesis method of M5, M50 was used to replace M4 to remove the Fmoc protecting group in a piperidine DMF solution to prepare M51, a white waxy solid, with a yield of about 68%. MS (EI) m/z: 692.43 [M + 2H]^{2 +} ; MS (ESI) m/z: 1383.8751 [M + H]⁺ .

步骤19-3：化合物-19的制备Step 19-3: Preparation of Compound-19

按照化合物-1的合成方法，采用M51替换M5与M12在HATU的作用下发生酰化反应，制备得到化合物-19，白色固体粉末，收率约为57％。MS(EI)m/z:868.89[M+2H]²⁺；MS(ESI)m/z:1736.9256[M+H]⁺.According to the synthesis method of compound-1, M51 was used to replace M5 and M12 to undergo acylation reaction under the action of HATU to prepare compound-19, a white solid powder, with a yield of about 57%. MS (EI) m/z: 868.89 [M + 2H]^{2 +} ; MS (ESI) m/z: 1736.9256 [M + H]⁺ .

实施例20：化合物-20的制备
Example 20: Preparation of Compound-20

步骤20-1：化合物-20的制备Step 20-1: Preparation of Compound-20

按照化合物-19的合成方法，采用M16替换M12与M51在HATU的作用下发生酰化反应，制备得到化合物-20，白色固体粉末，收率约为60％。MS(EI)m/z:936.02[M+2H]²⁺；MS(ESI)m/z:1870.9833[M+H]⁺.According to the synthesis method of compound-19, M16 was used to replace M12 and acylated with M51 under the action of HATU to prepare compound-20, a white solid powder, with a yield of about 60%. MS (EI) m/z: 936.02 [M + 2H]^{2 +} ; MS (ESI) m/z: 1870.9833 [M + H]⁺ .

实施例21：化合物-21的制备
Example 21: Preparation of Compound-21

步骤21-1：M52的制备Step 21-1: Preparation of M52

按照M1的合成方法，采用Boc-⁶N-L-Lys替换瓜氨酸与Fmoc-Val-NHS发生反应，制备得到M52，白色固体粉末。收率约为96％。MS(EI)m/z:568.35[M+H]⁺；MS(EI)m/z:590.28[M+Na]⁺.According to the synthesis method of M1, Boc^-6NL -Lys was used to replace citrulline and react with Fmoc-Val-NHS to prepare M52, a white solid powder. The yield was about 96%. MS (EI) m/z: 568.35 [M + H]⁺ ; MS (EI) m/z: 590.28 [M + Na]⁺ .

步骤21-2：M53的制备Step 21-2: Preparation of M53

按照M2的合成方法，采用M52替换M1在EEDQ的作用下与PAB发生缩合反应，制备得到M53，白色固体粉末。收率约为89％。MS(EI)m/z:673.33[M+H]⁺；MS(EI)m/z:695.31[M+Na]⁺.According to the synthesis method of M2, M52 was used to replace M1 and condensed with PAB under the action of EEDQ to prepare M53, a white solid powder. The yield was about 89%. MS (EI) m/z: 673.33 [M + H]⁺ ; MS (EI) m/z: 695.31 [M + Na]⁺ .

步骤21-3：M54的制备Step 21-3: Preparation of M54

按照M3的合成方法，采用M53替换M2在对硝基苯酚碳酸酯发生反应，制备得到M54，白色固体粉末。收率约为91％。MS(EI)m/z:838.35[M+H]⁺.According to the synthesis method of M3, M53 was used to replace M2 and react with p-nitrophenol carbonate to prepare M54, a white solid powder. The yield was about 91%. MS (EI) m/z: 838.35 [M + H]⁺ .

步骤21-4：M55的制备Step 21-4: Preparation of M55

按照M13的合成方法，采用M54替换M3在Dx-8951f发生酰化反应，制备得到M55，灰色固体粉末。收率约为49％。MS(EI)m/z:1134.52[M+H]⁺；MS(EI)m/z:1156.51[M+Na]⁺.According to the synthesis method of M13, M54 was used to replace M3 to undergo acylation reaction in Dx-8951f to prepare M55, a gray solid powder. The yield was about 49%. MS (EI) m/z: 1134.52 [M + H]⁺ ; MS (EI) m/z: 1156.51 [M + Na]⁺ .

步骤21-5：M56的制备Step 21-5: Preparation of M56

按照M14的合成方法，采用M55替换M13在哌啶的DMF溶液中脱除Fmoc保护基，制备得到M56，灰白色玻璃状固体。收率约为55％。MS(EI)m/z:912.43[M+H]⁺；MS(EI)m/z:934.41[M+Na]⁺.According to the synthesis method of M14, M55 was used to replace M13 to remove the Fmoc protecting group in a piperidine DMF solution to prepare M56, an off-white glassy solid. The yield was about 55%. MS (EI) m/z: 912.43 [M + H]⁺ ; MS (EI) m/z: 934.41 [M + Na]⁺ .

步骤21-6：M57的制备Step 21-6: Preparation of M57

按照化合物-2的合成方法，采用M56替换M14与M12在HATU的作用下发生酰化反应，制备得到M57，灰白色固体粉末，收率约为46％。MS(EI)m/z:633.28[M+2H]²⁺；MS(ESI)m/z:1265.4821[M+H]⁺.According to the synthesis method of compound-2, M56 was used to replace M14 and acylated with M12 under the action of HATU to prepare M57, a grayish white solid powder, with a yield of about 46%. MS (EI) m/z: 633.28 [M+2H]²⁺ ; MS (ESI) m/z: 1265.4821 [M+H]⁺ .

步骤21-7：化合物-21的制备Step 21-7: Preparation of Compound-21

将化合物M57(50mg，0.079mM)分散于DCM(10mL)中，搅拌状态下加入HCOOH(1mL)使M57全部溶解。室温下搅拌反应4小时后，减压浓缩去除溶剂，得到浅黄色的半固态油状物残渣，复溶后采用粗硅胶拌样，经硅胶柱层析进一步纯化得到化合物-21为灰白色玻璃状固体，47mg，收率约为51％。MS(EI)m/z:583.23[M+2H]²⁺；MS(ESI)m/z:1165.4287[M+H]⁺.Compound M57 (50 mg, 0.079 mM) was dispersed in DCM (10 mL), and HCOOH (1 mL) was added under stirring to dissolve all of M57. After stirring and reacting at room temperature for 4 hours, the solvent was removed by concentration under reduced pressure to obtain a light yellow semi-solid oily residue. After redissolution, the sample was mixed with crude silica gel and further purified by silica gel column chromatography to obtain compound-21 as an off-white glassy solid, 47 mg, with a yield of about 51%. MS (EI) m/z: 583.23 [M+2H]²⁺ ; MS (ESI) m/z: 1165.4287 [M+H]⁺ .

实施例22：化合物-22的制备
Example 22: Preparation of Compound-22

步骤22-1：M58的制备Step 22-1: Preparation of M58

按照M57的合成方法，采用M16替换M12与M56在HATU的作用下发生酰化反应，制备得到M58，灰白色玻璃状固体，收率约为49％。MS(EI)m/z:700.32[M+2H]²⁺；MS(ESI)m/z:1399.5392[M+H]⁺.According to the synthesis method of M57, M16 was used to replace M12 and acylated with M56 under the action of HATU to prepare M58, a grayish white glassy solid with a yield of about 49%. MS (EI) m/z: 700.32 [M+2H]²⁺ ; MS (ESI) m/z: 1399.5392 [M+H]⁺ .

步骤22-2：化合物-22的制备Step 22-2: Preparation of Compound-22

按照化合物-21的合成方法，采用M58替换M57在甲酸溶液中脱去Boc保护基，制备得到化合物-22，灰色玻璃状固体，收率约为52％。MS(EI)m/z:650.29[M+2H]²⁺；MS(ESI)m/z:1299.4873[M+H]⁺.According to the synthesis method of compound-21, M58 was used to replace M57 to remove the Boc protecting group in formic acid solution to prepare compound-22, a gray glassy solid, with a yield of about 52%. MS (EI) m/z: 650.29 [M + 2H]^{2 +} ; MS (ESI) m/z: 1299.4873 [M + H]⁺ .

实施例23：化合物-23的制备
Example 23: Preparation of Compound-23

步骤23-1：M59的制备Step 23-1: Preparation of M59

按照M4的合成方法，采用M54替换M3与MMAE反生酰化反应，制备得到M59，白色固体粉末，收率约为87％。MS(EI)m/z:708.95[M+2H]²⁺；MS(ESI)m/z:1416.843[M+H]⁺.According to the synthesis method of M4, M54 was used to replace M3 and react with MMAE to produce M59, a white solid powder, with a yield of about 87%. MS (EI) m/z: 708.95 [M + 2H]^{2 +} ; MS (ESI) m/z: 1416.843 [M + H]⁺ .

步骤23-2：M60的制备Step 23-2: Preparation of M60

按照M5的合成方法，采用M59替换M4在哌啶的DMF溶液中脱去Fmoc保护基，制备得到M60，白色固体粉末，收率约为68％。产物易吸潮，制备完成后直接用于下一步。MS(EI)m/z:597.84[M+2H]²⁺；MS(ESI)m/z:1194.7512[M+H]⁺.According to the synthesis method of M5, M59 was used to replace M4 to remove the Fmoc protecting group in a piperidine DMF solution to prepare M60, a white solid powder with a yield of about 68%. The product is easy to absorb moisture and is directly used in the next step after preparation. MS (EI) m/z: 597.84 [M + 2H]^{2 +} ; MS (ESI) m/z: 1194.7512 [M + H]⁺ .

步骤23-3：M61的制备Step 23-3: Preparation of M61

按照化合物-1的合成方法，采用M60替换M5与M12在HATU的催化下发生酰化反应，制备得到M61，白色固体粉末，收率约为49％。MS(EI)m/z:774.42[M+2H]²⁺；MS(ESI)m/z:1547.8262[M+H]⁺.According to the synthesis method of compound-1, M60 was used to replace M5 and acylated with M12 under the catalysis of HATU to prepare M61, a white solid powder, with a yield of about 49%. MS (EI) m/z: 774.42 [M + 2H]^{2 +} ; MS (ESI) m/z: 1547.8262 [M + H]⁺ .

步骤23-4：化合物-23的制备Step 23-4: Preparation of Compound-23

按照化合物-21的合成方法，采用M61替换M57在甲酸溶液中脱去Boc保护基，制备得到化合物-23，白色玻璃状固体，收率约为43％。MS(EI)m/z:724.43[M+2H]²⁺；MS(ESI)m/z:1447.7741[M+H]⁺.According to the synthesis method of compound-21, M61 was used to replace M57 to remove the Boc protecting group in formic acid solution to prepare compound-23, a white glassy solid, with a yield of about 43%. MS (EI) m/z: 724.43 [M + 2H]^{2 +} ; MS (ESI) m/z: 1447.7741 [M + H]⁺ .

实施例24：化合物-24的制备
Example 24: Preparation of Compound-24

步骤24-1：化合物-24的制备Step 24-1: Preparation of Compound-24

将化合物-23(45mg，～30μM)溶解于DMF(10mL)中，全部溶解后加入M36(20mg，60μM)的DMF溶液(5mL)，搅拌均匀后再加入催化量的DIPEA(10μL)，室温下搅拌反应过夜。待反应完成后，减压浓缩去除溶剂，所得油状物残渣进一步经硅胶柱层析纯化得到化合物-24，蜡状白色固体，31mg，收率约为62％。MS(EI)m/z:833.41[M+2H]²⁺；MS(ESI)m/z:1665.8876[M+H]⁺.Compound-23 (45 mg, ~30 μM) was dissolved in DMF (10 mL). After all the solution was dissolved, M36 (20 mg, 60 μM) in DMF solution (5 mL) was added. After stirring evenly, a catalytic amount of DIPEA (10 μL) was added. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The obtained oily residue was further purified by silica gel column chromatography to obtain compound-24, a waxy white solid, 31 mg, with a yield of about 62%. MS (EI) m/z: 833.41 [M+2H]²⁺ ; MS (ESI) m/z: 1665.8876 [M+H]⁺ .

实施例25：化合物-25的制备
Example 25: Preparation of Compound-25

步骤25-1：M62的制备Step 25-1: Preparation of M62

按照M59的合成方法，采用MMAF替换MMAE与M54反生酰化反应，制备得到M62，白色固体粉末，收率约为68％。MS(ESI)m/z:1430.8232[M+H]⁺.According to the synthesis method of M59, MMAF was used to replace MMAE and react with M54 to produce M62, a white solid powder, with a yield of about 68%. MS (ESI) m/z: 1430.8232 [M + H]⁺ .

步骤25-2：M63的制备Step 25-2: Preparation of M63

按照M5的合成方法，采用M62替换M4在哌啶的DMF溶液中脱去Fmoc保护基，制备得到M63，白色固体粉末，收率约为59％。制备完成后直接用于下一步。MS(ESI)m/z:1208.7549[M+H]⁺.According to the synthesis method of M5, M62 was used to replace M4 to remove the Fmoc protecting group in a piperidine DMF solution to prepare M63, a white solid powder with a yield of about 59%. After preparation, it was directly used in the next step. MS (ESI) m/z: 1208.7549 [M + H]⁺ .

步骤25-3：M64的制备Step 25-3: Preparation of M64

按照M61的合成方法，采用M63替换M60与M12在HATU的催化下发生酰化反应，制备得到M64，白色固体粉末，收率约为53％。MS(ESI)m/z:1561.8053[M+H]⁺.According to the synthesis method of M61, M63 was used to replace M60 and acylated with M12 under the catalysis of HATU to prepare M64, a white solid powder, with a yield of about 53%. MS (ESI) m/z: 1561.8053 [M + H]⁺ .

步骤25-4：化合物-25的制备Step 25-4: Preparation of Compound-25

按照化合物-23的合成方法，采用M64替换M61在甲酸溶液中脱去Boc保护基，制备得到化合物-25，白色玻璃状固体，收率约为37％。MS(EI)m/z:724.43[M+2H]²⁺；MS(ESI)m/z:1461.7533[M+H]⁺.According to the synthesis method of compound-23, M64 was used to replace M61 to remove the Boc protecting group in formic acid solution to prepare compound-25, a white glassy solid, with a yield of about 37%. MS (EI) m/z: 724.43 [M + 2H]^{2 +} ; MS (ESI) m/z: 1461.7533 [M + H]⁺ .

实施例26：化合物-26的制备
Example 26: Preparation of Compound-26

步骤26-1：化合物-26的制备Step 26-1: Preparation of Compound-26

按照化合物-24的合成方法，采用化合物-21替换化合物-23与M36发生酰化反应，制备得到化合物-26，白色固体粉末，收率约为47％。MS(ESI)m/z:1383.5443[M+H]⁺.According to the synthesis method of compound-24, compound-21 was used to replace compound-23 and acylated with M36 to prepare compound-26, a white solid powder, with a yield of about 47%. MS (ESI) m/z: 1383.5443 [M+H]⁺ .

实施例27：化合物-27的制备
Example 27: Preparation of Compound-27

步骤27-1：化合物-27的制备Step 27-1: Preparation of Compound-27

按照化合物-26的合成方法，采用乙酰八聚肌氨酸取代M36与化合物-21发生酰化反应，制备得到化合物-27，类白色固体粉末，收率约为61％。MS(ESI)m/z:1775.7363[M+H]⁺.According to the synthesis method of compound-26, acetyl octapolysarcosine was used to replace M36 and react with compound-21 to undergo acylation to prepare compound-27, an off-white solid powder, with a yield of about 61%. MS (ESI) m/z: 1775.7363 [M + H]⁺ .

实施例28：化合物-28的制备
Example 28: Preparation of Compound-28

步骤28-1：化合物-28的制备Step 28-1: Preparation of Compound-28

按照化合物-21(50mg，0.043mM)溶解于无水四氢呋喃(50mL)中，超声使全部溶解后，置于冰浴中降温至0℃，加入催化量的K₂CO₃并缓慢滴入MeI(12mg，0.086mM)的四氢呋喃溶液(10mL)，低温下搅拌反应1小时后，减压浓缩去除溶剂，所得残渣采用C-18反相色谱纯化制备得到化合物-28，类白色固体粉末，14.5mg，收率约为28％。MS(ESI)m/z:1193.4611[M+H]⁺.Compound-21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL), and after being completely dissolved by ultrasonication, the mixture was placed in an ice bath and cooled to 0°C. A catalytic amount of K₂ CO₃ was added and a tetrahydrofuran solution (10 mL) of MeI (12 mg, 0.086 mM) was slowly added dropwise. After stirring and reacting for 1 hour at low temperature, the mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by C-18 reverse phase chromatography to prepare compound-28, an off-white solid powder, 14.5 mg, with a yield of about 28%. MS (ESI) m/z: 1193.4611 [M+H]⁺ .

实施例29：化合物-29的制备
Example 29: Preparation of Compound-29

步骤29-1：化合物-29的制备Step 29-1: Preparation of Compound-29

按照化合物-21(50mg，0.043mM)溶解于无水四氢呋喃(50mL)中，超声使全部溶解后，置于冰浴中降温至0℃，滴加P(O)(OEt)₂Cl(0.045mM)，低温下搅拌反应1小时后转移至室温再搅拌反应2小时。待反应结束后，加入盐酸(10eq)并在室温下继续反应3小时。待反应完成后，减压浓缩去除溶剂，所得残渣采用C-18反相色谱纯化制备得到化合物-29，类白色固体粉末，17mg，收率约为32％。MS(ESI)m/z:1245.3962[M+H]⁺.Compound-21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL), and after being completely dissolved by ultrasound, it was placed in an ice bath and cooled to 0°C, and P(O)(OEt)₂ Cl (0.045 mM) was added dropwise. After stirring and reacting at low temperature for 1 hour, it was transferred to room temperature and stirred for another 2 hours. After the reaction was completed, hydrochloric acid (10 eq) was added and the reaction was continued at room temperature for 3 hours. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was purified by C-18 reverse phase chromatography to prepare compound-29, an off-white solid powder, 17 mg, with a yield of about 32%. MS (ESI) m/z: 1245.3962 [M + H]⁺ .

实施例30：化合物-30的制备
Example 30: Preparation of Compound-30

步骤30-1：化合物-30的制备Step 30-1: Preparation of Compound-30

按照参考文献(Carbohydrate Research(2006),341(8),947-956.)所述的方法制备得到4-(β-D-吡喃葡萄糖基氨基)-4-氧代丁酸(CAS No.:896730-79-3)，收率约为57％。不经久置而直接用于下一步反应。再按照化合物-26的合成方法，采用制备的4-(β-D-吡喃葡萄糖基氨基)-4-氧代丁酸替换M36与化合物-21发生酰化反应，制备得到化合物-30，白色固体粉末，收率约为45％。MS(ESI)m/z:1426.5133[M+H]⁺.4-(β-D-pyranose glucopyranose amino)-4-oxobutyric acid (CAS No.: 896730-79-3) was prepared according to the method described in the reference (Carbohydrate Research (2006), 341 (8), 947-956.) with a yield of about 57%. It was directly used in the next reaction without long-term storage. According to the synthesis method of compound-26, the prepared 4-(β-D-pyranose glucopyranose amino)-4-oxobutyric acid was used to replace M36 and acylate with compound-21 to prepare compound-30, a white solid powder with a yield of about 45%. MS (ESI) m/z: 1426.5133 [M+H]⁺ .

实施例31：化合物-31的制备
Example 31: Preparation of Compound-31

步骤31-1：化合物-31的制备Step 31-1: Preparation of Compound-31

按照化合物-21(50mg，0.043mM)溶解于无水四氢呋喃(50mL)中，超声使全部溶解后，加入吡啶(2eq)后置于冰浴中降温至0℃，缓慢滴加ClSO₃H(1eq)，低温下搅拌反应1小时后转移至室温再搅拌反应2小时。待反应结束后减压浓缩去除溶剂，所得残渣采用C-18反相色谱纯化制备得到化合物-31，灰白色固体粉末，15mg，收率约为28％。MS(ESI)m/z:1245.3857[M+H]⁺.Compound-21 (50 mg, 0.043 mM) was dissolved in anhydrous tetrahydrofuran (50 mL), and after being completely dissolved by ultrasound, pyridine (2 eq) was added and placed in an ice bath to cool to 0°C, ClSO₃ H (1 eq) was slowly added dropwise, and the mixture was stirred at low temperature for 1 hour and then transferred to room temperature and stirred for another 2 hours. After the reaction was completed, the solvent was removed by vacuum concentration, and the residue was purified by C-18 reverse phase chromatography to prepare compound-31, an off-white solid powder, 15 mg, with a yield of about 28%. MS (ESI) m/z: 1245.3857 [M+H]⁺ .

实施例32：化合物-35的制备

Example 32: Preparation of Compound-35

步骤35-1：M65的制备Step 35-1: Preparation of M65

将1-氨基-3,6,9,12-四氧十五烷-15-酸(5g，18.85mM)溶解于50mL的饱和NaHCO₃中，分散均匀后将反应体系置于冰浴中进行降温。加入2,5-二氧代-2,5-二氢-1H-吡咯-1-羧酸甲酯(3.22g，20.73g)，继续在低温下搅拌反应半小时后，转移至室温下继续反应2小时。采用HCl将反应液的pH调至～2，浓缩去除反应溶剂，采用甲醇复溶后采用粗硅胶拌样，乙酸乙酯洗脱，得到产物M65为无色油状物，5.31g，收率约为75％。MS(EI)m/z:346.16[M+H]⁺；MS(EI)m/z:368.15[M+Na]⁺.1-Amino-3,6,9,12-tetrahydropentadecan-15-acid (5 g, 18.85 mM) was dissolved in 50 mL of saturated NaHCO₃ , and the reaction system was placed in an ice bath for cooling after being evenly dispersed. 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester (3.22 g, 20.73 g) was added, and the reaction was continued to stir at low temperature for half an hour, and then transferred to room temperature for 2 hours. The pH of the reaction solution was adjusted to ~2 with HCl, and the reaction solvent was removed by concentration. After re-dissolving with methanol, the sample was mixed with crude silica gel, and eluted with ethyl acetate to obtain the product M65 as a colorless oil, 5.31 g, with a yield of about 75%. MS (EI) m/z: 346.16 [M + H]⁺ ; MS (EI) m/z: 368.15 [M + Na]⁺ .

步骤35-2：M66的制备Step 35-2: Preparation of M66

将M65(5g，14.48mM)溶解于四氢呋喃(40mL)中，依次加入NHS(2.17g，18.82mM)和DCC(3.88g，18.82mM)，反应液在低温下搅拌反应半小时后，转移至室温下继续反应2小时。待反应结束后，过滤去除不溶物DCU，滤液浓缩得到浅黄色油状物残渣。进一步经硅胶柱层析纯化得到M66为浅黄色粘稠油状物，4.70g，收率约为74％。MS(EI)m/z:443.19[M+H]⁺；MS(EI)m/z:465.20[M+Na]⁺.M65 (5 g, 14.48 mM) was dissolved in tetrahydrofuran (40 mL), and NHS (2.17 g, 18.82 mM) and DCC (3.88 g, 18.82 mM) were added in sequence. The reaction solution was stirred at low temperature for half an hour and then transferred to room temperature for further reaction for 2 hours. After the reaction was completed, the insoluble DCU was removed by filtration, and the filtrate was concentrated to obtain a light yellow oily residue. M66 was further purified by silica gel column chromatography to obtain a light yellow viscous oily substance, 4.70 g, with a yield of about 74%. MS (EI) m/z: 443.19 [M + H]⁺ ; MS (EI) m/z: 465.20 [M + Na]⁺ .

步骤35-3：化合物-35的制备Step 35-3: Preparation of Compound-35

按照化合物-14的合成方法，采用M66替代M16与M42发生缩合反应，制备得到化合物-35，类白色固体粉末，收率约为50％。MS(EI)m/z:757.74[M+2H]²⁺；MS(ESI)m/z:1514.7115[M+H]⁺.According to the synthesis method of compound-14, M66 was used to replace M16 and react with M42 to produce compound-35, an off-white solid powder, with a yield of about 50%. MS (EI) m/z: 757.74 [M + 2H]^{2 +} ; MS (ESI) m/z: 1514.7115 [M + H]⁺ .

实施例33：化合物-39的制备
Example 33: Preparation of Compound-39

步骤39-1：化合物-39的制备Step 39-1: Preparation of Compound-39

按照参考文献(WO2023125530A1；P44，化合物2i)所述的方法制备得到M70。再按照化合物-36的合成方法，采用M12替代McOSu与M70发生缩合反应，制备得到化合物-39，浅黄色固体粉末，收率约为39％。MS(EI)m/z:875.38[M+2H]²⁺；MS(ESI)m/z:1749.8582[M+H]⁺.M70 was prepared according to the method described in reference (WO2023125530A1; P44, compound 2i). Then, according to the synthesis method of compound-36, M12 was used to replace McOSu and react with M70 to obtain compound-39, a light yellow solid powder, with a yield of about 39%. MS (EI) m/z: 875.38 [M + 2H]^{2 +} ; MS (ESI) m/z: 1749.8582 [M + H]⁺ .

实施例34：化合物-40的制备
Example 34: Preparation of Compound-40

步骤40-1：化合物-40的制备Step 40-1: Preparation of Compound-40

按照参考文献(WO2023125530A1；P84，化合物27h)所述的方法制备得到M71。再按照化合物-39的合成方法，采用M71替代M70与M12发生缩合反应，制备得到化合物-40，浅黄色固体粉末，收率约为36％。MS(EI)m/z:734.77[M+2H]²⁺；MS(ESI)m/z:1468.4982[M+H]⁺.M71 was prepared according to the method described in reference (WO2023125530A1; P84, compound 27h). Then, according to the synthesis method of compound-39, M71 was used to replace M70 and react with M12 to prepare compound-40, a light yellow solid powder, with a yield of about 36%. MS (EI) m/z: 734.77 [M + 2H]^{2 +} ; MS (ESI) m/z: 1468.4982 [M + H]⁺ .

实施例35：化合物-41的结构及制备方法
Example 35: Structure and preparation of compound-41

步骤41-1：M72的制备Step 41-1: Preparation of M72

将苄氧乙酸(46.29mg，0.279mM)溶解于DMF(8mL)中，依次加入HATU(105.92mg，0.279mM)和DIPEA(36mg，0.279mM)，待固体全部溶解后，将反应体系置于冰浴中降温。加入MMAE(100mg，0.139mM)，在低温下反应1小时后，缓慢升至室温继续反应4小时。待反应完成后，减压浓缩去除溶剂得油状物残渣，经C-18反相柱层析纯化，进一步冻干后得到M72为白色固体粉末，73mg，收率约为61％。MS(EI)m/z:866.80[M+H]⁺.Benzyloxyacetic acid (46.29 mg, 0.279 mM) was dissolved in DMF (8 mL), and HATU (105.92 mg, 0.279 mM) and DIPEA (36 mg, 0.279 mM) were added in sequence. After all the solids were dissolved, the reaction system was placed in an ice bath to cool down. MMAE (100 mg, 0.139 mM) was added, and after reacting at low temperature for 1 hour, the temperature was slowly raised to room temperature and the reaction was continued for 4 hours. After the reaction was completed, the solvent was removed by vacuum concentration to obtain an oily residue, which was purified by C-18 reverse phase column chromatography and further freeze-dried to obtain M72 as a white solid powder, 73 mg, with a yield of about 61%. MS (EI) m/z: 866.80 [M + H]⁺ .

步骤41-2：化合物-41的制备Step 41-2: Preparation of Compound-41

将M72(70mg，0.081mM)溶解于甲醇(15mL)中，全部溶解后，加入10％的钯炭(0.5eq)，并在氢气的氛围中室温搅拌反应过夜。待反应完成后，减压浓缩去除溶剂，所得残渣经C-18反相柱层析纯化，进一步冻干后得到化合物-41为白色固体粉末，60mg，收率约为96％。MS(EI)m/z:776.75[M+H]⁺；MS(EI)m/z:798.66[M+Na]⁺.M72 (70 mg, 0.081 mM) was dissolved in methanol (15 mL). After all the solution was dissolved, 10% palladium carbon (0.5 eq) was added and the reaction was stirred overnight at room temperature in a hydrogen atmosphere. After the reaction was completed, the solvent was removed by concentration under reduced pressure. The residue was purified by C-18 reverse phase column chromatography and further freeze-dried to obtain compound-41 as a white solid powder, 60 mg, with a yield of about 96%. MS (EI) m/z: 776.75 [M + H]⁺ ; MS (EI) m/z: 798.66 [M + Na]⁺ .

实施例36：抗体偶联药物的制备Example 36: Preparation of Antibody-Drug Conjugates

在ADC的制备过程中，ADC的抗体选择了抗HER-2的人源化IgG抗体曲妥珠单抗(Trastuzumab)，以及抗B7-H3(CD276)的人源化IgG抗体CE4-Z3，以验证所制备的新型连接子-有效荷载的偶联性能、成药性以及体内外药效特征。During the preparation of ADC, the antibodies selected for ADC were humanized IgG antibodies Trastuzumab against HER-2 and CE4-Z3 against B7-H3 (CD276) to verify the coupling performance, drugability, and in vitro and in vivo efficacy characteristics of the prepared new linker-payload.

步骤36-1：DAR4 ADC的一般制备方法Step 36-1: General preparation method of DAR4 ADC

①抗体的置换：进行偶联之前，将所选择的抗体(IgG)经搭配G25葡聚糖凝胶柱的AKTA系统进行脱盐处理，将蛋白置换于偶联体系缓冲液中(PBS:20mM；EDTA:2mM；pH 6.5～8.0。或L-HIs:20mM；EDTA:2mM；pH 6.5～8.0)。通过紫外分光光度计对蛋白进行吸光度测定，并通过消光系数法计算换液后的抗体浓度。当抗体浓度低于偶联浓度时，先进行超滤浓缩以提高蛋白浓度。① Replacement of antibodies: Before coupling, the selected antibody (IgG) is desalted by the AKTA system with a G25 dextran gel column, and the protein is replaced in the coupling system buffer (PBS: 20mM; EDTA: 2mM; pH 6.5-8.0. Or L-HIs: 20mM; EDTA: 2mM; pH 6.5-8.0). The absorbance of the protein is measured by a UV spectrophotometer, and the antibody concentration after the liquid exchange is calculated by the extinction coefficient method. When the antibody concentration is lower than the coupling concentration, ultrafiltration concentration is first performed to increase the protein concentration.

②偶联反应液的准备：根据所需偶联抗体的量(1eq)，采用移液枪精确移取抗体对应的偶联体系缓冲液，使得抗体浓度为偶联初始浓度，通常1～10mg/mL。② Preparation of coupling reaction solution: According to the amount of coupled antibody required (1eq), use a pipette to accurately transfer the coupling system buffer corresponding to the antibody so that the antibody concentration is the initial coupling concentration, usually 1-10 mg/mL.

③抗体的还原：在缓慢搅拌下，向装有抗体溶液的反应小瓶中加入2.0～2.5eq的TCEP·HCl溶液(1.0mg/mL～5.73mg/mL)；加完后，室温下徐徐搅拌，反应60～180min。③ Antibody reduction: Add 2.0 to 2.5 eq of TCEP·HCl solution (1.0 mg/mL to 5.73 mg/mL) to the reaction vial containing the antibody solution under slow stirring; after the addition, stir slowly at room temperature and react for 60 to 180 min.

④抗体的偶联：计算需要加入的有机溶剂(DMAC或DMSO)的体积，使其占总体积的5％～15％(通常占比≤10％)；同时计算需要加入的小分子荷载(Linker-Payload)的质量，通常小分子荷载需要稍微过量(通常为8eq)，进而计算出所需加入的荷载的有机溶液的浓度。精确配制荷载的溶液后，将其缓慢滴加到已经还原的抗体反应液中。室温下继续缓慢搅拌，根据具体偶联情况反应0.25～1.0h。④ Antibody coupling: Calculate the volume of organic solvent (DMAC or DMSO) to be added so that it accounts for 5% to 15% of the total volume (usually ≤10%); at the same time, calculate the mass of the small molecule load (Linker-Payload) to be added. Usually, the small molecule load needs to be slightly excessive (usually 8eq), and then calculate the concentration of the organic solution of the load to be added. After accurately preparing the load solution, slowly add it dropwise to the reduced antibody reaction solution. Continue to stir slowly at room temperature and react for 0.25 to 1.0h depending on the specific coupling situation.

⑤反应的淬灭：反应液达到预定的偶联时间后，加入过量的包含还原性巯基的水溶性小分子N-乙酰基半胱氨酸(NAC)溶液(1.0～3.26mg/mL)，缓慢搅拌继续反应15～60min。⑤ Quenching of the reaction: After the reaction solution reaches the predetermined coupling time, add an excess of a water-soluble small molecule N-acetylcysteine (NAC) solution (1.0-3.26 mg/mL) containing a reducing thiol group, and continue the reaction with slow stirring for 15-60 minutes.

⑥ADC产物纯化：待偶联反应淬灭完成后，先将反应液经过滤，再采用搭配G25葡聚糖凝胶柱的AKTA系统进行脱盐处理，收集前段(约为80％)的组分流出液，再次经超滤浓缩后，无菌过滤并进行样品分装；除预留的用于分析的部分ADC样品置于4℃下短期存储外，其它ADC产品置于-80℃下存储待用。⑥ ADC product purification: After the coupling reaction is quenched, the reaction solution is first filtered, and then desalted using an AKTA system with a G25 dextran gel column. The effluent of the front section (about 80%) is collected, concentrated by ultrafiltration again, and then sterile filtered and sample packaged; except for some ADC samples reserved for analysis that are stored at 4°C for a short period of time, other ADC products are stored at -80°C for use.

步骤36-2：DAR8 ADC的一般制备方法Step 36-2: General preparation method of DAR8 ADC

按照步骤36-1中的操作，将③抗体的还原步骤中的TCEP·HCl用量调整为4.0～8.0eq，并将④抗体的偶联步骤中的连接子-有效荷载和⑤反应的淬灭步骤中的NAC用量调整为8～12eq。制备得到DAR≈8.0的ADC产物。According to the operation in step 36-1, the amount of TCEP·HCl in the reduction step of the antibody (③) was adjusted to 4.0-8.0 eq, and the amount of linker-payload in the coupling step of the antibody (④) and the amount of NAC in the quenching step of the reaction (⑤) were adjusted to 8-12 eq. An ADC product with DAR≈8.0 was prepared.

步骤36-3：ADC的DAR值分析的一般方法Step 36-3: General method for analyzing the DAR value of ADC

①以MMAE为有效荷载的ADC的DAR值分析① Analysis of DAR values of ADCs with MMAE as effective load

在室温下使用丁基HIC柱(TSK凝胶丁基NPR 4.6×35mm 2.5μm，Tosoh Bioscience)分离每个抗体具有不同药物数量的ADC。HIC也在Agilent 1260Infinity II HPLC系统或岛津高效液相色谱系统上进行，在280nm下进行UV检测。流动相A为1.5mmol/L(NH₄)₂SO₄、50mmol/L K₂HPO₄，pH 7.0，流动相B为21.3mmol/L KH₂PO₄、28.6mmol/L K₂HPO₃、25％(v/v)异丙醇，pH 7.0。梯度程序如下：B％：0％至25％(0-1分钟，0.8mL/分钟)、25％(1-3分钟，0.6mL/分钟)。对DAR＝0、DAR＝2、DAR＝4、DAR＝6、DAR＝8所对应色谱峰面积进行积分并加权，计算被测ADC的DAR值。ADCs with different drug amounts per antibody were separated using a butyl HIC column (TSK gel butyl NPR 4.6×35 mm 2.5 μm, Tosoh Bioscience) at room temperature. HIC was also performed on an Agilent 1260 Infinity II HPLC system or a Shimadzu HPLC system with UV detection at 280 nm. Mobile phase A was 1.5 mmol/L (NH₄ )₂ SO₄ , 50 mmol/L K₂ HPO₄ , pH 7.0, and mobile phase B was 21.3 mmol/L KH₂ PO₄ , 28.6 mmol/L K₂ HPO₃ , 25% (v/v) isopropanol, pH 7.0. The gradient program was as follows: B%: 0% to 25% (0-1 min, 0.8 mL/min), 25% (1-3 min, 0.6 mL/min). The chromatographic peak areas corresponding to DAR=0, DAR=2, DAR=4, DAR=6, and DAR=8 were integrated and weighted to calculate the DAR value of the measured ADC.

②以Dx-8951f或Dxd为有效荷载的ADC的DAR值分析② Analysis of DAR values of ADCs with Dx-8951f or Dxd as effective load

采用紫外分光光度计分别测定待测ADC在280nm和360nm波长下的吸光度，平行测定3次，然后分别计算两个波长下吸光度的平均值。由于抗体在连接子-有效荷载的特征吸收波长360nm下并无紫外吸收，采用标准曲线法计算出连接子-有效荷载的质量浓度和A360的关系(一元一次方程)，以及A360与A280的倍数关系。通过计算得到的A360计算出ADC中连接子-有效荷载的质量浓度，以及连接子-有效荷载的A280值。最后根据朗伯比尔定律，任何给定波长下的总吸光度等于系统中存在的所有吸光化学物质的在该波长下的吸光度之和，通过A_mAb＝A_平均-A_{连接子-有效荷载}，计算抗体在A280下对应的吸光度值A_mAb；最后通过计算出溶液中连接子-有效荷载与抗体的摩尔量比值，计算出ADC的DAR值。The absorbance of the ADC to be tested at 280nm and 360nm wavelengths was measured by ultraviolet spectrophotometer, and the measurement was performed three times in parallel, and then the average absorbance at the two wavelengths was calculated. Since the antibody has no ultraviolet absorption at the characteristic absorption wavelength of 360nm of the linker-payload, the standard curve method was used to calculate the relationship between the mass concentration of the linker-payload and A360 (a linear equation), as well as the multiple relationship between A360 and A280. The mass concentration of the linker-payload in the ADC and the A280 value of the linker-payload were calculated by the calculated A360. Finally, according to the Lambert-Beer law, the total absorbance at any given wavelength is equal to the sum of the absorbances of all light-absorbing chemicals present in the system at that wavelength. The absorbance value A_mAb corresponding to the antibody at A280 was calculated by A_mAb = A_average_{- A linker-payload} ; finally, the DAR value of the ADC was calculated by calculating the molar ratio of the linker-payload to the antibody in the solution.

③LC-MS法进行ADC-DAR值分析③ LC-MS method for ADC-DAR value analysis

取ADC或裸抗样品100μg，加入PNGaseF 500U 1μl，37℃酶切过夜，获得待测样品。液质联用参数设置：液相系统采用ACQUITY UPLC H-Class(Waters)，色谱柱为ACQUITY UPLC Protein,BEH SEC,1.7μm,2.1mm×100mm，柱温25℃，流动相为100mM乙酸铵水溶液，等度洗脱，流速为0.1ml/min。质谱检测系统为：Xevo G2-XS Qtof(Waters)，检测模式为正离子，全扫描方式。数据采集采用MassLynx 4.1(Waters)软件，并经过MaxEnt I去卷积处理。模型参数设置分辨率为2-3.5Da。最小强度比率设置60％，输出分辨率设置为1Da。算法迭代参数设置为20。通过对去卷积化处理后的ADC的DAR＝0、DAR＝2、DAR＝4、DAR＝6、DAR＝8所对应TIC峰高进行加权，计算被测ADC的DAR值。Take 100 μg of ADC or naked antibody sample, add 1 μl of PNGaseF 500U, and digest overnight at 37℃ to obtain the sample to be tested. LC-MS parameter setting: The liquid phase system uses ACQUITY UPLC H-Class (Waters), and the chromatographic column is ACQUITY UPLC Protein, BEH SEC, 1.7μm, 2.1mm×100mm, column temperature 25℃, mobile phase 100mM ammonium acetate aqueous solution, isocratic elution, flow rate 0.1ml/min. The mass spectrometry detection system is: Xevo G2-XS Qtof (Waters), the detection mode is positive ion, full scan mode. Data acquisition uses MassLynx 4.1 (Waters) software and is deconvoluted by MaxEnt I. The model parameter sets the resolution to 2-3.5Da. The minimum intensity ratio is set to 60%, and the output resolution is set to 1Da. The algorithm iteration parameter is set to 20. The DAR value of the ADC tested is calculated by weighting the TIC peak heights corresponding to DAR＝0, DAR＝2, DAR＝4, DAR＝6, and DAR＝8 of the ADC after deconvolution.

步骤36-4：ADC的聚合度分析的一般方法Step 36-4: General method for ADC polymerization analysis

通过尺寸排阻色谱法评估ADC产品的聚集，该色谱法在Agilent 1260Infinity II HPLC系统或岛津高效液相色谱系统上进行，在280nm处进行UV检测。该柱是TSKgel G3000SWXL柱(Tosoh Bioscience)。以20～50μg的负荷注入样品。流动相为200mmol/L磷酸钠和150mmol/L氯化钠(pH 7.0)。此外，在流动相中加入10％(v/v)异丙醇，以最大限度地减少与固定相的二次疏水相互作用，并防止细菌生长。柱温度设定为室温。对被测ADC的SEC色谱下对应的单体峰、碎片峰以及聚合物峰的峰面积进行积分，并通过面积归一化法计算被测ADC的单体含量所占的百分比。Aggregation of ADC products was evaluated by size exclusion chromatography, which was performed on an Agilent 1260 Infinity II HPLC system or a Shimadzu HPLC system with UV detection at 280 nm. The column was a TSKgel G3000SWXL column (Tosoh Bioscience). Samples were injected at a load of 20-50 μg. The mobile phase was 200 mmol/L sodium phosphate and 150 mmol/L sodium chloride (pH 7.0). In addition, 10% (v/v) isopropanol was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth. The column temperature was set to room temperature. The peak areas of the corresponding monomer peaks, fragment peaks, and polymer peaks under the SEC chromatogram of the tested ADC were integrated, and the percentage of monomer content of the tested ADC was calculated by area normalization.

所制备的ADC的结构如下表所示：

The structure of the prepared ADC is shown in the following table:

其中，in,

表示曲妥珠单抗或CE4-Z3。

Denotes trastuzumab or CE4-Z3.

其中，in,

表示曲妥珠单抗或CE4-Z3。 Denotes trastuzumab or CE4-Z3.

实施例37：抗体偶联药物的制备Example 37: Preparation of Antibody-Drug Conjugates

采用抗B7-H3(CD276)的人源化重链骆驼抗体(HCAb)替换实施例36中的传统IgG型抗体，与所制备的新型连接子-有效荷载相偶联，制备小型抗体HCAb偶联药物(HCAb-Drug conjugates，HDCs)，以验证上述连接子-有效荷载的普适性。相关偶联方法和偶联后表征按照实施例36所述进行。Humanized heavy chain camel antibody (HCAb) against B7-H3 (CD276) was used to replace the traditional IgG antibody in Example 36, and coupled with the prepared novel linker-payload to prepare small antibody HCAb-drug conjugates (HDCs) to verify the universality of the above linker-payload. The relevant coupling method and post-coupling characterization were carried out as described in Example 36.

为与传统结构的连接子-有效荷载做对比，通过伊诺凯试剂平台商业途径直接购买MC-VC-PABC-MMAE(Cas No.:646502-53-6)和MC-GGFG-Dxd(Cas No.:1599440-13-7)，直接与蛋白进行偶联。其中，MC-VC-PABC-MMAE和MC-GGFG-Dxd在本发明中对应的化合物代号分别为化合物-42和化合物-43。MC-VC-PABC-MMAE和MC-GGFG-Dxd的化学结构如下：
In order to compare with the linker-payload of the traditional structure, MC-VC-PABC-MMAE (Cas No.: 646502-53-6) and MC-GGFG-Dxd (Cas No.: 1599440-13-7) were purchased directly through the commercial channel of the Inokai reagent platform and directly coupled with the protein. Among them, the compound codes corresponding to MC-VC-PABC-MMAE and MC-GGFG-Dxd in the present invention are compound-42 and compound-43, respectively. The chemical structures of MC-VC-PABC-MMAE and MC-GGFG-Dxd are as follows:

所制备的HDC的结构如下：
The structure of the prepared HDC is as follows:

其中，in,

表示CA2-VHH25。 Indicates CA2-VHH25.

实施例38：抗体偶联药物的亲水性/疏水性研究Example 38: Hydrophilicity/hydrophobicity study of antibody-drug conjugates

为直接对比本发明中的基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC与基于传统琥珀酰亚胺接头的ADC的亲/疏水性差异，本发明采用结构更简单的HCAb偶联物替代IgG偶联物率先开展了包含2-(甲基磺酰基)噻唑[5,4-b]吡啶接头和包含传统琥珀酰亚胺接头的对比研究。In order to directly compare the hydrophilicity/hydrophobicity differences between the ADC based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and the ADC based on the traditional succinimide linker in the present invention, the present invention uses a simpler HCAb conjugate to replace the IgG conjugate and first conducts a comparative study containing a 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and a traditional succinimide linker.

(一)基于HCAb偶联的HDC的亲水性/疏水性分析(I) Hydrophilicity/hydrophobicity analysis of HDC based on HCAb coupling

在相同的偶联条件下，通常疏水性较强时，通常会导致更高的ADC聚合。在DAR值基本相当的情况下，通常连接子-有效荷载的亲水性越高，偶联后产物的单体占比也越高。本发明首先检测了所制备的HDC的聚合度水平，以初步判定所制备的连接子-有效荷载的亲水性/疏水性。通常，偶联后要求单体占比不低于95％。
Under the same coupling conditions, when the hydrophobicity is stronger, it usually leads to higher ADC polymerization. When the DAR values are basically the same, the higher the hydrophilicity of the linker-payload, the higher the monomer ratio of the coupled product. The present invention first detects the polymerization degree level of the prepared HDC to preliminarily determine the hydrophilicity/hydrophobicity of the prepared linker-payload. Generally, the monomer ratio is required to be not less than 95% after coupling.

分析以上表中数据可以看出：Analyzing the data in the above table, we can see that:

①通过对比HDC-14E和HDC-14F的聚合度数据可以看出，降低HCAb的DAR值可以提高HDC的单体含量。① By comparing the polymerization degree data of HDC-14E and HDC-14F, it can be seen that reducing the DAR value of HCAb can increase the monomer content of HDC.

②通过对比HDC-14E和HDC-35E聚合度数据可以看出，基于2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC比基于传统琥珀酰亚胺接头的ADC具有更高的亲水性。② By comparing the polymerization degree data of HDC-14E and HDC-35E, it can be seen that ADC based on 2-(methylsulfonyl)thiazole[5,4-b]pyridine linker has higher hydrophilicity than ADC based on traditional succinimide linker.

(二)基于HCAb偶联的HDC的HIC分析(II) HIC analysis based on HCAb-coupled HDC

通常，在疏水作用色谱(HPLC-HIC)中，偶联不同有效荷载的抗体偶联药物HDC的各个组分也会根据组分的亲水性大小先后出峰(裸抗(DAR＝0)保留时间最短，其次是DAR＝2，DAR＝4的组分)。因此，可以根据HDC在HPLC-HIC上的保留时间，判断对应的连接子-有效荷载的疏水性强弱。
Generally, in hydrophobic interaction chromatography (HPLC-HIC), the components of the antibody-drug conjugate HDC coupled with different payloads will also peak in sequence according to the hydrophilicity of the components (the naked antibody (DAR=0) has the shortest retention time, followed by the components with DAR=2 and DAR=4). Therefore, the hydrophobicity of the corresponding linker-payload can be judged based on the retention time of HDC on HPLC-HIC.

分析以上表中数据可以看出：在DAR值基本相当的情况下(DAR＝4)，基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的HDC-14E比基于传统琥珀酰亚胺接头的HDC-35E在HPLC-HIC上具有显著更接近于裸抗(DAR＝0)的保留时间，证明基于2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的连接子较基于传统琥珀酰亚胺接头的连接子具有更高的亲水性。Analysis of the data in the above table shows that: when the DAR values are basically the same (DAR=4), HDC-14E based on the new 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has a retention time on HPLC-HIC that is significantly closer to that of the naked antibody (DAR=0) than HDC-35E based on the traditional succinimide linker, proving that the linker based on the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has a higher hydrophilicity than the linker based on the traditional succinimide linker.

实施例39：ADC的稳定性研究Example 39: Stability study of ADC

基于新接头ADC的稳定性改善Improved ADC stability based on new connector

为验证基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC的稳定性特征，本发明以基于传统琥珀酰亚胺接头的ADC做对比，选择了ADC-14B和ADC-35B为模型ADC分子，开展了上述两种基于不同接头结构的ADC在人血浆和小鼠血浆中的稳定性研究。评估中的ADC的连接子中均包含PEG片段，评估ADC接头稳定性的参数为抗体偶联的药物(acDrug)的剩余含量随着血浆孵育时间的含量变化。结果如图2和图3所示。In order to verify the stability characteristics of ADC based on the new 2-(methylsulfonyl)thiazole[5,4-b]pyridine linker, the present invention compared the ADC based on the traditional succinimide linker, selected ADC-14B and ADC-35B as model ADC molecules, and carried out the stability study of the above two ADCs based on different linker structures in human plasma and mouse plasma. The linkers of the ADCs under evaluation all contain PEG fragments, and the parameters for evaluating the stability of the ADC linker are the changes in the residual content of the antibody-coupled drug (acDrug) with the plasma incubation time. The results are shown in Figures 2 and 3.

上述ADC-14B和ADC-35B在人血浆和小鼠血浆中的稳定性试验结果显示：在连接子中的抗体端接头位置，采用本发明开发的新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头替代传统琥珀酰亚胺接头，可以显著增加抗体偶联的药物的含量(P值分别为0.0006和0.0124)，即基于2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC在血浆中具有更低的连接子-有效荷载脱落率。The stability test results of the above ADC-14B and ADC-35B in human plasma and mouse plasma show that: at the antibody end linker position in the linker, the use of the new 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker developed by the present invention to replace the traditional succinimide linker can significantly increase the content of the antibody-conjugated drug (P values are 0.0006 and 0.0124, respectively), that is, the ADC based on the 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has a lower linker-payload shedding rate in plasma.

实施例40：ADC的体外细胞毒性研究Example 40: In vitro cytotoxicity study of ADC

本发明为基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头筛选和匹配更合理的连接子-有效荷载组合。The present invention is based on the screening and matching of more reasonable linker-payload combinations based on a novel 2-(methylsulfonyl)thiazole[5,4-b]pyridine linker.

本发明评估了游离MMAE、Dxd、化合物-41和星形孢菌素(作为对照使用)在上述肿瘤细胞系上的体外活性。在细胞系的选择方面，HER2高表达的细胞系包括乳腺癌细胞系BT-474、SK-BR-3和胃癌细胞系NCI-N87，HER2中表达的细胞系有MDA-MB-453，HER2低表达的细胞系有MCF-7，HER2不表达的细胞系有MDA-MB-231。The present invention evaluates the in vitro activity of free MMAE, Dxd, compound-41 and staurosporine (used as a control) on the above tumor cell lines. In terms of the selection of cell lines, HER2 highly expressed cell lines include breast cancer cell lines BT-474, SK-BR-3 and gastric cancer cell line NCI-N87, HER2 medium expressed cell lines include MDA-MB-453, HER2 low expressed cell lines include MCF-7, and HER2 non-expressed cell lines include MDA-MB-231.

如图4所示，游离形式的有效荷载活性强弱顺序为：MMAE>Dxd>化合物-41。由于游离化合物-41的EC50值远差于Dxd，而化合物-41经偶联后得到的ADC(DAR4)与基于Dxd的ADC(DAR8)的体外活性差异较小，因此，基于本发明中的化合物-41作为ADC有效荷载时具有提高药物治疗窗口的应用潜力。As shown in Figure 4, the order of the activity of the free form of the effective load is: MMAE>Dxd>Compound-41. Since the EC50 value of the free compound-41 is much worse than that of Dxd, and the in vitro activity difference between the ADC (DAR4) obtained after coupling with compound-41 and the ADC (DAR8) based on Dxd is small, therefore, the compound-41 in the present invention has the potential to improve the drug therapeutic window when used as an ADC effective load.

本发明基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头，制备了化合物-1～化合物-31，以及化合物-39和化合物-40，并将其与多种类型的抗体(以anti-HER2和anti-CD276的IgG型抗体，和/或HCAb为例)相偶联，制备得到抗体偶联药物(ADC或HCAb)，并用于开展更进一步的成药性研究。The present invention is based on a novel 2-(methylsulfonyl)thiazole[5,4-b]pyridine linker to prepare compounds-1 to -31, as well as compounds-39 and -40, and couple them with various types of antibodies (taking anti-HER2 and anti-CD276 IgG antibodies, and/or HCAb as examples) to prepare antibody-drug conjugates (ADC or HCAb), which are used to carry out further drugability studies.

实施例41：ADC的在小鼠模型上的体内药效研究Example 41: In vivo efficacy study of ADC in mouse model

为了验证所制备的新型连接子-有效荷载所制备的抗体偶联药物的体内药效，本发明分别选择了抗B7-H3的人源化IgG1型抗体CE4-Z3(简写为Z3)和人源化HCAb型抗体CA2-VHH25(简写为VHH25)所对应的抗体偶联药物，在B7-H3高表达的人肺癌Calu-6异种移植CDX模型上评估了这些ADC(含HDC)的体内药效特征。In order to verify the in vivo efficacy of the antibody-drug conjugates prepared by the prepared novel linker-payload, the present invention selected the antibody-drug conjugates corresponding to the humanized IgG1 antibody CE4-Z3 (abbreviated as Z3) against B7-H3 and the humanized HCAb antibody CA2-VHH25 (abbreviated as VHH25), and evaluated the in vivo efficacy characteristics of these ADCs (including HDC) in the human lung cancer Calu-6 xenograft CDX model with high expression of B7-H3.

该体内药效试验旨在研究受试ADC在人肺癌Calu-6细胞株皮下异种移植的雌性小鼠动物模型中的抗肿瘤作用。受试动物选用的是6～8周龄的雌性Balb/c-nude小鼠，在温度20～26℃，湿度40～70％的环境中饲养，每受试组/盒分开饲养。Calu-6细胞在MEM(+0.01mM NEAA)+10％FBS的培养基中传代，收集指数生长期的细胞，重悬细胞至合适浓度后用于小鼠皮下肿瘤接种。实验小鼠分别于右侧背部皮下接种5×10⁶的Calu-6细胞，待肿瘤生长至平均体积约为100mm³时，根据肿瘤大小和小鼠体重，采用随机分组设计的方法(采用StudyDirector^TM软件进行分组，版本号3.1.399.19，供应商Studylog System,Inc.,S.San Francisco,CA,USA)对小鼠进行分组，并于分组后当天按设定的剂量进行给药，给药体积根据V＝10μL/g计算。肿瘤体积按照V(mm³)＝1/2×(长径×短径²)进行计算，同时，每周测量两次动物的体重。每次测量肿瘤体积和小鼠体重时，同时观察和记录受试药物对动物正常行为的影响。数据以平均值±SD表示，指标采用非配对双尾t检验进行统计分析，统计显著性水平设定为p<0.05。使用GraphPad软件(Version8.0.2,La Jolla,CA,USA)进行统计分析。This in vivo efficacy test is designed to study the anti-tumor effect of the tested ADC in a female mouse animal model of subcutaneous xenografts of human lung cancer Calu-6 cell lines. The test animals are 6-8 week old female Balb/c-nude mice, which are raised in an environment with a temperature of 20-26°C and a humidity of 40-70%, and each test group/box is raised separately. Calu-6 cells are passaged in a medium of MEM (+0.01mM NEAA) + 10% FBS, and cells in the exponential growth phase are collected and resuspended to an appropriate concentration for subcutaneous tumor inoculation in mice. The experimental mice were subcutaneously inoculated with 5×10⁶ Calu-6 cells on the right back. When the tumor grew to an average volume of about 100 mm³ , the mice were grouped according to the tumor size and mouse weight using a random grouping design method (using StudyDirector^TM software for grouping, version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA). The mice were administered according to the set dose on the day after grouping, and the administration volume was calculated according to V=10 μL/g. The tumor volume was calculated according to V (mm³ )=1/2×(long diameter×short diameter² ). At the same time, the weight of the animals was measured twice a week. Each time the tumor volume and mouse weight were measured, the effects of the test drug on the normal behavior of the animals were observed and recorded. The data were expressed as mean ± SD, and the indicators were statistically analyzed using an unpaired two-tailed t-test, with the statistical significance level set at p<0.05. GraphPad software (Version 8.0.2, La Jolla, CA, USA) was used for statistical analysis.

本发明开展了ADC-14B的体内药效研究。结果显示，与溶媒Vehicle组相比，ADC-14B在所有剂量下均具有显著性的体内抑瘤药效(P<0.0001)。同时，ADC-14B在低、中、高三个剂量下(1.5mpk、3.0mpk和6.0mpk)体内抑瘤药效表现出明显的量效依赖关系。其中，1.5mpk剂量组与3.0mpk剂量组相比，3.0mpk剂量组的体内药效具有显著性优势(P＝0.0092)；1.5mpk剂量组与6.0mpk剂量组相比，6.0mpk剂量组的体内药效具有显著性优势(P＝0.0028)；3.0mpk剂量组与6.0mpk剂量组相比，6.0mpk剂量组的体内药效具有显著性优势(P＝0.0428)。结果如图5所示。The present invention has carried out an in vivo efficacy study of ADC-14B. The results show that compared with the vehicle group, ADC-14B has significant in vivo anti-tumor efficacy at all doses (P<0.0001). At the same time, the in vivo anti-tumor efficacy of ADC-14B at low, medium and high doses (1.5mpk, 3.0mpk and 6.0mpk) shows an obvious dose-effect dependence. Among them, compared with the 3.0mpk dose group, the in vivo efficacy of the 1.5mpk dose group has a significant advantage (P=0.0092); compared with the 6.0mpk dose group, the in vivo efficacy of the 6.0mpk dose group has a significant advantage (P=0.0028); compared with the 6.0mpk dose group, the in vivo efficacy of the 3.0mpk dose group has a significant advantage (P=0.0428). The results are shown in Figure 5.

将购买得到的MC-GGFG-Dxd(化合物代号：43)与第一三共株式会社报道的DS7300相偶联制备得到DAR＝4的ADC(代号：DS7300-43)。同时，将非特异性的同型IgG1与化合物14相偶联得到的DAR4的ADC(代号：IgG-14B)。The purchased MC-GGFG-Dxd (compound code: 43) was coupled with DS7300 reported by Daiichi Sankyo Co., Ltd. to prepare an ADC with DAR=4 (code: DS7300-43). At the same time, a non-specific isotype IgG1 was coupled with compound 14 to obtain an ADC with DAR4 (code: IgG-14B).

更进一步地，本发明在抗原B7-H3中度表达的PC3前列腺癌细胞的异种移植小鼠模型上，对比了ADC-14B、DS7300-43以及IgG-14B的体内药效特征。一方面，如图6A所示，与溶媒Vehicle组相比，ADC-14B在所有剂量下均具有显著性的体内抑瘤药效(P<0.0001)。同时，ADC-14B在低、中、高三个剂量下(1.5mpk、3.0mpk和6.0mpk)体内抑瘤药效表现出明显的量效依赖关系。其中，1.5mpk剂量组与3.0mpk剂量组相比，3.0mpk剂量组的体内药效具有显著性优势(P＝0.0038)；3.0mpk剂量组与6.0mpk剂量组相比，6.0mpk剂量组的体内药效具有显著性优势(P＝0.0356)。另一方面，如图6B所示，在相同剂量下(3.0mpk)，ADC-14B较DS7300-43和IgG-14B具有显著的体内药效优势(P值分别为0.0076和<0.0001)。Furthermore, the present invention compares the in vivo efficacy characteristics of ADC-14B, DS7300-43 and IgG-14B in a xenograft mouse model of PC3 prostate cancer cells with moderate expression of antigen B7-H3. On the one hand, as shown in FIG6A, compared with the vehicle group, ADC-14B has significant in vivo anti-tumor efficacy at all doses (P<0.0001). At the same time, the in vivo anti-tumor efficacy of ADC-14B at low, medium and high doses (1.5mpk, 3.0mpk and 6.0mpk) shows a significant dose-effect dependence. Among them, the in vivo efficacy of the 1.5mpk dose group compared with the 3.0mpk dose group has a significant advantage (P=0.0038); the in vivo efficacy of the 3.0mpk dose group compared with the 6.0mpk dose group has a significant advantage (P=0.0356). On the other hand, as shown in FIG6B , at the same dose (3.0 mpk), ADC-14B had a significant in vivo efficacy advantage over DS7300-43 and IgG-14B (P values were 0.0076 and <0.0001, respectively).

将本发明中的化合物-14与我方筛选得到的抗B7-H3人源化HCAb CA2-VHH25相偶联，制备得到DAR≈4的HDC-14E。体内药效结果显示，与溶媒Vehicle组相比，所有HDC-14E(DAR4)受试组中肿瘤生长得到显著抑制(P<0.0001)，并且，HDC-14E(DAR4)在低、中、高三个剂量下(1.5mpk、3.0mpk和6.0mpk)体内抑瘤药效表现出明显的量效依赖关系。其中，1.5mpk剂量组与3.0mpk剂量组相比，3.0mpk剂量组具有显著性优势(P＝0.0001)；1.5mpk剂量组与6.0mpk剂量组相比，6.0mpk剂量组具有显著性优势(P<0.0001)；3.0mpk剂量组与6.0mpk剂量组相比，6.0mpk剂量组具有显著性优势(P<0.0001)。结果如图7所示。Compound-14 of the present invention was coupled with the anti-B7-H3 humanized HCAb CA2-VHH25 screened by us to prepare HDC-14E with DAR≈4. The in vivo efficacy results showed that compared with the vehicle group, the tumor growth in all HDC-14E (DAR4) test groups was significantly inhibited (P<0.0001), and the in vivo anti-tumor efficacy of HDC-14E (DAR4) at low, medium and high doses (1.5mpk, 3.0mpk and 6.0mpk) showed a significant dose-effect dependence. Among them, the 1.5 mpk dose group was compared with the 3.0 mpk dose group, and the 3.0 mpk dose group had a significant advantage (P = 0.0001); the 1.5 mpk dose group was compared with the 6.0 mpk dose group, and the 6.0 mpk dose group had a significant advantage (P < 0.0001); the 3.0 mpk dose group was compared with the 6.0 mpk dose group, and the 6.0 mpk dose group had a significant advantage (P < 0.0001). The results are shown in Figure 7.

实施例42：抗B7-H3 ADC在小鼠模型上的安全性研究Example 42: Safety study of anti-B7-H3 ADC in mouse model

为了直接对比本发明中的基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC与基于传统琥珀酰亚胺接头的ADC的用药安全性差异，本发明采用基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC-14B(DAR4)和基于传统琥珀酰亚胺接头的ADC-35B(DAR4)为模型ADC分子，初步对比了其在B-hB7-H3(C57BL6-Cd276^tm1(CD276)/Bcgen，BIOCYTOGEN，Beijing，CN)转基因小鼠上高剂量(200mg/kg)下的耐受性研究试验，每个受试组包含3只动物。In order to directly compare the difference in drug safety between the ADC based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and the ADC based on the traditional succinimide linker in the present invention, the present invention uses ADC-14B (DAR4) based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker and ADC-35B (DAR4) based on the traditional succinimide linker as model ADC molecules, and preliminarily compares their tolerance study experiments at a high dose (200 mg/kg) on B-hB7-H3 (C57BL6-Cd276^tm1(CD276) /Bcgen, BIOCYTOGEN, Beijing, CN) transgenic mice, and each test group contains 3 animals.

试验结果显示，给药4天后受试动物在给药后均未发生死亡，同时所有受试动物均未表现出严重的体征和行为上的异常。The test results showed that none of the test animals died 4 days after administration, and all the test animals showed no serious physical signs or behavioral abnormalities.

给药4天后，ADC-14B组受试动物的平均体重较给药前下降11.0％，而ADC-35B组受试动物的平均体重较给药前下降18.4％，ADC-14B受试组动物体重下降的百分比数据(9.4％、9.4％、14.4％)与ADC-35B受试组动物体重下降的百分比数据(18.6％、19.5％、17.1％)相比，具有显著性差异(P＝0.0154)。Four days after administration, the average body weight of the test animals in the ADC-14B group decreased by 11.0% compared with that before administration, while the average body weight of the test animals in the ADC-35B group decreased by 18.4% compared with that before administration. The percentage data of body weight loss of the animals in the ADC-14B test group (9.4%, 9.4%, 14.4%) were significantly different from the percentage data of body weight loss of the animals in the ADC-35B test group (18.6%, 19.5%, 17.1%) (P=0.0154).

给药7天后，ADC-14B组受试动物和ADC-35B受试组动物平均体重较给药前分别恢复至98.53％和68.73％，ADC-14B受试组动物体重下降的百分比数据(-3.53％、1.56％、6.38％)与ADC-35B受试组动物体重下降的百分比数据(32.79％、31.61％、29.41％)相比，具有显著性差异(P＝0.0006)。After 7 days of administration, the average body weights of the animals in the ADC-14B group and the ADC-35B group recovered to 98.53% and 68.73% respectively compared with those before administration. The percentage of body weight loss in the ADC-14B group (-3.53%, 1.56%, 6.38%) was significantly different from that in the ADC-35B group (32.79%, 31.61%, 29.41%) (P=0.0006).

另外，观测到ADC-35B受试组动物出现步态不稳、被毛无光泽、肛周污秽，动作迟缓等严重不良反应，而ADC-14B组受试动物未见明显异常。In addition, serious adverse reactions were observed in the animals in the ADC-35B test group, such as unstable gait, dull fur, perianal dirt, and slow movements, while no obvious abnormalities were observed in the animals in the ADC-14B test group.

以上数据说明，200mg/kg的剂量尚未到达ADC-14B的最大耐受剂量，而ADC-35B的最大耐受剂量为低于200mg/kg，证明基于新型2-(甲基磺酰基)噻唑[5,4-b]吡啶接头的ADC-14B与基于传统琥珀酰亚胺接头的ADC-35B相比具有潜在的安全性优势。

The above data show that the dose of 200 mg/kg has not yet reached the maximum tolerated dose of ADC-14B, while the maximum tolerated dose of ADC-35B is less than 200 mg/kg, proving that ADC-14B based on the novel 2-(methylsulfonyl)thiazolyl[5,4-b]pyridine linker has potential safety advantages over ADC-35B based on the traditional succinimide linker.

以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.