WO2019154120A1

WO2019154120A1 - Cytotoxic agent and conjugate thereof, preparation method therefor and use thereof

Info

Publication number: WO2019154120A1
Application number: PCT/CN2019/073148
Authority: WO
Inventors: 肖亮; 田强; 宋帅; 钟国庆; 王川; 唐祖建; 刘登念; 胡瑞斌; 汪静; 薛彤彤; 蔡家强; 王利春; 王晶翼
Original assignee: 四川科伦博泰生物医药股份有限公司
Priority date: 2018-02-11
Filing date: 2019-01-25
Publication date: 2019-08-15
Also published as: CN111542324A; CN111542324B

Abstract

A novel cytotoxic agent (which is a camptothecin derivative), a conjugate thereof with a target, a preparation method for the cytotoxic agent and the conjugate, and a use of the conjugate in the prevention and/or treatment of proliferative diseases associated with cellular activity abnormalities (including but not limited to cancers).

Description

Cytotoxic agent and conjugate thereof, preparation method and use thereof

Field of invention

The invention belongs to the field of medical technology. In particular, the present invention relates to a novel cytotoxic agent which is a camptothecin derivative, a conjugate thereof to a target, a preparation method of the cytotoxic agent and conjugate, and the conjugate Use in the prevention and/or treatment of proliferative diseases associated with abnormal cellular activity, including but not limited to cancer diseases.

Background of the invention

Chemotherapy was once the standard treatment for cancer, but high-killing bioactive molecules can kill normal cells, causing serious side effects. Targeted anti-tumor drugs have become a hot spot in the field of cancer research due to their simultaneous targeting and anti-tumor activity. Since the 20th century, breakthroughs have been made in the use of biomacromolecules such as therapeutic antibodies or antibody fragments for targeted therapy of tumors. However, although biomacromolecules are highly targeted, they have limited therapeutic effects on solid tumors. Although bioactive molecules have high killing efficacy against cancer cells, they lack targeting and often accidentally injure normal cells, causing serious toxicity. side effect.

In recent years, studies have found that therapeutic antibodies can be linked to biologically active molecules to form antibody-drug conjugates (ADCs). The ADC combines the targeting of antibodies with the high activity of biologically active molecules to become a "bio-missile". The antibody directs the ADC to bind to the target cell, which is then internalized by the cell, releasing the drug to treat the disease. Because the antibody has specificity and targeting to tumor cell related targets, its application value is not only reflected in the treatment, but also becomes an ideal carrier for drug targeted delivery, which reduces the side effects of the drug.

Typically, ADC drugs consist of antibodies, biologically active molecules, and linkers. The biologically active molecule is covalently coupled to the antibody via a linker. Antibodies (eg, monoclonal antibodies) are capable of specifically recognizing specific targets on the surface of cancer cells, thereby directing the ADC to the surface of the cancer cells and allowing the ADC to enter the cancer cells through the endocytosis effect. Bioactive molecules can be released intracellularly, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

Small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs) are designed with the same principle of chemically binding biologically active molecules and small molecule ligands that selectively bind to tumor cell surface receptors. Coupling, thereby increasing the targeting of effector molecules (bioactive molecules) to tumor cells. The chemical structure of SMDC is almost identical to that of ADC, but small molecule ligands are used instead of antibodies. Currently, there is no SMDC listed.

Currently, there are four ADCs already on the market: Mylotarg (Gemtuzumab Ozogamicin, gemtuzumab oxazoic acid), Adcetris (Brentuximab Vedotin, CD30 mAb-MMAE), Kadcyla (Trastuzumab Emtansine, trastuzumab-美Dessert alkaloids and Besponsa (Inotuzumab ozogamicin, CD22 mAb-caccimycin).

Summary of invention

After intensive research, the inventors have unexpectedly discovered a conjugate comprising a target moiety and a cytotoxic agent moiety (camptothecin derivative) having an antitumor effect, which has good inhibitory ability against tumor cells. .

A first aspect of the invention provides a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

among them:

R ₁ is each independently selected from H (hydrogen), D (oxime), halogen, nitro, -NR _a R _b , C _1-6 alkyl, halo C _1-6 alkyl at each occurrence (for example -CF ₃ ), C _1-6 alkoxy, halo C _1-6 alkoxy (for example -OCF ₃ ) and C _3-6 cycloalkyl; R _a and R _b are each independently selected from H (hydrogen) ), D(氘), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C _1-6 alkylene-C _3-6 Cycloalkyl, -C _1-6 alkylene-(6-10 membered aryl) and -C _1-6 alkylene group (5-12 membered heteroaryl); n ₁ is 1, 2, 3 or 4;

R _{2 is} selected from the group consisting of H (hydrogen), D (oxime), C _1-6 alkyl, -NR _c R _d and a 3-7 membered nitrogen-containing heterocyclic group; R _c and R _d are each independently selected from H (hydrogen) ), D(氘), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C _1-6 alkylene-C _3-6 Cycloalkyl, -C _1-6 alkylene-(6-10 membered aryl), -C _1-6 alkylene-(5-12 membered heteroaryl), C _1-6 alkyl-C ( =O)-, halogenated C _1-6 alkyl-C(=O)-, 6-10 membered aryl-C(=O)-, 5-12 membered heteroaryl-C(=O)-, 6-10 membered aryl-C _1-6 alkylene-C(=O)-, 5-12 membered heteroaryl-C _1-6 alkylene-C(=O)-, C _1-6 alkane a base-SO ₂ -, a halogenated C _1-6 alkyl-SO ₂ -, a 6-10 membered aryl-SO ₂ - and a 5-12 membered heteroaryl-SO ₂ -; the 6-10 membered aryl group And a 5-12 membered heteroaryl group is optionally substituted by H (hydrogen), D (hydrazine), halogen or C _1-6 alkyl; n ₂ is 0, 1, 2, 3 or 4;

R is each independently selected from H (hydrogen), D (oxime), halogen, nitro, cyano, C _1-6 alkyl, halo C _1-6 alkyl (eg, -CF ₃ ). , C _1-6 alkoxy, halo C _1-6 alkoxy (eg -OCF ₃ ), C _3-6 cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is 1, 2, 3 or 4;

AA is selected from the group consisting of an amino acid group and a peptide comprising 2-6 amino acids;

L _{1 is} selected from -(CH ₂ ) _t1 -,

(Such as

),

with

The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L ₂ by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L _{2 is} attached; R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C _1-6 alkyl, halogenated C _1-6 alkyl, cyano substituted C _1-6 alkyl, C _1-6 alkoxy and C _3-6 cycloalkyl, or R ₃ and R ₄ , R ₅ and R ₆ or R ₃ R ₅ together with the atom/group to which it is attached forms a 3-8 membered ring; t1, t2, y ₁ and y ₂ are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L ₂ does not exist or is

One of its two positions marks to L ₁ via 1 or 2, and L ₃ is connected by a further position, preferably it is connected to L ₁ via a marked location, and by the marked position 2 and L ₃ Connected, Z _{1 is} selected from O, S and -NH-, Z ₂ is absent or selected from O, S and -NH-, and m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L ₃ is absent or is selected from -NR ₇ - and optionally substituted by one or more R ₇ : C _3-8 cycloalkylene, 6-10 membered arylene (eg phenylene or Naphthylene), 5-12 membered heteroarylene, 3-8 membered heterocyclylene, 6-12 membered subbridged heterocyclic group, 6-12 membered snail heterocyclic group, 6-12 membered sub-fused a cyclic group, -NR ₈ -C _3-8 cycloalkylene- and -C _3-8 cycloalkylene-NR ₈ -, when the above group contains a nitrogen atom, the nitrogen atom is optionally a quaternary ammonium Each of R ₇ is independently selected from H (hydrogen), D (oxime), halogen, =O, cyano, carboxyl, sulfonate, C _1-6 alkyl, halo C ₁ at each occurrence. _{a -6} alkyl group, a cyano substituted C _1-6 alkyl group, a C _1-6 alkoxy group, a C _2-6 alkenyl group and a C _2-6 alkynyl group, and R _{8 is} selected from the group consisting of H (hydrogen) and D (氘) ), C _1-6 alkyl, C _3-6 cycloalkyl and C _1-6 alkoxy;

L ₄ is absent or is selected from C _1-6 alkylene, -NR ₉ -C _1-6 alkylene-, -C _1-6 alkylene-NR ₉ -, -NR ₉ - and is one or more R ₉ substituted 3-7 membered nitrogen-containing heterocyclylene; R _{9 is} independently selected from H (hydrogen), D (oxime), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 a aryl group (e.g., phenyl or naphthyl) and a 5-12 membered heteroaryl; said C _1-6 alkyl or C _1-6 alkylene optionally being one or more H (hydrogen), D (氘) or halogen substituted;

L _{5 is} selected from

r is 0, 1, 2, 3, 4 or 5.

A second aspect of the invention provides a conjugate of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

Wherein, A is a group obtained by removing γ thiol groups in the target or a group obtained by reducing a disulfide bond in the target and then removing γ thiol groups;

L ₅ ' is selected from

It is linked to L ₄ by one of the two positions labeled 1 or 2 and to the thiol group of the target by another position; preferably it is linked to L ₄ by the position of the 1 mark and passes the position of the 2 mark Attached to the thiol group of the target; r is 0, 1, 2, 3, 4 or 5;

γ is an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, γ is an integer of 4 to 8, for example, γ is 4, 5 , 6, 7 or 8; and

The remaining groups are as defined above.

A third aspect of the invention provides a pharmaceutical composition comprising one or more (preferably two or more) conjugates of formula (II) of the invention, or a pharmaceutically acceptable salt, solvate thereof, hydrate thereof Or an isomer, or any crystalline form or racemate thereof, and optionally one or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more other An anticancer drug such as a chemotherapeutic agent and/or an antibody, wherein the pharmaceutical composition has an average DAR value of an integer or a decimal of 1 to 10, such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2, 4.3. 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 or 10.0, preferably an integer or fraction of 4 to 8, such as 4.0, 4.1, 4.2, 4.3 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

A fourth aspect of the present invention provides a conjugate of the formula (II) of the present invention or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, which is used in the preparation Use in medicines for the prevention or treatment of cancer diseases.

A fifth aspect of the present invention provides a conjugate of the formula (II) of the present invention or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, which is used for Prevent or treat cancer diseases.

A sixth aspect of the invention provides a method for preventing or treating a cancer disease, which comprises administering an effective amount of a conjugate of the formula (II) of the present invention or a pharmaceutically acceptable salt or solvate thereof to an individual in need thereof , hydrates, isomers, or any of their crystal forms or racemates.

A seventh aspect of the invention provides a process for the preparation of a compound of formula (I'), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof:

among them:

R _d " is R _d or hydrogen,

R _d ' is R _d or PG ₁ ,

PG ₁ and PG ₂ are each independently an amino protecting group, preferably Boc or MMT,

The remaining groups are as defined above,

The method includes the following steps:

Step 1: In the presence of a base, functionalization or protection of a nitrogen atom (such as acetylation, mesylation, Boc protection), the compound (Ia)-a is reacted to obtain a compound of (Ia)-b, conditions When R _d " and R _d ' are both R _d , the reaction of the first step is not required;

Step 2: reacting the compound (Ia)-b with the compound (Ia)-c in the presence of a condensation reagent and a base to obtain a compound of (Ia)-d;

Step 3: reacting the compound (Ia)-d with the compound (Ia)-e in the presence of a metal catalyst to obtain a compound of the formula (Ia), and

Step 4: The compound of the formula (Ia) is reacted under conditions to remove the amino protecting group to give a compound of the formula (I') or a pharmaceutically acceptable salt thereof.

An eighth aspect of the invention provides a process for the preparation of a compound of formula (I"), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof:

among them:

R _d ' is R _d or PG ₁ ,

R ₁₀ is absent or is C _1-6 alkylene, a C _1-6 alkylene group optionally substituted by one or more of H (hydrogen), D (deuterium) or halogen,

LG is a leaving group, preferably a hydroxyl group or a succinimide-N-oxy group,

The remaining groups are as defined above,

The method includes the following steps:

Step 1: in the presence of a reducing agent, the compound (Ia)-d is reacted to obtain the compound (Ib)-a;

Step 2: reacting compound (Ib)-a with compound (Ib)-b in the presence of a base, optionally in the presence of a condensation reagent, to give a compound of formula (Ib), and

Step 3: The compound of the formula (Ib) is reacted under conditions to remove the amino protecting group to give a compound of the formula (I") or a pharmaceutically acceptable salt thereof.

A ninth aspect of the invention provides a process for the preparation of a conjugate of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

The method comprises coupling a compound of formula (I) to a target,

Each of the groups is as defined above.

DRAWINGS

Figure 1 II-1-A light chain deconvolution diagram.

Figure 2 II-1-A heavy chain deconvolution diagram.

Figure 3 II-5-A light chain deconvolution diagram.

Figure 4 II-5-A heavy chain deconvolution diagram.

Figure 5 II-6-A light chain deconvolution diagram.

Figure 6 II-6-A heavy chain deconvolution diagram.

Figure 7 II-7-A light chain deconvolution diagram.

Figure 8 II-7-A heavy chain deconvolution diagram.

Figure 9 II-8-A light chain deconvolution diagram.

Figure 10 II-8-A heavy chain deconvolution diagram.

Figure 11 II-9-A light chain deconvolution diagram.

Figure 12 II-9-A heavy chain deconvolution diagram.

Figure 13 II-10-A light chain deconvolution diagram.

Figure 14 II-10-A heavy chain deconvolution plot.

Figure 15 II-11-A light chain deconvolution diagram.

Figure 16 II-11-A heavy chain deconvolution plot.

Figure 17 II-12-A light chain deconvolution diagram.

Figure 18 II-12-A heavy chain deconvolution diagram.

Figure 19 II-13-A light chain deconvolution diagram.

Figure 20 II-13-A heavy chain deconvolution diagram.

Figure 21 II-15-A light chain deconvolution diagram.

Figure 22 II-15-A heavy chain deconvolution diagram.

Figure 23 SEC chromatogram of II-1-A.

Figure 24 SEC chromatogram of II-5-A.

Figure 25 SEC chromatogram of II-7-A.

Figure 26 SEC chromatogram of II-8-A.

Figure 27 SEC chromatogram of II-10-A.

Figure 28 SEC chromatogram of II-14-A.

Figure 29 Release of cytotoxic agents in the plasma of cynomolgus monkeys II-1-A, II-7-A and II-8-A.

Figure 30. Cytotoxic agent release in rat plasma of II-1-A, II-7-A and II-8-A.

Figure 31. Cytotoxic agent release of II-1-A, II-7-A and II-8-A in 5% bovine serum albumin (BSA) PBS buffer.

Detailed description of the invention

Definition of Terms

In the present invention, the terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise stated. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all routine steps widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

In the present invention, the term "conjugate" means a substance obtained by linking a cytotoxic agent to a target. In some embodiments of the invention, the cytotoxic agent is linked to the target by a linker. The linker is capable of cleavage in a particular environment (e.g., an intracellular low pH environment) or a specific action (e.g., the action of a lysosomal protease) to separate the cytotoxic agent from the target. In some embodiments of the invention, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the invention, the cytotoxic agent is linked directly to the target by a covalent bond that is capable of cleavage under a particular environment or action, thereby isolating the cytotoxic agent from the target.

In the present invention, the term "cytotoxic agent" means a substance capable of exerting a detrimental effect on the growth or proliferation of cells, and also means a substance which inhibits or hinders the function of cells and/or causes cell death or destruction.

A "small molecule" is defined herein as a biologically active small molecule drug. In certain embodiments, the small molecule has a molecular weight of no greater than 2000 Da, such as no greater than 1500 Da, 1000 Da, or 500 Da.

In the present invention, the term "linker" refers to a structural fragment that links a cytotoxic agent to a target.

In the present invention, the term "target" refers to a substance that is capable of specifically binding to a target (or a portion of a target) on a cell surface. The conjugate can be delivered to a particular cell population by interaction of the target moiety with the target.

In the present invention, when the target is an antibody, the conjugate may be referred to as an "antibody-drug conjugate." In the present invention, "antibody-drug conjugate" and "immunoconjugate" are used interchangeably.

In the present invention, the term "antibody" is used in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, as long as they have The required biological activity. As used herein, "antibody" and "immunoglobulin" are used interchangeably.

In the present invention, the term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the cluster are identical except for a small number of natural mutations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of the antigen, while polyclonal antibodies relative thereto contain different antibodies for different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being free from contamination by other antibodies during synthesis. The modifier "monoclonal" as used herein means that the antibody is characterized by a substantially homogeneous population of antibodies and is not to be construed as being prepared by a particular method.

In some embodiments of the invention, the monoclonal antibody also specifically includes a chimeric antibody, ie, a portion of the heavy and/or light chain is identical or homologous to a certain, certain or a subset of antibodies, and the remainder is One, another or another subclass of antibodies are identical or homologous as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies useful in the present invention include primatized antibodies comprising variable region antigen binding sequences from human non-human primates (e.g., ancient monkeys or orangutans, etc.) and human constant region sequences.

The term "antibody fragment" refers to a portion of an antibody, preferably an antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb and complementarity determining region fragments; diabodies; linear antibodies; and single chain antibody molecules.

The term "bispecific antibody" is used interchangeably with "bifunctional antibody conjugate" and refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) by a coupling arm. The conjugate retains the activity of the respective antibody and is therefore bifunctional and bispecific.

The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies having three different antigen binding specificities and the latter being antibodies having four different antigen binding specificities.

The term "intact antibody" refers to an antibody comprising an antigen binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region can be a native sequence (eg, a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

The term "Probody" is a modified antibody comprising an antibody or an antibody fragment that specifically binds to its target and is capable of coupling to a masking group, wherein the masking group refers to an antibody or antibody fragment thereof The cleavage constant of the binding ability of the target is at least 100-fold, 1000-fold or 10,000-fold greater than the cleavage constant of the binding ability of the antibody or antibody fragment without the coupling masking group to its target.

In the present invention, a "humanized" form of a non-human (eg, murine) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequences. Most humanized antibodies are human receptors. The hypervariable region residues of immunoglobulins are replaced by non-humans (eg, mice, rats, rabbits, or non-human primates) with the desired specificity, affinity, and function. Hypervariable region residues (donor antibodies). In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally comprise at least one, usually two variable regions, wherein all or nearly all of the hypervanable loops correspond to non-human immunoglobulins, while FR is completely or almost entirely human immunoglobulin. The sequence of the protein. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details, see, for example, Jones et al, 1986, Nature, 321 :522-525; Riechmann et al, 1988, Nature, 332:323-329; and Presta, 1992, CurrOp Struct Bwl 2:593-596.

Intact antibodies can be divided into different "classes" based on the amino acid sequence of the heavy chain constant region. The main five classes are IgA, IgD, IgE, IgG and IgM, several of which can also be divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions of different classes of antibodies are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

In the present invention, although the amino acid substitution in the antibody is substituted by the L-amino acid in most cases, it is not limited thereto. In some embodiments, one or more D-amino acids can be included in the antibody peptide chain. Peptides comprising D-amino acids are more stable and less susceptible to degradation in the oral, intestinal or plasma than peptides comprising only L-amino acids.

The monoclonal antibodies used in the present invention can be produced by a number of methods. For example, monoclonal antibodies for use in the present invention can be obtained by hybridoma methods using a variety of species including cells of mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495). Alternatively by recombinant DNA techniques (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al, 1991, Nature, 352: 624-628; and Marks et al, 1991, Journal of Molecular Biology , 222: 581-597). Monoclonal antibodies useful in the present invention include, but are not limited to, monoclonal antibodies against Trop-2, such as Sacituzumab (i.e., Isactuzumab or hRS7 antibody), or monoclonal antibodies against Her2, such as trastuzumab or Patuxin. Monoclonal antibody (Pertuzumab).

In some embodiments of the invention, the target anti-Trop-2 antibody is RS7 as described in U.S. Patent No. 7,517,964; and hRS7 (i.e., Sacituzumab of the present invention) as described in US 2012/0237518. Anti-Trop-2 antibodies useful in the present invention can also be obtained by screening methods for designing, constructing, and constructing antibody libraries displaying antibodies, as disclosed in CN103476941A.

In the present invention, Trop-2 or TROP2 refers to human trophoblast cell surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is composed of many humans. A cell surface receptor expressed by a tumor (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, or cervical cancer). In some embodiments, a compound or conjugate of the invention is capable of inhibiting or killing a cell that expresses a TROP2 receptor, such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, or cervical cancer.

In some embodiments of the invention, the target is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her2 whose amino acid sequence is known to those skilled in the art, the schematic sequence of which can be found, for example, in CN103319599. Lys at the end of the heavy chain of trastuzumab is readily deleted, but this deletion does not affect biological activity, see Dick, L. W. et al, Biotechnol. Bioeng., 100: 1132-1143. Exemplary heavy chain and light chain sequences of pertuzumab can be found in SEQ ID No. 16 and SEQ ID No. 15 of US7560111.

In the present invention, ErbB2 and Her2 are used interchangeably, both of which represent the human HER2 protein of the native sequence (Genebank accession number: X03363, see, for example, Semba et al., 1985, PNAS, 82: 6497-6501; and Yamamoto et al. , 1986, Nature, 319: 230-234) and functional derivatives thereof, such as amino acid sequence variants. ErbB2 represents the gene encoding human Her2, and neu represents the gene encoding rat pl85neu. In some embodiments, the compounds or conjugates of the invention are capable of inhibiting or killing cells expressing the ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, Renal cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells or liver cancer cells.

In the present invention, the term "amino acid" includes natural amino acids and unnatural amino acids. The above natural amino acid and unnatural amino acid may be in the L configuration or the D configuration. Specific examples include, but are not limited to, L-lysine, D-lysine, L-ornithine, D-ornithine, L-valine, D-valine, L-citrulline, D - citrulline, L-alanine, D-alanine, L-phenylalanine, D-phenylalanine, glycine, L-leucine, D-leucine, L-isoleucine Acid, D-isoleucine, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-glutamic acid, D-glutamic acid, L-Valley Aminoamide, D-glutamine, L-arginine, D-arginine, L-histidine, D-histidine, L-tyrosine, D-tyrosine, L-methylthioamide Acid, D-methionine, and the like.

In the present invention, the term "C _1-6 alkyl" means a straight or branched alkyl group having 1 to 6 carbon atoms, and includes, for example, "C _1-4 alkyl" or "C _1-3 alkyl". Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2- Methyl butyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethyl Butyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethyl Butyl, 2-ethylbutyl and 1,2-dimethylpropyl, and the like. The term "C _1-6 alkylene" is a corresponding divalent group, and includes, for example, "C _1-4 alkylene", "C _1-3 alkylene", and the like, and specific examples include, but are not limited to, Base, ethylene, propylene, butylene, pentylene and hexylene.

In the present invention, the term "C _2-10 alkenyl" means a straight-chain, branched or cyclic alkenyl group having at least one double bond and having 2 to 10 carbon atoms, and includes, for example, "C _2-6 alkene". ",""C ₂₄ alkenyl" and the like. Examples thereof include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexyl Dienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, and 1,4-cyclohexadienyl, and the like. The term "C _2-10 alkenylene" is a corresponding divalent group including, for example, "C _2-6 alkenylene", "C _2-4 alkenylene" and the like. Examples thereof include, but are not limited to, a vinylidene group, a propylene group, a butenylene group, a pentenylene group, a hexylene group, a cyclopentylene group, a cyclohexylene group, and the like.

In the present invention, the term "C _2-10 alkynyl" means a straight or branched alkynyl group having at least one triple bond and having 2 to 10 carbon atoms, and includes, for example, "C _2-6 alkynyl group", "C _2-4 alkynyl" and the like. Examples thereof include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 - hexynyl and 5-methyl-2-hexynyl and the like. The term "C _2-10 alkynylene" is a corresponding divalent group including, for example, "C _2-6 alkynylene", "C _2-4 alkynylene" and the like. Examples thereof include, but are not limited to, ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

In the present invention, the term "halogen" includes fluorine, chlorine, bromine and iodine.

In the present invention, the term "3-10 membered cycloalkyl" or "C _3-10 cycloalkyl" means a saturated cyclic alkyl group having 3 to 10 carbon atoms, and includes, for example, "C _3-8 cycloalkane". ",""C _3-6 cycloalkyl", "C _4-6 cycloalkyl", "C _5-7 cycloalkyl" or "C _5-6 cycloalkyl" and the like. Specific examples include, but are not limited to, cyclopropyl, cyclobutane, cyclopentyl, cyclohexane, cycloheptyl, cyclooctyl, and the like. The term "3-10 membered cycloalkylene" or "C _3-10 cycloalkylene" is a corresponding divalent group including, for example, "C _3-8 cycloalkylene", "C _3-6 cyclo ring"Alkyl","C _4-6 cycloalkylene", "C _5-7 cycloalkylene" or "C _5-6 cycloalkylene" and the like. Specific examples include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.

In the present invention, the term "3-8 membered heterocyclic group" means a cyclic group having 3 to 8 ring atoms in which at least one ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "3-8 membered heterocyclic group" includes, for example, "3-8 membered nitrogen-containing heterocyclic group", "3-8 membered oxygen-containing heterocyclic group", "3-6 membered heterocyclic group", and "3-6 member". "Oxygen-containing heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered heterocyclic group", "5- 6-membered nitrogen-containing heterocyclic group", preferably including but not limited to oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and gaoprene Azinyl and the like. The term "3-8 membered heterocyclylene" is a corresponding divalent group and includes, for example, "3-8 membered nitrogen-containing heterocyclylene", "3-7 membered nitrogen-containing heterocyclylene", "3- 8-membered oxygen-containing heterocyclylene", "3-6 membered heterocyclylene", "3-6 membered oxyheterocyclylene", "4-7 membered heterocyclylene", "4-6 member" "heterocyclylene", "5-7 membered heterocyclylene", "5-6 membered heterocyclylene", "5-6 membered nitrogen-containing heterocyclylene", preferably including but not limited to ethylene oxide B Alkyl, oxycyclobutanealkyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and xagopiperazinyl, and the like.

As used herein, the term "6-12 membered spirocyclic group" means a cyclic structure having 6 to 12 ring carbon atoms formed by two or more cyclic structures sharing one carbon atom with each other. Optionally, the carbon atoms in the cyclic structure can be replaced by oxo. The "6-12 membered spiro group" includes, for example, "6-11 membered spiro group", "6-10 membered spiro group", "7-10 membered spiro group", and "7-9 membered spiro group" , "7-8 yuan spiro group", "9-10 yuan spiro group" and "8-10 yuan spiro group" and so on. Specific examples include, but are not limited to:

Wait. The term "6-12 membered spirocyclyl" is a corresponding divalent group and includes, for example, "6-11 membered spirocyclyl", "6-10 membered spirocyclyl", and "7-10 membered snail". "Cycloalkyl", "7-9 membered spirocyclyl", "7-8 membered spirocyclyl", "9-10 membered spirocyclyl" and "8-10 membered spirocyclyl" and the like.

As used herein, the term "6-12 membered bridged ring group" refers to a ring of 6 to 12 ring carbon atoms formed by two or more ring structures sharing two non-adjacent carbon atoms with each other. structure. Optionally, the carbon atoms in the cyclic structure can be replaced by oxo. "6-12 Yuan Bridged Ring Group" includes, for example, "6-11 Yuan Bridged Ring Group", "6-10 Yuan Bridged Ring Group", "7-10 Yuan Bridged Ring Group", "7-9 Yuan Bridged Ring Group" , "7-8 yuan bridge ring base", "9-10 yuan bridge ring base" and "8-10 yuan bridge ring base" and so on. Specific examples include, but are not limited to:

Wait. The term "6-12 membered subbridged ring group" is a corresponding divalent group including, for example, "6-11 membered subbridged ring group", "6-10 membered subbridged ring group", and "7-10 membered subbridge" Ring group", "7-9 member subbridge ring group", "7-8 member subbridge ring group", "9-10 yuan subbridge ring group" and "8-10 yuan subbridge ring group".

As used herein, the term "6-12 membered fused ring group" refers to a cyclic structure having 6 to 12 ring carbon atoms formed by two or more ring structures sharing two adjacent atoms with each other. , including "6-11 membered fused ring group", "6-10 membered fused ring group", "6-8 membered fused ring group", "10-12 membered fused ring group" and "7-10 membered fused ring group"". Examples include, but are not limited to:

Wait. The term "6-12 membered fused ring group" is a corresponding divalent group, and includes, for example, "6-11 membered fused ring group", "6-10 membered fused ring group", and "6-8 membered subcondensed group". Ring group", "10-12 membered fused ring group" and "7-10 membered fused ring group".

In the present invention, the term "6-12 membered spiroheterocyclyl" means a group of 6 to 12 ring atoms (having at least one ring atom) formed by two or more ring structures sharing one ring atom with each other. It is a cyclic structure of a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered spiroheterocyclyl" includes, for example, "6-12 membered nitrogen-containing spiroheterocyclyl", "6-11 membered spiroheterocyclyl", "6-10 membered spiroheterocyclyl", and "7- 11-membered spiroheterocyclyl", "7-10-membered spiroheterocyclyl", "7-9-membered spiroheterocyclyl", "7-8-membered spiroheterocyclyl", and "9-10-membered spiroheterocyclyl" And "8-10 yuan spiroheterocyclyl" and the like. Specific examples include, but are not limited to:

Wait. The term "6-12 membered spiroheterocyclyl" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing spiroheterocyclyl", "8-11 membered nitrogen-containing spiroheterocyclyl", "11-membered nitrogen-containing snail heterocyclic group", "6-11 membered snail heterocyclic group", "6-10 membered snail heterocyclic group", "7-11 membered snail heterocyclic group", "7 -10 membered snail heterocyclic group, "7-9 membered snail heterocyclic group", "7-8 membered snail heterocyclic group", "9-10 membered snail heterocyclic group" and "8-10" Yuan Yaspiroheterocyclic group" and the like.

In the present invention, the term "6-12 membered bridged heterocyclic group" means a group of 6 to 12 ring atoms formed by two or more ring structures sharing two non-adjacent ring atoms with each other ( A ring structure in which at least one of the ring atoms is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered bridged heterocyclic group" includes, for example, "6-12 membered nitrogen-containing bridged heterocyclic group", "6-11 membered bridged heterocyclic group", "6-9 membered bridged heterocyclic group", and "6- 10-membered bridged heterocyclic group, "7-10 membered bridged heterocyclic group", "7-9 membered bridged heterocyclic group", "7-8 membered bridged heterocyclic group", "8-membered bridged heterocyclic group", "9-10 membered bridged heterocyclic group" and "8-10 membered bridged heterocyclic group" and the like. Specific examples include, but are not limited to:

Wait. The term "6-12 membered subbridged heterocyclic group" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing ylidene heterocyclic group", "8-11 membered nitrogen-containing ylidene heterocyclic group", "8-membered nitrogen-containing subbridge heterocyclic group", "6-11 membered subbridge heterocyclic group", "6-9 membered subbridge heterocyclic group", "6-10 membered subbridged heterocyclic group", "7 -10 membered subbridge heterocyclic group", "7-9 membered subbridged heterocyclic group", "7-8 membered subbridged heterocyclic group", "8 membered subbridged heterocyclic group", "9-10 member Bridged heterocyclic group" and "8-10 membered subbridge heterocyclic group" and the like. Specific examples include but are not limited to

In the present invention, the term "6-12 membered fused heterocyclic group" means a group of 6 to 12 ring atoms formed by two or more ring structures sharing two adjacent atoms with each other (at least A ring structure in which a ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. The "6-12 membered fused heterocyclic group" includes, for example, "6-12 membered nitrogen-containing fused heterocyclic group", "6-11 membered fused heterocyclic group", "6-10 membered fused heterocyclic group", and "7- 10 membered fused heterocyclic group", "8-10 membered fused heterocyclic group", "8-10 membered nitrogen-containing fused heterocyclic group", "9-10 membered fused heterocyclic group", "9-10 membered nitrogen-containing group" A fused heterocyclic group" and a "6-12 membered oxygen-containing fused heterocyclic group" and the like. Specific examples include, but are not limited to, tetrahydroimidazo[4,5-c]pyridyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo[d][1 , 3]dioxolyl, 1,3-dihydroisobenzofuranyl, 4H-1,3-benzoxazinyl, 4,6-dihydro-1H-furo[3,4 -d]imidazolyl, 3a,4,6,6a-tetrahydro-1H-furo[3,4-d]imidazolyl, 4,6-dihydro-1H-thieno[3,4-d]imidazole , 4,6-dihydro-1H-pyrrolo[3,4-d]imidazolyl, benzimidazolylalkyl, octahydro-benzo[d]imidazolyl, decahydroquinolyl, hexahydrothieno Imidazolyl, hexahydrofurfurimidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahydrocyclopenta[c]pyrrolyl, indanyl, di Hydroisoisoindolyl, benzoxazole alkyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl and 4H- 1,3-benzoxazinyl and the like. The term "6-12 membered heterofused heterocyclic group" is a corresponding divalent group and includes, for example, "6-12 membered nitrogen-containing sulfene heterocyclic group", "6-11 membered heterofused heterocyclic group", "6" -10 membered fused heterocyclic group", "7-10 membered fused heterocyclic group", "8-10 membered fused heterocyclic group", "8-11 membered nitrogen-containing fused heterocyclic group", "8" -10-membered nitrogen-containing fused heterocyclic group", "8-membered nitrogen-containing fused heterocyclic group", "9-10 membered fused heterocyclic group", "9-10 membered nitrogen-containing fused heterocyclic group" and "6-12 yuan oxygen-containing fused heterocyclic group" and the like.

As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having an aromatic character, such as a 6-20 membered aryl group, a 6-10 membered aryl group, a 5-8 membered aryl group, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl and the like. The "6-20 membered aryl group" means an aryl group having 6 to 20 ring atoms. The term "arylene" is a corresponding divalent group, such as a 6-10 membered arylene group, a 5-8 membered arylene group, and the like. Specific examples include, but are not limited to, phenylene, naphthylene, anthracenylene, phenanthrylene, and the like.

In the present invention, the term "heteroaryl" refers to a cyclic group having an aromatic character in which at least one ring atom is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (eg, a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be substituted by oxo. Specific examples include, but are not limited to, a 5-10 membered heteroaryl group, a 5-10 membered nitrogen-containing heteroaryl group, a 6-10 membered oxygen-containing heteroaryl group, a 6-8 membered nitrogen-containing heteroaryl group, and a 5-8 membered oxygen group. a heteroaryl group or the like, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1, 2,3-Triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl 1,3,4-oxadiazolyl, pyridyl, 2-pyridinone, 4-pyridinone, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxa Azinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4- Azinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, azepanyl And 1,3-diazacycloheptatrienyl and azacyclotetradecenyl and the like. The term "heteroarylene" is a corresponding divalent group, and specific examples include, but are not limited to, nitrogen-containing heteroarylene, 5-12 membered heteroarylene, 5-10 membered heteroarylene, 5-10. a nitrogen-containing heteroarylene group, a 5-6 membered nitrogen-containing heteroarylene group, a 6-membered nitrogen-containing heteroarylene group, a 6-10 membered oxygen-containing heteroarylene group, a 6-8 membered nitrogen-containing heteroarylene group, and 5 -8-membered oxygen-containing heteroarylene, etc., such as furanyl, thienylene, pyrrolylene, thiazolyl, oxazolyl, imidazolyl, pyrazolyl, arazolyl, pyridylene , pyrimidinyl, oxazinyl, pyridazinyl, pyrazine, tritriazinyl and tetrazinylene.

In the present invention, the term "counter ion" means an ion that is accompanied by an ionic species to maintain electrical neutrality, for example, in sodium chloride, a sodium cation is a counter ion of a chloride anion, and vice versa.

Optionally, the hydrogen in the groups involved in the present invention may be substituted by deuterium.

The group referred to in the present invention is obtained by substituting 1, 2 or 3 hydrogen atoms of the compound/conjugate in the group with other atoms, and the number of the substituted hydrogen atoms It can be determined based on the number of valence bonds formed by the group in the compound or conjugate of the invention. For example, an alkyl group is a group obtained by substituting one hydrogen atom in an alkane, and an alkylene group is a group obtained by substituting two hydrogen atoms in an alkane, and the methyl group and the ethyl group are respectively one of methane and ethane. The group obtained by substituting a hydrogen atom, a methylene group and an ethylene group are respectively a group obtained by substituting two hydrogen atoms in methane and ethane.

As used herein, the term "substituted" means that one or more (eg, one, two, three, or four) hydrogens on the designated atom are replaced by the selection of the indicated group, provided that the The normal valence of the specified atom in the present case and the substitution form a stable compound/conjugate. Combinations of substituents and/or variables are permissible only if such combinations form stable compounds/conjugates.

If a substituent is described as "optionally substituted," the substituent may be unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the carbon (to the extent of any hydrogen present) may be independently and/or together independently The optional substituents selected are substituted. If the nitrogen of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected. Substitute substitution.

If a substituent is described as being "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.

The substituents of the substituents, as used herein, may be derived from any suitable position of the substituent, unless otherwise indicated.

When a bond of a substituent is shown to pass through a bond connecting two atoms in the ring, such a substituent may be bonded to any of the ring-forming atoms in the substitutable ring.

As used herein, the term "solvate" refers to a substance formed by association of a compound/conjugate with a solvent molecule. The solvent may be an organic solvent (for example, methanol, ethanol, propanol, acetonitrile, etc.) or the like. For example, a compound/conjugate of the invention can form an ethanolate with ethanol.

In the present invention, the term "hydrate" means a substance formed by association of a compound/conjugate with a water molecule.

In an embodiment of the invention, if a chiral carbon is present in the compound/conjugate, the invention includes isomers formed based on any stereo configuration of the chiral carbon, for example including racemates or any Mirror isomer. Moreover, the invention includes all other stereoisomers that may be present. That is, the compounds/conjugates of the present invention include all enantiomers, diastereomers, cis and trans isomers, racemates and the like.

Can be used in this article

The chemical linkages of the compounds/conjugates of the invention are depicted. use

The bond depicted to the asymmetric carbon atom is intended to include all possible stereoisomers at the carbon atom (e.g., specific enantiomers, racemic mixtures, etc.). use

To characterize the bond to the asymmetric carbon atom, it is indicated that the stereoisomer shown is present. When present in a racemic mixture, use

To define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds/conjugates of the invention are intended to be stereoisomers (including cis and trans isomers, optical isomers (eg, R and S enantiomers), diastereomeric Forms of isomers, geometric isomers, rotamers, conformers, atropisomers, and mixtures thereof exist. The compounds/conjugates of the invention may exhibit more than one type of isomerism and consist of a mixture thereof (e.g., a racemic mixture and a diastereomeric pair).

The invention encompasses all possible crystalline forms or polymorphs of the compounds/conjugates of the invention, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

Pharmaceutically acceptable salts of the compounds/conjugates of the invention include the acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, glucoheptonate, gluconate, orotate, palmitate, and other similar salts.

Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts, and other similar salts.

For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds/conjugates of the present invention are known to those skilled in the art.

Also included within the scope of the invention are metabolites of the compounds/conjugates of the invention, ie, substances formed in vivo upon administration of the compounds/conjugates of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, and the like of the administered compound/conjugate. Accordingly, the invention includes metabolites of the compounds/conjugates of the invention, including compounds/conjugates prepared by contacting a compound/conjugate of the invention with a mammal for a time sufficient to produce a metabolic product thereof.

As used herein, "DAR" means the drug antibody ratio. The average DAR value refers to the average of the DAR of each conjugate in the pharmaceutical composition.

Conjugate

In some embodiments, the invention provides a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

among them:

R ₁ is each independently selected from H (hydrogen), D (oxime), halogen, nitro, -NR _a R _b , C _1-6 alkyl, halo C _1-6 alkyl at each occurrence (for example -CF ₃ ), C _1-6 alkoxy, halo C _1-6 alkoxy (for example -OCF ₃ ) and C _3-6 cycloalkyl; R _a and R _b are each independently selected from H (hydrogen) ), D(氘), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C _1-6 alkylene-C _3-6 Cycloalkyl, -C _1-6 alkylene-(6-10 membered aryl) and -C _1-6 alkylene group (5-12 membered heteroaryl); n _{1 1,} 2, 3 or 4 ;

L _{1 is} selected from -(CH ₂ ) _t1 -,

(Such as

),

L ₂ does not exist or is

L _{5 is} selected from

r is 0, 1, 2, 3, 4 or 5.

In a preferred embodiment, R ₁ is each independently selected from H (hydrogen), D (氘), F, C1, nitro, -NR _a R _b , C _1-6 alkyl, at each occurrence. CF ₃ , -OCH ₃ and -OCF ₃ ; R _a and R _b are each independently selected from H (hydrogen), D (oxime), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10. Aryl, 5-12 membered heteroaryl, -C _1-6 alkylene-C _3-6 cycloalkyl, -C _1-6 alkylene-(6-10 membered aryl) and -C _{1- 6} alkylene-(5-12 membered heteroaryl); n ₁ is 1, 2 or 3.

Preferably, each occurrence of R ₁ is independently selected from the group consisting of H (hydrogen), F, nitro, -NR _a R _b , C _1-4 alkyl, -CF ₃ , -OCH ₃ and -OCF ₃ ; R _a and R _b are each independently selected from the group consisting of H (hydrogen), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 membered aryl, and 5-12 membered heteroaryl; n ₁ is 1 or 2.

More preferably, each occurrence of R ₁ is independently selected from the group consisting of H (hydrogen), nitro, -NH ₂ , -N(CH ₃ ) ₂ and -OCH ₃ ; n ₁ is 1 or 2.

In a preferred embodiment, R _{2 is} selected from the group consisting of H (hydrogen), D (氘), C _1-6 alkyl, and -NR _c R _d ; R _c and R _d are each independently selected from H (hydrogen), D (氘), C _1-6 alkyl, C _3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C _1-6 alkylene-C _3-6 cycloalkyl , -C _1-6 alkylene-(6-10 membered aryl), -C _1-6 alkylene group (5-12 membered heteroaryl), C _1-6 alkyl-C(=O) -, halogenated C _1-6 alkyl-C(=O)-, 6-10 membered aryl-C(=O)-, 5-12 membered heteroaryl-C(=O)-, C _{1- 6-} alkyl-SO ₂ -, halogenated C _1-6 alkyl-SO ₂ -, 6-10 membered aryl-SO ₂ - and 5-12 membered heteroaryl-SO ₂ -; 6-10 yuan aryl and 5-12 membered heteroaryl is optionally substituted with H (hydrogen), D (deuterium), halogen or C _1-6 alkyl substituents; n ₂ is 0, 1 or 4.

Preferably, R _{2 is} selected from the group consisting of H (hydrogen), C _1-6 alkyl and -NR _c R _d ; R _c and R _d are each independently selected from H (hydrogen), D (氘), C _{1 -} ₆ alkane , C _3-6 cycloalkyl, C _1-6 alkyl-C(=O)-, halogenated C _1-6 alkyl-C(=O)-, 6-10 member aryl-C (= O)-, 5-12 membered heteroaryl-C(=O)-, C _1-6 alkyl-SO ₂ -, halogenated C _1-6 alkyl-SO ₂ -, 6-10 membered aryl- SO ₂ - and 5-12 membered heteroaryl-SO ₂ -; the 6-10 membered aryl group and the 5-12 membered heteroaryl group are optionally H (hydrogen), D (oxime), halogen or C _{1 -6} alkyl substituted; n ₂ is 0, 1, 2 or 3.

More preferably, R _{2 is} selected from the group consisting of H (hydrogen), C _1-6 alkyl and -NR _c R _d ; R _c and R _d are each independently selected from H (hydrogen), C _1-6 alkyl, C _{1 -6-} alkyl-C(=O)-, halogenated C _1-6 alkyl-C(=O)-, 6-10 membered aryl-C(=O)-, C _1-6 alkyl-SO _2- , halo C _1-6 alkyl-SO ₂ - and 6-10 membered aryl-SO ₂ -; the 6-10 membered aryl group optionally being H (hydrogen) or C _1-6 alkyl Substituted; n ₂ is 0, 1, 2 or 3.

More preferably, R _{2 is} selected from the group consisting of H (hydrogen), C _1-6 alkyl and -NR _c R _d ; R _c and R _d are each independently selected from H (hydrogen), C _1-6 alkyl, CH ₃ C(=O)-, CF ₃ C(=O)-, benzoyl, CH ₃ SO ₂ -, CF ₃ SO ₂ - and p-toluenesulfonyl; n ₂ is 1 or 2.

Most preferably, R _{2 is} selected from the group consisting of H (hydrogen), C _1-6 alkyl and -NR _c R _d ; R _c and R _d are each independently selected from H (hydrogen), methyl, isopropyl, CH ₃ C(=O)-, benzoyl, CH ₃ SO ₂ - and p-toluenesulfonyl; n ₂ is 2.

In a preferred embodiment, each R is independently selected from H (hydrogen), D (oxime), C _1-6 alkyl, halo C _{1 -} ₆ alkyl (eg, -CF ₃ ), C _1-6 alkoxy and halo C _1-6 alkoxy (e.g., -OCF ₃ ); n is 1, 2 or 3.

Preferably, R is each independently selected from H (hydrogen), C _1-6 alkyl, and halo C _1-6 alkyl (eg, -CF ₃ ); n is 1 or 2.

More preferably, R is each independently selected from H (hydrogen), methyl and -CF ₃ at each occurrence; n is 1 or 2.

In a preferred embodiment, AA is selected from

The above group is attached to the -L1- group by one of the two positions marked by 1 or 2, and passes through another position

Linking, preferably, the above group is attached to the -L ₁ - group via the position of the 1 label, and the position of the label is 2

connection.

More preferably, AA is selected from

Linking, preferably, the above group is attached to the -L1- group through the position of the 1 label, and the position of the label is 2

connection.

Most preferably, AA is selected from

with

connection.

In a preferred embodiment, L _{1 is} selected from

(Such as

),

The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L ₂ by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L _{2 is} attached; R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C _1-6 alkyl, halogenated C _1-6 alkyl, cyano substituted C _1-6 alkyl, C _1-6 alkoxy and C _3-6 cycloalkyl; y1 and y2 are each independently 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10.

Preferably, L _{1 is} selected from

(Such as

),

The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L ₂ by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L _{2 is} attached; R ₃ , R ₄ , R ₅ and R ₆ are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C _1-6 alkyl, halogenated C _1-6 alkyl and cyano substituted C _1-6 alkyl; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

More preferably, L _{1 is} selected from

(Such as

),

Most preferably, L _{1 is} selected from

It is linked to AA by one of the two positions labeled 1 or 2 and to L ₂ by another position, preferably the above group is linked to AA by the position of 1 mark, and the position of L _{2 is} passed by ₂ connection.

In a preferred embodiment, L ₂ is absent or

One of its two positions marks to L ₁ via 1 or 2, and L ₃ is connected by a further position, preferably it is connected to L ₁ via a marked location, and by the marked position 2 and L ₃ Connected, Z _{1 is} selected from O and -NH-, Z ₂ is absent or selected from O and -NH-, and m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Preferably, L ₂ is absent or is

One of its two positions marks to L ₁ via 1 or 2, and L ₃ is connected by a further position, preferably it is connected to L ₁ via a marked location, and by the marked position 2 and L ₃ For the connection, Z ₁ is -NH-, Z ₂ is absent or is -NH-, and m1 and m2 are each independently 0, ₁ , ₂ , 3, 4, 5, 6, 7, 8, 9, or 10.

More preferably, L ₂ is absent or selected from

One of its two positions marks to L ₁ via 1 or 2, and L ₃ is connected by a further position, preferably it is connected to L ₁ via a marked location, and by the marked position 2 and L ₃ connection.

In a preferred embodiment, L ₃ is absent or selected from -NR ₇ - and optionally substituted by one or more R ₇ : C _3-8 cycloalkylene, phenylene, naphthalene a group, a 5-12 membered heteroarylene group, a 3-8 membered heterocyclylene group, a 6-12 membered subbridged heterocyclic group, a 6-12 membered snail heterocyclic group, and a 6-12 membered heterofused heterocyclic group. When the above group contains a nitrogen atom, the nitrogen atom is optionally quaternized; R ₇ is each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonic acid. Base, C _1-4 alkyl, -CF ₃ , C _1-4 alkoxy, C _2-6 alkenyl and C _2-6 alkynyl.

Preferably, L ₃ is absent or is selected from -NR ₇ - and the following groups optionally substituted by one or more R ₇ :

E is a counterion, each of R ₇ being independently selected from H (hydrogen), D (oxime), methyl and -CF ₃ at each occurrence, and the above group is one of two positions labeled by 1 or 2 and L _{2 is} joined and connected to L ₄ by another position, preferably, the above group is linked to L ₂ by the position of 1 mark, and is connected to L ₄ by the position of 2 mark.

More preferably, L ₃ is absent or selected from:

Most preferably, L ₃ is absent or selected from

E is a counter ion, preferably a halogen anion, more preferably a chloride ion, a bromide ion or an iodide ion, the above group being linked to L ₂ by one of two positions labeled 1 or 2, and connected to L ₄ through another position Preferably, the above group is attached to L ₂ through the position of the 1 mark and to the L ₄ through the position of the 2 mark.

In a preferred embodiment, L ₄ is absent or selected from the group consisting of C _1-6 alkylene, -NR ₉ -C _1-6 alkylene-, -C _1-6 alkylene-NR ₉ - and -NR ₉ -; R _{9 is} independently selected from H (hydrogen), D (oxime), C _1-6 alkyl and C _3-6 cycloalkyl; the C _1-6 alkyl or C _1-6 alkylene Optionally substituted with one or more H(hydrogen), D(氘) or halogen.

Preferably, L ₄ is absent or selected from the group consisting of -CH ₂ CH ₂ -, -NHCH ₂ -, -CH ₂ NH- and -NH-.

In a preferred embodiment, L _{5 is} selected from

Preferably, L _{5 is} selected from

In a preferred embodiment, the compound of formula (I) is selected from the group consisting of

In some embodiments, the invention provides a conjugate of Formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

L ₅ ' is selected from

γ is an integer from 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, γ is an integer from 4 to 8, for example, γ is 4, 5, 6 , 7 or 8; and

The remaining groups are as defined above.

In a preferred embodiment, L ₅ ' is selected from

It is linked to L ₄ by one of the two positions labeled 1 or 2 and to the thiol group of the target by another position; preferably it is linked to L ₄ by the position of the 1 mark and passes the position of the 2 mark Attached to the thiol group of the target.

Preferably, L ₅ ' is selected from

In a preferred embodiment, the target is selected from the group consisting of a small molecule ligand, a protein, a polypeptide, and a non-proteinaceous agent (such as a sugar, RNA or DNA).

Preferably, the target is a substance that targets the following targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEA CAM5, mucin (eg mucin) 1 and mucin 16), CD138, CD20, CD19, CD30, SLTRK6, connexin 4, tissue factor, endothelin receptor, STEAP1, SLC39A6, guanylate cyclase C, PSMA, CCD79b, CD22, sodium phosphate synergy Transporter 2B, GPNMB, trophoblastic glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, short proteoglycan, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1 , IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3 , sealing protein 18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1 lc, CD40, CD45 (P TPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2 CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD11b, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF- 1R, RNF43 and NaPi2b.

Preferably, the target is selected from the group consisting of small molecule ligands such as folic acid derivatives, glutamate urea derivatives, somatostatin derivatives, aryl sulfonamide derivatives (eg, carbonic anhydrase IX inhibitors), a combination of two aliphatic anthraquinone polyenes, a cyanine dye, and IR-783 or a derivative thereof; an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (eg, scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, pro-antibody, Bispecific antibody or multispecific antibody; RGD peptide that recognizes cell surface integrin receptor; growth factor that recognizes cell surface growth factor receptor such as EGF, PDGF or VEGF; recognizes functional cell surface plasminogen activation a peptide of factor, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, DR5 ligand and Trop-2 ligand.

More preferably, the target is a monoclonal antibody against Trop-2 or a monoclonal antibody against Her2, such as Sacituzumab, trastuzumab or pertuzumab.

In a preferred embodiment, the conjugate is selected from the group consisting of

Wherein, A is a group obtained by removing γ sulfhydryl groups in Sacituzumab, trastuzumab or pertuzumab or a disulfide bond in Sacituzumab, trastuzumab or pertuzumab is reduced The group obtained after removing γ thiol groups, γ is an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Preferably, the conjugate is selected from the group consisting of:

Wherein, A1 is a group obtained by removing γ thiol groups in Sacituzumab or a group obtained by reducing γ thiol groups after reduction of a disulfide bond in Sacituzumab, and γ is an integer of 4 to 8, for example, γ is 4. 5, 6, 7 or 8.

Pharmaceutical compositions and methods of treatment

In some embodiments, the invention provides a pharmaceutical composition comprising one or more (preferably two or more) conjugates of formula (II) of the invention, or a pharmaceutically acceptable salt, solvate thereof a hydrate, an isomer, or any crystalline form or racemate thereof, and optionally one or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more Other anticancer drugs, such as chemotherapeutic agents and/or antibodies, wherein the pharmaceutical composition has an average DAR value of an integer or fraction from 1 to 10, such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2. , 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 or 10.0, preferably an integer or fraction of 4 to 8, such as 4.0, 4.1, 4.2 , 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

In some embodiments, the invention provides a conjugate of formula (II) of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, in the preparation Use in medicines for preventing or treating cancer diseases.

In some embodiments, the invention provides a conjugate of formula (II) of the invention, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, For the prevention or treatment of cancer diseases.

In some embodiments, the invention provides a method of preventing or treating a cancer disease, the method comprising administering to an individual in need thereof an effective amount of a conjugate of Formula (II) of the present invention, or a pharmaceutically acceptable salt thereof, Solvates, hydrates, isomers, or any crystal form or racemate thereof.

Preferably, the cancer disease is selected from the group consisting of breast cancer (eg, triple negative breast cancer), gastric cancer, esophageal cancer (eg, esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, salivary gland cancer, lung cancer (eg, small cell lung cancer and Non-small cell lung cancer), squamous cell carcinoma, bladder cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, colon cancer, entity Tumor, non-Hodgkin's lymphoma, central nervous system tumor (eg glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

More preferably, the cancer disease is selected from the group consisting of breast cancer (eg, triple negative breast cancer) and gastric cancer.

Further preferably, the cancer disease is selected from the group consisting of gastric cancer.

"Pharmaceutically acceptable carrier" in the context of the invention means a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting humans and/or others within the scope of sound medical judgment. Animal tissue without excessive toxicity, irritation, allergic reactions or other problems or complications corresponding to a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil. , sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol and the like. The composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents as needed. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical compositions of the invention may act systemically and/or locally. For this purpose, they may be administered in a suitable route, for example by injection (for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or by oral, buccal, or oral administration. Nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

The pharmaceutical composition can be formulated into a variety of suitable dosage forms depending on the route of administration. For example, tablets, capsules, troches, hard candy, granules, oral solutions, oral suspensions, oral emulsions, powders, elixirs, syrups, injections, suppositories, gels, ointments, creams, Pastes, lotions, tinctures, aqueous suspensions, elixirs, ophthalmic preparations, pills, implants, aerosols, powders, sprays, and the like.

When administered orally, the pharmaceutical composition can be formulated into any orally acceptable preparation including, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, and oral emulsions. Wait. Among them, the carrier used for the tablet generally includes lactose and corn starch, and a lubricant such as magnesium stearate may also be added. The diluent used in the capsules generally includes lactose and dried corn starch. Oral suspensions are usually prepared by admixing the active ingredient with suitable emulsifiers and suspensions. Optionally, some sweeteners, fragrances or colorants may also be added to the above oral formulation forms.

When administered transdermally or topically, the pharmaceutical compositions may be in the form of a suitable ointment, lotion or lozenge, wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers which can be used in ointment preparations include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; and carriers which can be used for lotions or elixirs include, but are not limited to, minerals Oil, sorbitan monostearate, Tween 60, cetyl esters wax, hexadecene aryl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutical composition can also be administered in the form of an injection, including an injection, a sterile powder for injection, and a concentrated solution for injection. Among them, carriers and solvents which can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may also be employed as a solvent or suspension medium such as a monoglyceride or a diglyceride.

The term "effective amount" as used herein refers to the amount of a conjugate that, after administration, will alleviate one or more symptoms of the condition being treated to some extent.

The dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent need for treatment. It is noted that the dose value can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. It is to be further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or the composition of the supervised composition.

The amount of conjugate of the invention administered will depend on the severity of the individual, condition or condition being treated, the rate of administration, the treatment of the conjugate, and the judgment of the prescribing physician. Generally, an effective dose will be from about 0.0001 to about 50 mg per kg body weight per day, for example from about 0.01 to about 10 mg/kg/day (single or divided doses). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, such as from about 0.7 mg/day to about 700 mg/day. In some cases, a dose level that is not higher than the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any harmful side effects, provided that the larger The dose is divided into several smaller doses to be administered throughout the day.

The conjugate of the present invention may be included in the pharmaceutical composition or a suitable dosage form in an amount of from about 0.01 mg to about 1000 mg, suitably from 0.1 to 800 mg, preferably from 0.5 to 500 mg, more preferably from 0.5 to 350 mg, particularly preferably from 1 to 1. 250mg.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of a condition or condition to which such a term applies or one or more symptoms of such a condition or condition, or Prevention of such a condition or condition or one or more symptoms of such condition or condition.

"Individual" as used herein includes human or non-human animals. Exemplary human individuals include a human individual (referred to as a patient) or a normal individual having a disease, such as the disease described herein. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (eg, birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (eg, sheep, dogs). , cats, cows, pigs, etc.).

Preparation

In some embodiments, the invention provides a method of preparing a compound of Formula (I'), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof:

among them:

R _d ' is R _d or PG ₁ ,

The remaining groups are as defined above,

The method includes the following steps:

Step 1: reacting the compound (Ia)-d with the compound (Ia)-e in the presence of a metal catalyst to obtain a compound of the formula (Ia), and

Step 2: The compound of the formula (Ia) is reacted under conditions to remove the amino protecting group to give a compound of the formula (I') or a pharmaceutically acceptable salt thereof.

Preferably, the metal catalyst is a monovalent copper salt such as a cuprous halide (such as cuprous chloride, cuprous bromide or cuprous iodide); or a divalent copper salt/reducing agent such as copper sulfate/sodium ascorbate .

Preferably, said step 1 of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C.

Preferably, in the first step of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-d to the metal catalyst is 1: (1-15), preferably 1: (1-10), Most preferred is 1: (1-5).

Preferably, said step 1 of the process for the preparation of a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the conditions for the removal of the amino protecting group are acidic conditions, more preferably the conditions in which trifluoroacetic acid is present.

Preferably, said step two of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 °C to 60 °C, preferably from 0 °C to 35 °C, more preferably from 0 °C to 25 °C.

Preferably, said step 2 of the process for the preparation of a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the method of preparing a compound of formula (I'), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, is as follows:

among them:

R _d " is R _d or hydrogen,

R _d ' is R _d or PG ₁ ,

The remaining groups are as defined above,

The method includes the following steps:

Preferably, the base for the preparation of the compound of the formula (I') is selected from the group consisting of an organic base and an inorganic base such as triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, sodium hydroxide or potassium hydroxide. Preferred is triethylamine or diisopropylethylamine.

Preferably, said step 1 of the process for the preparation of a compound of formula (I') is carried out at a temperature between 0 ° C and 40 ° C, preferably between 10 ° C and 30 ° C.

Preferably, the first step of the process for preparing the compound of the formula (I') is carried out in an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methyl Pyrrolidone, a saturated hydrocarbon (such as cyclohexane or hexane), a halogenated hydrocarbon (such as dichloromethane, chloroform or 1,2-dichloroethane), a nitrile (such as acetonitrile), and any combination thereof, preferably two Methyl chloride.

Preferably, the condensing agent of the second step of the process for preparing the compound of the formula (I') is selected from the group consisting of carbonyldiimidazole, triphosgene, preferably triphosgene.

Preferably, the base of the second step of the process for preparing the compound of the formula (I') is selected from the group consisting of an organic base and an inorganic base such as triethylamine, diisopropylethylamine, p-dimethylaminopyridine, pyridine, 2,4,6. -Trimethylpyridine or the like, preferably p-dimethylaminopyridine.

Preferably, in the second step of the process for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-b to (Ia)-c is 1: (0.3-2), preferably 1: (0.8-1.5).

Preferably, in the step 2 of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-b to the condensing agent and the base is 1: (0.3-1): (2-10), preferably 1: ( 0.4-0.7): (3-6).

Preferably, said step 2 of the process for preparing a compound of formula (I') is carried out in an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methyl Pyrrolidone, a saturated hydrocarbon (such as cyclohexane or hexane), a halogenated hydrocarbon (such as dichloromethane, chloroform or 1,2-dichloroethane), a nitrile (such as acetonitrile), and any combination thereof, preferably two Methyl chloride.

Preferably, the metal catalyst of the third step of the method for preparing the compound of the formula (I') is a monovalent copper salt, such as a cuprous halide (such as cuprous chloride, cuprous bromide or cuprous iodide); or a divalent Copper salt/reducing agent, such as copper sulfate/sodium ascorbate.

Preferably, the third step of the process for preparing the compound of the formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C.

Preferably, in the step (3) of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-d to the metal catalyst is 1: (1-15), preferably 1: (1-10), most preferably 1 :(1-5).

Preferably, the method of preparing the compound of the formula (I') is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-A Pyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, dioxane or 1,2-Dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the step of removing the amino protecting group described in the fourth step of the method for preparing the compound of the formula (I') is an acidic condition, more preferably a condition in which trifluoroacetic acid is present.

Preferably, said step 4 of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 °C to 60 °C, preferably from 0 °C to 35 °C, more preferably from 0 °C to 25 °C.

Preferably, said step 4 of the process for preparing a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

In some embodiments, the invention provides a method of preparing a compound of Formula (I"), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof:

among them:

R _d ' is R _d or PG ₁ ,

The remaining groups are as defined above,

The method includes the following steps:

Step 1: reacting compound (Ib)-a with compound (Ib)-b in the presence of a base, optionally in the presence of a condensing reagent, to obtain a compound of formula (Ib), and

Step 2: The compound of the formula (Ib) is reacted under conditions to remove the amino protecting group to give a compound of the formula (I") or a pharmaceutically acceptable salt thereof.

Preferably, the base is an organic base or an inorganic base; the organic base is selected from the group consisting of triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from the group consisting of NaH, NaOH, Na ₂ CO ₃ and K ₂ CO ₃ .

Preferably, the base is DIPEA.

Preferably, the condensation reagent is selected from the group consisting of phosgene, solid phosgene,

HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP and PyBOP.

Preferably, said step 1 of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 °C to 60 °C, preferably from 0 °C to 35 °C, more preferably from 0 °C to 25 °C.

Preferably, in the step (1) of the method for preparing the compound of the formula (I"), the molar ratio of the compound (Ib)-a to the base is 1: (0.5-4), preferably 1: (0.8-3), more Preferably 1: (1-2).

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, said step two of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 °C to 60 °C, preferably from 0 °C to 35 °C, more preferably from 0 °C to 25 °C.

Preferably, said step 2 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the method of preparing a compound of formula (I"), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, is as follows:

among them:

R _d ' is R _d or PG ₁ ,

The remaining groups are as defined above,

The method includes the following steps:

Preferably, the reducing agent is lithium aluminum hydride, palladium carbon, palladium hydroxide, platinum dioxide, triphenylphosphine, preferably platinum dioxide, triphenylphosphine.

Preferably, the base is DIPEA.

HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP and PyBOP.

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out at a temperature of from -10 ° C to 50 ° C, preferably from 10 ° C to 30 ° C.

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out in an organic solvent; said organic solvent being selected from ethers (for example tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxy) Ethane), an alcohol (for example methanol, ethanol, isopropanol or tert-butanol), ethyl acetate and any combination thereof, preferably tetrahydrofuran.

Preferably, in the second step of the method for preparing the compound of the formula (I"), the molar ratio of the compound (Ib)-a to the base is 1: (0.5-4), preferably 1: (0.8-3), more Preferably 1: (1-2).

Preferably, said step three of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 °C to 60 °C, preferably from 0 °C to 35 °C, more preferably from 0 °C to 25 °C.

Preferably, said step 3 of the process for preparing a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

In some embodiments, the invention provides a method of preparing a conjugate of Formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

The method comprises coupling a compound of formula (I) to a target,

Each of the groups is as defined above.

Preferably, the method comprises: mixing a compound of formula (I) with a target; optionally reducing a disulfide bond in the target prior to mixing.

Preferably, the disulfide bond reduction is carried out by using a thiol reducing agent; the thiol reducing agent is preferably TCEP.

Preferably, the method comprises mixing a solution comprising a target with a compound of formula (I).

Preferably, the molar ratio of the target to the compound of formula (I) is 1: (1-20).

Preferably, the process is carried out at a temperature between 0 ° C and 50 ° C, preferably between 15 ° C and 25 ° C.

Preferably, the method is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and N- Methyl pyrrolidone.

Preferably, the method further comprises purifying by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, and affinity chromatography.

Advantageous effects of the invention

The compound of the formula (I) of the present invention can be efficiently coupled with a target such as an antibody, and the coupling efficiency is high, and a target (such as an antibody) to which no coupling occurs is small. The obtained conjugate has remarkable in vitro tumor cell growth inhibitory activity and has an excellent antitumor effect.

Detailed ways

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

The structure of the compound/conjugate described in the following examples was determined by nuclear magnetic resonance spectroscopy ( ¹ H NMR) and/or mass spectrometry (MS).

The nuclear magnetic resonance spectroscopy ( ¹ H NMR) was measured using a Bruker 400 MHz NMR spectrometer; the solvent was deuterated methanol (CD ₃ OD), deuterated chloroform (CDCl ₃ ) or hexamethyl dimethyl sulfoxide (DMSO- d ₆ ); The internal standard substance is tetramethylsilane (TMS).

The abbreviations in the nuclear magnetic resonance (NMR) spectrum data of the examples are as follows:

s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quadruple Quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet , br: broad peak, J: coupling constant, Hz: Hertz, DMSO-d ₆ : hexamethylene dimethyl sulfoxide.

All δ values are expressed in ppm values.

The mass spectrometer (MS) assay instrument used an Agilent (ESI) mass spectrometer, model Agilent 6120B.

The method for preparing the high-performance liquid phase is as follows:

Method A:

Column: Waters Bridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: acetonitrile; mobile phase B: water

时间[min]Time [min]	流动相A[％]Mobile phase A[%]	流动相B[％]Mobile phase B [%]	流速[mL/min]Flow rate [mL/min]
0.000.00	10.010.0	90.090.0	24twenty four
16.0016.00	90.090.0	10.010.0	24twenty four

Method B:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: acetonitrile; mobile phase B: water (containing 0.05% trifluoroacetic acid)

时间[min]Time [min]	流动相A[％]Mobile phase A[%]	流动相B[％]Mobile phase B [%]	流速[mL/min]Flow rate [mL/min]
0.000.00	10.010.0	90.090.0	2828
16.0016.00	90.090.0	10.010.0	2828

Method C:

Column: Waters SunFire Prep C18 OBD 5μm 19x150mm

Mobile phase A: acetonitrile; mobile phase B: water (containing 0.05% formic acid)

Method D:

Column: Waters Bridge Prep C18 OBD 5μm 19x150mm

Mobile phase A: acetonitrile; mobile phase B: water (containing 0.05% ammonium bicarbonate)

The abbreviation in the present invention has the following meanings:

Boc tert-butoxycarbonyl

BOP benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate

DCC N,N'-dicyclohexylcarbodiimide

DCM dichloromethane

DEPC cyanophosphate

DIC N, N'-diisopropylcarbodiimide

DIPEA N,N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMSO-d ₆ hexamethylene dimethyl sulfoxide

DTT dithiothreitol

EDC 1,2-dichloroethane

EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

FA formic acid

HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate

HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate

MMT p-methoxytrityl

MeOH methanol

NMM N-methylmorpholine

PyAOP (3H-1,2,3-triazolo[4,5-b]pyridin-3-yloxy)tri-1-pyrrolidinium hexafluorophosphate

PyBOP 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate

TCEP tris(2-carboxyethyl)phosphine

TMS tetramethylsilane

Example 1

4-((S)-2-(4-Aminobutyl)-35-(4-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H) -pyrrol-1-yl)methyl)cyclohexanecarboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15 ,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(iso) Propylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazine[1, Synthesis of 2-b]quinolin-4-yl)carbonate (Compound I-1)

step one:

(S)-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6, 7] Synthesis of tert-butyl pyridazine[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate (Compound 1-2)

Di-tert-butyl dicarbonate (288 mg, 1.32 mmol) was added dropwise to a solution of compound 1-1 (310 mg, 0.66 mmol) and triethylamine (200 mg, 1.98 mmol) in dichloromethane (10 mL). . The title compound (412 mg) was obtained. ESI-MS (m/z): 534.2 [M+H] ⁺ .

Step two:

(2-((S)-4-((((4-(()))-)-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino) Butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide)benzyl Oxy)carbonyl)oxy)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7 Synthesis of tert-butyl phthalazino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate (Compound 1-3)

A solution of triphosgene (85 mg, 0.29 mmol) in dichloromethane (1 mL) was added dropwise to a solution of 4-dimethylaminopyridine (234 mg, 1.92 mmol) and compound 1-2 (256 mg, 0.48 mmol) at 0 ° C under a nitrogen atmosphere. In a mixed solution of methyl chloride (4 mL), the reaction was carried out for 1 h. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6 under nitrogen protection at 0 °C -azadodecanamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (212.05 Mg, 0.2 mmol) was added to the reaction solution, and the mixture was reacted at 0 ° C for 2 h. The reaction mixture was purified by preparative high-purpur chromatography (Method A). ESI-MS (m/z): 1619.8 [M+H] ⁺ .

Step three:

(2-((S)-4-((((((r)))))) Dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxamide)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4) -Methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-

dioxo

6,12,15,18,21,24,27,30,33-nonaoxa-3, 9-diazatridecanoylamino)benzyl)oxy)carbonyl)oxy)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H- Pyrano[3',4':6,7]pyridazino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamic acid tert-butyl ester (Compounds 1-4 )Synthesis

Add cuprous bromide (18.67 mg, 0.13 mmol) and water (0.5 mL) to (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-) at room temperature. Pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexanecarboxamide (25 mg, 0.09 mmol) and compound 1-3 (75 mg, 0.046 mmol) in dimethyl In a solution of sulfone (3 mL), the reaction was carried out for 2 h. The reaction mixture was purified by preparative EtOAc (EtOAc) ESI-MS (m/z): 1893.9 [M+H] ⁺ .

Step four:

Synthesis of Compound I-1

Compound 1-4 (75 mg, 0.04 mmol) was added to a mixed solvent of trifluoroacetic acid (0.2 mL), dichloromethane (0.05 mL) and water (0.025 mL), and the mixture was stirred for 5 min. The reaction mixture was purified by preparative high-purpur chromatography (Method B). ESI-MS (m/z): 1521.7 [M+H] ⁺ .

Example 2

4-((S)-2-(4-Aminobutyl)-35-(4-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)) Hexanoyl)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33- Nonaza-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(isopropylamino)ethyl)-3,14-di Oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate Synthesis of (Compound I-3)

step one:

(2-((S)-4-(((((((((((((((((((((((((((((((( 1-yl)hexanoamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl) Amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide) Benzyl)oxy)carbonyl)oxy)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6, 7] Synthesis of tert-butyl pyridazine-[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamate (Compound 2-1)

Using a synthetic method similar to that of Step 3 of Example 1, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yn-1 -yl)hexanoic acid instead of (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2- Purification by preparative high performance liquid chromatography (Method A) ESI-MS (m/z): 1867.9 [M+H] ⁺ .

Step two:

Preparation of Compound I-3

Substituting compound 2-1 for compound 1-4 to give the title compound as trifluoroacetic acid salt, 5.18 mg. ESI-MS (m/z): 1495.7 [M+H] ⁺ .

Example 3

4-((S)-2-(4-Aminobutyl)-35-(4-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1) -yl)methyl)piperidin-1-yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12, 15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-( Isopropylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazine[1] Synthesis of 2-b]quinolin-4-yl)carbonate (Compound I-4)

step one:

Synthesis of 1-(piperidin-4-ylmethyl)-1H-pyrrole-2,5-dione (compound 3-2)

Trifluoroacetic acid (1 mL) was added to a solution of Compound 3-1 in dichloromethane (3 mL). The reaction was carried out for 2 h at room temperature. The reaction mixture was concentrated to give crystall The product was used in the next step without purification. ESI-MS (m/z): 195.2 [M+H] ⁺ .

Step two:

Synthesis of 1-((1-(pent-4-ynyl)piperidin-4-yl)methyl)-1H-pyrrole-2,5-dione (Compound 3-3)

N,N-diisopropylethylamine (67 mg, 0.52 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-four at room temperature Methylurea hexafluorophosphate (235 mg, 0.62 mmol) was added to a solution of pent-4-ynoic acid (55 mg, 0.56 mmol) and compound 3-2 (100 mg, 0.52 mmol) in dichloromethane (5 mL). 3h. The reaction mixture was purified by preparative EtOAc (EtOAc) ESI-MS (m/z): 275.1 [M+H] ⁺ .

Step three:

(2-((S)-4-(((((((((((((((((((((((((((()))) -pyrrol-1-yl)methyl)piperidin-1-yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-2-(4-((( 4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa- 3,9-diazatridecanoylamino)benzyl)oxy)carbonyl)oxy)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3',4':6,7]pyridazino[1,2-b]quinolin-11-yl)ethyl)(isopropyl)carbamic acid tert-butyl ester (Compound 3 -4) synthesis

Substitution of compound 3-3 for (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) was carried out in a similar manner to that in the first step of Example 1. Methyl)-N-(prop-2-yn-1-yl)cyclohexanecarboxamide gave the title compound, 26 mg. ESI-MS (m/z): 1893.9 [M+H] ⁺ .

Step four:

Synthesis of Compound I-4

The title compound was obtained as the trifluoroacetic acid salt of the title compound (yield: 2.39 mg). ESI-MS (m/z): 1521.7 [M+H] ⁺ .

Example 4

4-((S)-2-(4-Aminobutyl)-35-(4-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H) -pyrrol-1-yl)methyl)cyclohexanecarboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15 ,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(N -isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]吲Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound I-5)

step one:

(S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3',4':6 ,7]Synthesis of pyridazino[1,2-b]quinolin-11-yl)ethyl)N-isopropylmethanesulfonamide (Compound 4-2)

Compound 4-1 hydrochloride (150 mg, 0.33 mmol) was dissolved in dichloromethane (4 mL), EtOAc (EtOAc) Purification by preparative high performance liquid chromatography (Method C) gave the title compound 75 mg. ESI-MS (m/z): 512.3 [M+H] ⁺ .

Step two:

4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 ,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-( 2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6 ,7]Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 4-3)

A solution of triphosgene (41 mg, 0.14 mmol) in dichloromethane (1 mL) was added dropwise to 4-dimethylaminopyridine (136.5 mg, 1.12 mmol) and compound 4-2 (71.7 mg, 0.14 mmol). The mixed solution of dichloromethane (4 mL) was reacted for 1 h. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6 under nitrogen protection at 0 °C -azadodecanamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (14.0 Mg, 0.14 mmol) was added to the reaction solution, and the mixture was reacted at 0 ° C for 2 h. The reaction mixture was purified by preparative EtOAc (EtOAc) ESI-MS (m/z): 1597.7 [M+H] ⁺ .

Step three:

4-((S)-35-(4-(((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)) Cyclohexanecarboxamide)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl)amino Butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide)benzyl (S)-4-Ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro Synthesis of -1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 4-4).

Compound 4-3 (43 mg, 0.025 mmol) and (1r, 4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl at room temperature -N-(prop-2-yn-1-yl)cyclohexanecarboxamide (14.0 mg, 0.050 mmol) was added to a mixed solvent of H ₂ O (0.4 ml) and DMSO (1.6 ml), followed by bromine Cuprous (10.8 mg, 0.075 mmol) was stirred for 2 h. Filtration and purification of the filtrate by preparative HPLC (Method A) ESI-MS (m/z): 1871.8 [M+H] ⁺ .

Step four:

Synthesis of Compound I-5

Compound 4-4 (30 mg, 0.016 mmol) was added to a mixed solvent of trifluoroacetic acid (0.2 mL), dichloromethane (0.05 mL) and water (0.025 mL), and the mixture was stirred for 5 min. The reaction mixture was purified by preparative high-purpur chromatography (Method B).

^{1 H NMR (400MHz, DMSO-} d 6) δ10.19 (s, 1H), 8.33 (d, J = 8.3Hz, 1H), 8.23-8.21 (m, 3H), 8.10 (t, J = 5.6Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.82 (s, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.68 (s, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.06 (s, 1H), 7.01 (s, 2H), 5.54 (d, J = 3.9 Hz, 2H), 5.46 (s, 2H), 5.11 ( q, J = 12.2 Hz, 2H), 4.47 (t, J = 4.9 Hz, 3H), 4.25 (d, J = 5.5 Hz, 2H), 4.04 (d, J = 3.6 Hz, 2H), 4.00 (d, J=2.5 Hz, 2H), 3.99-3.93 (m, 1H), 3.78 (t, J = 5.2 Hz, 2H), 3.50-3.43 (m, 32H), 3.39-3.35 (m, 2H), 3.28 (q) , J=5.6 Hz, 2H), 3.23 (d, J=7.0 Hz, 2H), 3.00 (s, 3H), 2.82-2.74 (m, 2H), 2.21-2.15 (m, 2H), 2.09-2.02 ( m,1H), 1.82-1.66 (m, 4H), 1.63-1.47 (m, 5H), 1.42-1.23 (m, 4H), 1.15 (q, J = 6.9 Hz, 6H), 0.91 (t, J = ESI-MS (m/z): 800.5 [M/2+H] ⁺ .

Example 5

4-((S)-2-(4-Aminobutyl)-35-(4-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H) -pyrrol-1-yl)methyl)cyclohexanecarboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15 ,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(N -isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazine Synthesis of [1,2-b]quinolin-4-yl)carbonate (Compound I-6)

step one:

(S)-N-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4': Synthesis of 6,7]oxazino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide (Compound 5-2)

Triethylamine (300 mg, 3.0 mmol) and acetyl chloride (100 mg, 1.2 mmol) were added dropwise to a solution of Compound 5-1 (440 mg, 1.0 mmol) in dichloromethane (30 mL). The reaction mixture was concentrated to give purified crystals crystals crystals eluted ESI-MS (m/z): 476.3 [M+H] ⁺ .

Step two:

4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 ,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-( 2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7 Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 5-3)

Compound 5-2 (60 mg, 0.13 mmol) was dissolved in dry dichloromethane <RTI ID=0.0>(2 </RTI><RTIID=0.0></RTI></RTI><RTIgt; A solution of methane (0.5 mL) was added dropwise a solution of triphos (37 mg, 0.13 mmol) in dry dichloromethane (0.5 mL), and the mixture was stirred and stirred at 0 ° C for 1 hour. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriene was added to the reaction solution. Dodecanoylamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (118 mg, 0.11 mmol) A dry dichloromethane (0.5 mL) solution was added and the mixture was stirred at 0 ° C for 2 h. Purification by preparative high performance liquid chromatography (Method A) gave the title compound. ESI-MS (m/z): 1562.5 [M+H] ⁺ .

Step three:

4-((2S)-35-(4-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)) Cyclohexanecarboxamide)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl)amino Butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide)benzyl (S)-4-Ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H Synthesis of pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 5-4)

Compound 5-3 (20 mg, 0.012 mmol) and (1r, 4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl at room temperature -N-(prop-2-yn-1-yl)cyclohexanecarboxamide (5.3 mg, 0.019 mmol) was dissolved in dimethyl sulfoxide (0.5 mL) and water (0.1 mL). Copper (3.67 mg, 0.026 mmol) was stirred for 1 h. Filtration and purification of the filtrate by preparative HPLC (Method A) ESI-MS (m/z): 1836.5 [M+H] ⁺ .

Step four:

Synthesis of Compound I-6

Compound 5-4 (18 mg, 0.011 mmol) was dissolved in EtOAc (EtOAc) (EtOAc) The reaction mixture was purified by preparative high-purpur chromatography (Method B).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.19 (s, 1H), 8.57 (d, J = 8.5 Hz, 1H), 8.21. (t, J = 6.4 Hz, 3H), 8.10 (t, J = 5.6 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.82 (s, 1H), 7.79 (t, J = 7.3 Hz, 1H), 7.69 (s, 3H), 7.61 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 7.05 (s, 1H), 7.01 (s, 2H), 5.54 (d, J = 4.0 Hz, 2H), 5.45 (s, 2H) , 5.11 (q, J = 12.2 Hz, 2H), 4.47 (t, J = 5.1 Hz, 3H), 4.25 (d, J = 5.6 Hz, 2H), 4.13-4.06 (m, 1H), 4.04 (d, J = 3.6 Hz, 2H), 4.00 (d, J = 2.6 Hz, 2H), 3.78 (t, J = 5.3 Hz, 2H), 3.50-3.40 (m, 34H), 3.28 (q, J = 6.0 Hz, 2H), 3.23 (d, J = 7.1 Hz, 2H), 2.82-2.74 (m, 2H), 2.23-2.16 (m, 2H), 2.14 (s, 3H), 2.10-2.02 (m, 1H), 1.79 -1.66 (m, 4H), 1.63-1.47 (m, 5H), 1.42-1.22 (m, 4H), 1.19 (q, J = 3.1 Hz, 6H), 0.91 (t, J = 7.3 Hz, 3H), ESI-MS (m/z): 1563.7 [M+H] ⁺ .

Example 6

4-((S)-2-(4-Aminobutyl)-35-(4-((6-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) Caproamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33 -nonaoxa-3,9-diazatridecanoylamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)- 3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazino[1,2-b]quinoline-4 Synthesis of -base)carbonate (compound I-7)

step one:

4-((S)-35-(4-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)methyl)-1H- 1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo- 6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11- (2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6, 7] Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 6-1)

Using a synthetic procedure similar to that of Step 3 of Example 5, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)N-(prop-2-yn-1- Substituted hexamide to replace (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yne) Purification by preparative high performance liquid chromatography (method A) gave the title compound, 10 mg. ESI-MS (m/z): 1810.5 [M+H] ⁺ .

Step two:

4-((S)-2-(4-Aminobutyl)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) Acetamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33- Nonaza-3,9-diazatridecylamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3 ,14-Dioxy-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazino[1,2-b]quinolin-4-yl Synthesis of Carbonate Trifluoroacetate (Compound I-7)

The title compound was purified by preparative high-performance liquid chromatography (Method B) to give the title compound as a trifluoroacetate, 1.67 mg. ESI-MS (m/z): 1537.7 [M+H] ⁺ .

Example 7

4-((S)-2-(4-Aminobutyl)-35-(4-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1) -yl)methyl)piperidin-1-yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12, 15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-( N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7] Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound I-8)

step one:

4-((S)-35-(4-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)piperidine-1 -yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl) Amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide) Benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetra Synthesis of Hydrogen-1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 7-1)

Substitution with 1-((1-(pent-4-ynyl)piperidin-4-yl)methyl)-1H-pyrrole-2,5-dione by a similar method to the third step of Example 5 ( 1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl) ring The title compound was obtained after purification by preparative high-purity chromatography (Method A). ESI-MS (m/z): 1871.8 [M+H] ⁺ .

Step two:

Preparation of Compound I-8

The title compound was obtained as a trifluoroacetic acid salt of the title compound (yield: EtOAc). ESI-MS (m/z): 1599.7 [M+H] ⁺ .

Example 8

4-((S)-2-(4-Aminobutyl)-35-(4-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1) -yl)methyl)piperidin-1-yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12, 15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-( N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]吲哚Synthesis of oxazolo[1,2-b]quinolin-4-yl)carbonate (Compound I-9)

step one:

4-((S)-35-(4-(3-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)piperidine-1 -yl)-3-oxopropyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl) Amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide) Benzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro- Synthesis of 1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 8-1)

Substituting 1-((1-(pent-4-ynyl)piperidin-4-yl)methyl)-1H-pyrrole-2,5-dione (1r) in a similar manner to the third step of Example 5 , 4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane The title compound was obtained after purification by preparative HPLC (Method A). ESI-MS (m/z): 1835.8 [M+H] ⁺ .

Step two:

Preparation of Compound I-9

The title compound was obtained as a trifluoroacetic acid salt of the title compound (23.3 mg). ESI-MS (m/z): 1563.7 [M+H] ⁺ .

Example 9

4-((S)-36-(4-aminobutyl)-1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1) -yl)methyl)cyclohexyl)-1,30,34-trioxo-5,8,11,14,17,20,23,26,32-nonaoxa-2,29,35-triazole Hetero-heptadecanoylamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3 ,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound I- 10) Synthesis

step one:

4-((S)-35-Amino-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12 ,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2- (N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7 Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 9-1)

Compound 4-3 (30 mg, 0.019 mmol) was dissolved in tetrahydrofuran (6.0 mL) at room temperature. Platinum dioxide (5 mg) was added under N ₂ and the mixture was replaced with hydrogen three times and reacted at room temperature for 3 h. The insoluble material was filtered off, and the filtrate was concentrated. ESI-MS (m/z): 650.3 [(M-273) / 2+H] ⁺ .

Step two:

4-((S)-1-((1,5r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)- 36-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-1,30,34-trioxo-5,8,11,14,17,20, 23,26,32-nonaoxa-2,29,35-triazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(N-isopropyl) Alkylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazine[1] Synthesis of 2-b]quinolin-4-yl)carbonate (Compound 9-2)

Compound 9-1 (20 mg, 0.013 mmol) and (1r, 4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl at room temperature Methyl cyclohexanecarboxylate (6 mg, 0.019 mmol) was dissolved in dichloromethane (2 mL), and N,N-diisopropylethylamine (3.0 mg, 0.025 mmol) was added dropwise. The reaction solution was used directly for the next reaction.

Step three:

Synthesis of Compound I-10

Trifluoroacetic acid (0.1 mL) was added to a reaction mixture of Compound 9-2 (20 mg, 0.011 mmol The reaction mixture was concentrated and purified by preparative EtOAc (EtOAc) ESI-MS (m/z): 760.0 [M / 2+H] ⁺ .

Example 10

4-((S)-36-(4-aminobutyl)-1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1) -yl)methyl)cyclohexyl)-1,30,34-trioxo-5,8,11,14,17,20,23,26,32-nonaoxa-2,29,35-triazole Hetero-heptadecanolamide)benzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4 ,12,14-tetrahydro-1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound I-11) Synthesis

step one:

4-((S)-35-Amino-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12 ,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2- (N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]吲Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 10-1)

The title compound was obtained by the preparative high-performance liquid chromatography (method D). ESI-MS (m/z): 632.5 [(M-273) / 2+H] ⁺ .

Step two:

4-((S)-1-((1,5r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)- 36-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-1,30,34-trioxo-5,8,11,14,17,20, 23,26,32-nonaoxa-2,29,35-triazatridecanoylamino)benzyl ((S)-4-ethyl-11-(2-(N-isopropyl) Amido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazine[1,2 Synthesis of -b]quinolin-4-yl)carbonate (Compound 10-2)

The synthesis method similar to the second step of Example 9 was carried out, and the compound 10-1 was used instead of the compound 9-1, and the reaction liquid was directly used for the next reaction.

Step three:

Synthesis of Compound I-11

Purification by high performance liquid chromatography (method B) gave the title compound as a trifluoroacetic acid salt, 1.5 mg. ESI-MS (m/z): 742.0 [M / 2+H] ⁺ .

Example 11

4-((S)-2-(4-Aminobutyl)-35-(4-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H) -pyrrol-1-yl)methyl)cyclohexanecarboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15 ,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)-2-methylbenzyl ((S)-4-ethyl-11- (2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6, 7] Synthesis of oxazino[1,2-b]quinolin-4-yl)carbonate (Compound I-12)

step one:

(S)-2-Amino-N-(4-(hydroxymethyl)-3-methylphenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide Synthesis of (Compound 11-2)

4-Amino-2-methylbenzyl alcohol (107 mg, 0.78 mmol) and compound 11-1 (500 mg, 0.78 mmol) were dissolved in dichloromethane (5 mL). Ethoxycarbonyl-1,2-dihydroquinoline (289 mg, 1.17 mmol) was stirred at 35 ° C for 5 h. After dropping to room temperature, diethylamine (1 mL) was added to the reaction mixture and stirring was continued for 2 h. The reaction mixture was concentrated and purified mjjjjjjjj

Step two:

(S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxaoxa-6-azatridodecanoylamide -N-(4-(hydroxymethyl)-3-methylphenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (Compound 11-3) Synthesis

Compound 11-2 (100 mg, 0.19 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-nitrogen at room temperature Hetero-d-dodecane-1-acid (103 mg, 0.19 mmol) was dissolved in dichloromethane (5 mL) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (69 mg, 0.28) Methyl), the reaction was stirred at 30 ° C for 5 h. The reaction mixture was concentrated and purified tolulululululu ESI-MS (m/z): 1074.5 [M+H] ⁺ .

Step three:

4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 ,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)-2-methylbenzyl ((S)-4-B -11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4 Synthesis of ':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 11-4)

Substituting the compound 11-3 for the substitution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27 by a similar synthesis method in the second step of Example 5. , 30-nonaoxa-6-azatridodecanoylamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenyl) Purification by preparative high performance liquid chromatography (Method A) gave the title compound, 60 mg. ESI-MS (m/z): 1575.6 [M+H] ⁺ .

Step four:

4-((S)-35-(4-(((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)) Cyclohexanecarboxamide)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-((4-methoxyphenyl)diphenylmethyl)amino Butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazatridecylamide)- 2-methylbenzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14 Synthesis of tetrahydro-1H-pyrano[3',4':6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 11-5)

The title compound was obtained by the preparative high-performance liquid chromatography (method A). ESI-MS (m/z): 1824.7 [M+H] ⁺ .

Step five:

Synthesis of Compound I-12

Compound 11-5 (15 mg, 0.008 mmol) was dissolved in acetonitrile (0.4 mL) and water (0.1 mL), and a solution of trifluoroacetic acid (0.5 mL) in acetonitrile (0.5 mL). Stir at room temperature for 2 h. Purification by preparative high performance liquid chromatography (Method B) gave the title compound. ESI-MS (m/z): 1577.7 [M+H] ⁺ .

Example 12

4-((S)-2-(4-Aminobutyl)-35-(4-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)) Hexanoyl)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33- Nonaza-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl) -3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridazino[1,2-b]quinoline- Synthesis of 4-yl)carbonate (Compound I-13)

step one:

4-((S)-35-(4-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)methyl)-1H- 1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo- 6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatridecylamide)benzyl ((S)-4-ethyl-11- (2-(N-Isopropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4': Synthesis of 6,7]oxazino[1,2-b]quinolin-4-yl)carbonate (Compound 12-1)

Using a similar synthetic procedure as in step 3 of Example 4, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(prop-2-yne-1 -yl)hexanoic acid instead of (1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2- Purification by preparative high performance liquid chromatography (method A) gave the title compound, 30 mg. ESI-MS (m/z): 1846.7 [M+H] ⁺ .

Step two:

Synthesis of Compound I-13

The title compound was obtained as a trifluoroacetic acid salt of the title compound (19.2 mg). ESI-MS (m/z): 1574.6 [M+H] ⁺ .

Preparation of conjugate

Example 13 Preparation of II-1-A

Take 5 mg of Sacituzumab, dilute with a diluent (20 mM PB + 150 mM NaCl + 20 mM sodium edetate solution, pH 7.6) (final concentration of sodium edetate 5 mM, final antibody concentration of 10 mg / mL), mix; then use 1M The Na ₂ PO4 solution was adjusted to pH 7.4, mixed with 10 mM TCEP solution, and allowed to stand at room temperature for 30 min; I-1 (I-1 to antibody molar ratio of 9:1) dissolved in dimethyl sulfoxide was added to the above solution system. , mix, and let stand at room temperature for 2 h. Finally, the buffer was replaced with a PBS solution of pH 6.5 using a G-25 gel column to obtain a product of I-1 and antibody coupling, designated as II-1-A.

Example 14 Preparation of II-5-A

In a similar manner to Example 13, I-1 was replaced with I-3 to give the product of I-3 and antibody coupling, designated II-5-A.

Example 15 Preparation of II-6-A

In a similar manner to Example 13, I-1 was replaced with I-4 to give the product of I-4 and antibody coupling, designated II-6-A.

Example 16 Preparation of II-7-A

In a similar manner to Example 13, I-1 was replaced with I-5 to give the product of I-5 and antibody coupling, designated II-7-A.

Example 17 Preparation of II-8-A

In a similar manner to Example 13, I-1 was replaced with I-6 to give the product of I-6 and antibody coupling, designated II-8-A.

Example 18 Preparation of II-9-A

In a similar manner to Example 13, I-1 was replaced with I-7 to give the product of I-7 and antibody coupling, designated II-9-A.

Example 19 Preparation of II-10-A

In a similar manner to Example 13, I-1 was replaced with I-8 to give the product of I-8 and antibody coupling, designated II-10-A.

Example 20 Preparation of II-11-A

In a similar manner to Example 13, I-1 was replaced with I-9 to give the product of I-9 and antibody coupling, designated II-11-A.

Example 21 Preparation of II-12-A

In a similar manner to Example 13, I-1 was replaced with I-10 to give the product of I-10 and antibody coupling, designated as II-12-A.

Example 22 Preparation of II-13-A

In a similar manner to Example 13, I-1 was replaced with I-11 to give the product of I-11 and antibody coupling, designated II-13-A.

Example 23 Preparation of II-14-A

In a similar manner to Example 13, I-1 was replaced with I-12 to give the product of I-12 and antibody coupling, designated II-14-A.

Example 24 Preparation of II-15-A

In a similar manner to Example 13, I-1 was replaced with I-13 to give the product of I-13 and antibody coupling, designated II-15-A.

Molecular weight determination and DAR value analysis of conjugates

In the following examples, LC represents the antibody light chain, HC represents the antibody heavy chain, DAR1 represents a conjugate comprising an antibody light or heavy chain and a cytotoxic agent, and DAR2 represents an antibody light or heavy chain and two a conjugate of a cytotoxic agent, DAR3 represents a conjugate comprising one antibody light or heavy chain and three cytotoxic agents, and DAR4 represents a conjugate comprising one antibody light or heavy chain and four cytotoxic agents .

Example 25 LC-MS molecular weight analysis of the conjugate obtained after coupling

Sample treatment: Dilute the sample to 1 mg/mL, add 1M DTT (dithiothreitol) to a final DTT concentration of 20 mmol/L, and mix at 37 ° C for 30 min.

The chromatographic conditions are as follows:

LC column: ACQUITU

Protein BEH C18 1.7μm, 2.1mm×100mm

Mobile phase A: 0.1% formic acid (FA) / 98% H ₂ O / 2% acetonitrile (ACN); mobile phase B: 0.1% FA / 2% H ₂ O / 98% ACN

Flow rate: 0.25 mL/min; sample chamber temperature: 8 ° C; column temperature: 60 ° C; injection volume: 1 μg

Elution conditions:

时间(分钟)Time (minutes)	22	2020	22twenty two	2525	2626	3030
流动相B(体积％)Mobile phase B (% by volume)	2020	4040	9090	9090	2020	2020

Mass spectrometry conditions:

Mass Spectrometer Model: Triple TOF 5600+

GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m/z 600-5000

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-1-A formed by coupling of the compound I-1 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 1 and 2. DAR: Light chain coupled with 0 to 2 cytotoxic agents (LC, DAR1 and DAR2 ratios of 22%, 72%, respectively) were calculated based on the ratio of signal intensity after mass deconvolution. 6%), heavy chain coupling of 1 to 3 cytotoxic agents (DAR1, DAR2 and DAR3 ratios of 49%, 27% and 24%, respectively), thereby calculating the coupling ratio of antibody to cytotoxic agent (average DAR) The value) is 5.0.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-5-A formed by coupling of the compound I-3 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 3 and 4. DAR: Light chain coupled with 0 to 1 cytotoxic agent (LC and DAR1 ratios of 18% and 82%, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. The heavy chain was coupled with 1 to 3 cytotoxic agents (the ratios of DAR1, DAR2, and DAR3 were 46%, 20%, and 34%, respectively), and the coupling ratio (average DAR value) of the antibody to the cytotoxic agent was calculated to be 5.4.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-6-A formed by coupling of the compound I-4 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 5 and 6. DAR: Light chain coupled with 0 to 1 cytotoxic agent (LC and DAR1 ratios of 16% and 84%, respectively) were calculated based on the ratio of signal intensity after mass deconvolution. The heavy chain was coupled with 1 to 3 cytotoxic agents (the ratios of DAR1, DAR2, and DAR3 were 31%, 23%, and 46%, respectively), and the coupling ratio (average DAR value) of the antibody to the cytotoxic agent was calculated to be 6.0.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-7-A formed by coupling of the compound I-5 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 7 and 8. DAR: Light chain coupled with 0 to 1 cytotoxic agent (24% and 76% for LC and DAR1, respectively), based on the ratio of signal intensity after deconvolution by mass spectrometry, Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 6%, 43%, 20%, 25%, and 6%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) was 5.2.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-8-A formed by coupling of the compound I-6 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 9 and 10. DAR: Light chain coupled with 0 to 1 cytotoxic agent (19% and 81% for LC and DAR1, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 6%, 37%, 18%, 32%, and 7%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) is 5.5.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-9-A formed by coupling of the compound I-7 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 11 and 12. DAR: Light chain coupled with 0 to 1 cytotoxic agent (19% and 81% for LC and DAR1, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. The heavy chain is coupled with 0 to 3 cytotoxic agents (HC, DAR1, DAR2, and DAR3 ratios of 3%, 45%, 18%, and 34%, respectively), thereby calculating the coupling ratio of the antibody to the cytotoxic agent (average DAR) The value) is 5.3.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-10-A formed by coupling of the compound I-8 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 13 and 14. The DAR: light chain coupled with 0 to 1 cytotoxic agent (LC and DAR1 ratios of 20% and 80%, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 4%, 43%, 20%, 29%, and 4%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) is 5.3.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-11-A formed by coupling of the compound I-9 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 15 and 16. DAR: Light chain coupled with 0 to 1 cytotoxic agent (19% and 81% for LC and DAR1, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 5%, 44%, 17%, 31%, and 3%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) is 5.3.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-12-A formed by coupling of the compound I-10 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 17 and 18. DAR: Light chain coupled with 0 to 1 cytotoxic agent (LC and DAR1 ratios of 26% and 74%, respectively) was calculated based on the ratio of signal intensity after mass deconvolution. Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 6%, 41%, 21%, 23%, and 9%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) was 5.2.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-13-A formed by coupling of the compound I-11 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 19 and 20. DAR: Light chain coupled with 0 to 1 cytotoxic agent (19% and 81% for LC and DAR1, respectively), based on the ratio of signal intensity after deconvolution of mass spectrometry, The heavy chain is coupled with 0 to 3 cytotoxic agents (HC, DAR1, DAR2, and DAR3 ratios of 3%, 47%, 18%, and 32%, respectively), thereby calculating the coupling ratio of the antibody to the cytotoxic agent (average DAR) The value) is 5.2.

The theoretical molecular weight of the light chain and heavy chain of the conjugate II-15-A formed by coupling of the compound I-13 with the antibody and the measured molecular weight (calculated as the main glycoform G0F) are shown in the following table:

Mass spectrometry deconvolution results are shown in Figures 21 and 22, and DAR is calculated based on the ratio of signal intensity after deconvolution of mass spectrometry: light chain coupled with 0 to 1 cytotoxic agent (LC and DAR1 ratios are 28% and 72%, respectively). Heavy chain coupling of 0 to 4 cytotoxic agents (HC, DAR1, DAR2, DAR3, and DAR4 ratios of 9%, 48%, 19%, 18%, and 6%, respectively), thereby calculating the antibody and cytotoxic agent The joint ratio (average DAR value) is 4.7.

Size exclusion chromatography (SEC) analysis

Example 26 The coupling reaction was monitored by SEC-HPLC for SEC detection.

Chromatographic conditions:

Liquid chromatography column: TOSOH TSKgel G3000 SWXL 7.8×300mm

Mobile phase: 25 mmol/L Na ₂ HPO ₄ , 25 mmol/L NaH ₂ PO ₄ , 300 mmol/L NaCl, pH 6.5

Flow rate: 0.5 mL/min; detection wavelength: 280 nm; column temperature: room temperature; sample chamber temperature: 8 ° C

Injection volume: 30μg; isocratic operation for 30min

Based on the SEC retention time and peak area ratio (Fig. 23), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-1-A obtained by coupling I-1 with the antibody, and still maintained the intact structure of the antibody.

Based on the SEC retention time and peak area ratio (Fig. 24), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-5-A obtained by coupling I-3 with the antibody, and still maintained the intact structure of the antibody.

Based on the SEC retention time and peak area ratio (Fig. 25), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-7-A obtained by coupling I-5 with the antibody, and still maintained the intact structure of the antibody.

Based on the SEC retention time and peak area ratio (Fig. 26), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-8-A obtained by coupling I-6 with the antibody, and still maintained the intact structure of the antibody.

Based on the SEC retention time and peak area ratio (Fig. 27), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-10-A obtained by coupling I-8 with the antibody, and still maintained the intact structure of the antibody.

Based on the SEC retention time and peak area ratio (Fig. 28), it was confirmed that the main coupling product had a molecular weight of about 150 kD, that is, II-14-A obtained by coupling I-12 with the antibody, and still maintained the intact structure of the antibody.

Size exclusion chromatography (SEC) analysis showed that after the antibody was conjugated to the cytotoxic agent, the light and heavy chains of the antibody were still bound by hydrophobic interaction, etc., maintaining the intact structure of the antibody and maintaining the activity of the antibody well.

Biological test

Example 27 conjugated object external drug efficacy test

This test is carried out by using the cytotoxic agent of the present invention (Compound 1-1 in Example 1, Compound 4-2 in Example 4, and Compound 5-2 in Example 5) and antibody-drug conjugate, respectively. Tumor cells were co-cultured and the activity of dehydrogenase in the mitochondria was examined to evaluate the inhibitory effect of the conjugate on cell proliferation. Test procedure: The conjugate was diluted with the corresponding detection medium (RPMI 1640 containing 2% FBS), and the cytotoxic agent was used as a control, starting at 1000 nM, diluted 4 times, and diluting 12 concentration gradients. The breast cancer cells HCC1806 (Trop-2 positive cell line, Nanjing Branch) were digested by trypsin using conventional methods, and tumor cells were collected and resuspended in the corresponding assay medium (RPMI1640 containing 2% FBS). The diluted conjugate and cytotoxic agent were separately added to a 96-well plate and then added to the cells. After 3 days of cell culture, 20 μL of CCK8 reagent (RHINO BIO) was added to each well for 2 hours, and the activity of dehydrogenase in the mitochondria was measured using a microplate reader (manufactured by Molecular Devices, model: SpectraMax M2, detection wavelength: 450 nm). Based on this, the EC ₅₀ value of the growth inhibitory effect of the cytotoxic agent and the conjugate on the breast cancer cell HCC1806 was calculated. The results of the growth inhibition of conjugates on breast cancer cells HCC1806 are shown in Table 1 below:

Table 1. Growth inhibition results of conjugates on breast cancer cells HCC1806

As can be seen from Table 1, the antibody-drug conjugate of the present invention has a significant growth inhibitory effect on HCC1806 (breast cancer cells).

Further, the EC ₅₀ values of the growth inhibitory action of the compound 1-1 in Example 1, the compound 4-2 in the Example 4, and the compound 5-2 in the Example 5 on the breast cancer cell HCC1806 were 4.07 nM and 7.56 nM, respectively. And 8.72 nM, and the corresponding antibody-drug conjugates II-1-A, II-7-A and II-8-A have EC ₅₀ values of 1.54 nM, 1.63 nM and 1.15 nM, respectively, which are significantly lower than corresponding The EC ₅₀ value of the cytotoxic agent. This further demonstrates that after the cytotoxic agent is conjugated to the antibody, its tumor inhibiting activity is significantly enhanced due to increased targeting.

Example 28 Conjugate Plasma Stability Test

Experimental methods: samples of II-1-A, II-7-A and II-8-A were prepared from rat plasma, cynomolgus plasma and 0.5% bovine serum albumin (BSA) PBS buffer, respectively, and placed at 37 ° C. In the biochemical incubator, they were incubated for 0h, 1h, 4h, 24h, 48h and 72h respectively, and then stored at -80 °C. The release of cytotoxic agent in the conjugate was determined by LC/MS to determine the conjugate. Plasma stability. The cytotoxic agents corresponding to II-1-A, II-7-A and II-8-A are the compound 1-1 in Example 1, the compound 4-2 in Example 4, and the compound in Example 5, respectively. 5-2.

As can be seen from Figures 29-31, both II-1-A, II-7-A and II-8-A have good plasma stability in cynomolgus plasma, rat plasma and 0.5% BSA PBS buffer.

Example 29 Pharmacodynamic test in a coupled object

Experimental method: NCI-N87 cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, and NCI-N87 cells in exponential growth phase were collected. Resuspend in PBS to the appropriate concentration and inoculate subcutaneously on the right scapula of Balb/c nude mice. After approximately 100-200mm ³ volumes of inoculation of tumor growth, tumor volume removed too small (less than 100mm ³⁾ or too large (greater than 200mm ³⁾ mice, the remaining mice were randomized into groups (8 / group) and to Drug conjugate or solvent control (saline), tail vein injection, 2 times a week for a total of 3 weeks. After 3 weeks of administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5 a ² × b, where a and b represent the long and short diameters of the tumor, respectively. Animal deaths were recorded daily.

The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the antitumor efficacy of antibody-drug conjugates:

TGI (%) = [1- (VT end - VT start) / (VC end - VC start)] × 100%

Among them, the end of VT: the mean volume of the tumor at the end of the experiment in the conjugate treatment group,

VT start: the mean volume of the tumor at the start of administration of the conjugate treatment group,

End of VC: the mean volume of the tumor at the end of the experiment in the solvent control group,

Beginning of VC: mean volume of tumor at the beginning of drug control group administration.

The experimental results are shown in Table 2 below:

Table 2. Results of drug efficacy test in coupled objects

As can be seen from Table 2, the conjugates of the present invention (e.g., II-8-A and II-10-A) have significant antitumor effects, and the P value results indicate that the conjugate exhibits significant contrast compared with the physiological saline group. Anti-tumor efficacy trend.

In addition, there was no animal death or weight loss in the conjugate treatment group during the evaluation period, indicating that the conjugates of the present invention (for example, II-8-A and II-10-A) not only have remarkable antitumor effects, but also have excellent properties. Security.

Although the invention has been illustrated by the foregoing specific embodiments, it should be understood that The present invention covers the general aspects of the invention, and various modifications and changes in the details of the invention may be made without departing from the spirit and scope of the invention. Accordingly, the description is for illustrative purposes only and not for purposes of limitation.

Claims

a compound of formula (I): or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof,

among them:

R 1 is each independently selected from H (hydrogen), D (oxime), halogen, nitro, -NR a R b , C 1-6 alkyl, halo C 1-6 alkyl at each occurrence (for example -CF 3 ), C 1-6 alkoxy, halo C 1-6 alkoxy (for example -OCF 3 ) and C 3-6 cycloalkyl; R a and R b are each independently selected from H (hydrogen) ), D(氘), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C 1-6 alkylene-C 3-6 Cycloalkyl, -C 1-6 alkylene-(6-10 membered aryl) and -C 1-6 alkylene group (5-12 membered heteroaryl); n 1 is 1, 2, 3 or 4;

R 2 is selected from the group consisting of H (hydrogen), D (oxime), C 1-6 alkyl, -NR c R d and a 3-7 membered nitrogen-containing heterocyclic group; R c and R d are each independently selected from H (hydrogen) ), D(氘), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C 1-6 alkylene-C 3-6 Cycloalkyl, -C 1-6 alkylene-(6-10 membered aryl), -C 1-6 alkylene-(5-12 membered heteroaryl), C 1-6 alkyl-C ( =O)-, halogenated C 1-6 alkyl-C(=O)-, 6-10 membered aryl-C(=O)-, 5-12 membered heteroaryl-C(=O)-, 6-10 membered aryl-C 1-6 alkylene-C(=O)-, 5-12 membered heteroaryl-C 1-6 alkylene-C(=O)-, C 1-6 alkane a base-SO 2 -, a halogenated C 1-6 alkyl-SO 2 -, a 6-10 membered aryl-SO 2 - and a 5-12 membered heteroaryl-SO 2 -; the 6-10 membered aryl group And a 5-12 membered heteroaryl group is optionally substituted by H (hydrogen), D (hydrazine), halogen or C 1-6 alkyl; n 2 is 0, 1, 2, 3 or 4;

R is each independently selected from H (hydrogen), D (oxime), halogen, nitro, cyano, C 1-6 alkyl, halo C 1-6 alkyl (eg, -CF 3 ). , C 1-6 alkoxy, halo C 1-6 alkoxy (eg -OCF 3 ), C 3-6 cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is 1, 2, 3 or 4;

AA is selected from the group consisting of an amino acid group and a peptide comprising 2-6 amino acids;

L 1 is selected from -(CH 2 ) t1 -,

(Such as
),

The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L 2 by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L 2 is attached; R 3 , R 4 , R 5 and R 6 are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano substituted C 1-6 alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl, or R 3 and R 4 , R 5 and R 6 or R 3 R 5 together with the atom/group to which it is attached forms a 3-8 membered ring; t1, t2, y 1 and y 2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L 2 does not exist or is
One of its two positions marks to L 1 via 1 or 2, and L 3 is connected by a further position, preferably it is connected to L 1 via a marked location, and by the marked position 2 and L 3 Connected, Z 1 is selected from O, S and -NH-, Z 2 is absent or selected from O, S and -NH-, and m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L 3 is absent or is selected from -NR 7 - and optionally substituted by one or more R 7 : C 3-8 cycloalkylene, 6-10 membered arylene (eg phenylene or Naphthylene), 5-12 membered heteroarylene, 3-8 membered heterocyclylene, 6-12 membered subbridged heterocyclic group, 6-12 membered snail heterocyclic group, 6-12 membered sub-fused a cyclic group, -NR 8 -C 3-8 cycloalkylene- and -C 3-8 cycloalkylene-NR 8 -, when the above group contains a nitrogen atom, the nitrogen atom is optionally a quaternary ammonium Each of R 7 is independently selected from H (hydrogen), D (oxime), halogen, =O, cyano, carboxyl, sulfonate, C 1-6 alkyl, halo C 1 at each occurrence. a -6 alkyl group, a cyano substituted C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-6 alkenyl group and a C 2-6 alkynyl group, and R 8 is selected from the group consisting of H (hydrogen) and D (氘) ), C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 alkoxy;

L 4 is absent or is selected from C 1-6 alkylene, -NR 9 -C 1-6 alkylene-, -C 1-6 alkylene-NR 9 -, -NR 9 - and is one or more R 9 substituted 3-7 membered nitrogen-containing heterocyclylene; R 9 is independently selected from H (hydrogen), D (oxime), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 a aryl group (e.g., phenyl or naphthyl) and a 5-12 membered heteroaryl; said C 1-6 alkyl or C 1-6 alkylene optionally being one or more H (hydrogen), D (氘) or halogen substituted;

L 5 is selected from

r is 0, 1, 2, 3, 4 or 5.
The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein each occurrence of R 1 is independently selected from H ( Hydrogen), D(氘), F, Cl, nitro, -NR a R b , C 1-6 alkyl, -CF 3 , -OCH 3 and -OCF 3 ; R a and R b are each independently selected from H (hydrogen), D (氘), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C 1-6 alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-(6-10 membered aryl) and -C 1 6 alkylene-(5-12 membered heteroaryl); n 1 is 1, 2 Or 3;

Preferably, each occurrence of R 1 is independently selected from the group consisting of H (hydrogen), F, nitro, -NR a R b , C 1-4 alkyl, -CF 3 , -OCH 3 and -OCF 3 ; R a and R b are each independently selected from the group consisting of H (hydrogen), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, and 5-12 membered heteroaryl; n 1 is 1 or 2; and

More preferably, each occurrence of R 1 is independently selected from the group consisting of H (hydrogen), nitro, -NH 2 , -N(CH 3 ) 2 and -OCH 3 ; n 1 is 1 or 2.
The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein R 2 is selected from the group consisting of H (hydrogen) and D (氘) , C 1-6 alkyl and -NR c R d ; R c and R d are each independently selected from H (hydrogen), D (氘), C 1-6 alkyl, C 3-6 cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C 1-6 alkylene-C 3-6 cycloalkyl, -C 1-6 alkylene-(6-10 membered aryl), -C 1-6 alkylene-(5-12 membered heteroaryl), C 1-6 alkyl-C(=O)-, halogenated C 1-6 alkyl-C(=O)-, 6 -10-membered aryl-C(=O)-, 5-12 membered heteroaryl-C(=O)-, C 1-6 alkyl-SO 2 -, halogenated C 1-6 alkyl-SO 2 a 6-10 membered aryl-SO 2 - and 5-12 membered heteroaryl-SO 2 -; the 6-10 membered aryl group and the 5-12 membered heteroaryl group are optionally H (hydrogen), D (氘), halogen or C 1-6 alkyl substituted; n 2 is 0, 1, 2, 3 or 4;

Preferably, R 2 is selected from the group consisting of H (hydrogen), C 1-6 alkyl and -NR c R d ; R c and R d are each independently selected from H (hydrogen), D (氘), C 1 - 6 alkane , C 3-6 cycloalkyl, C 1-6 alkyl-C(=O)-, halogenated C 1-6 alkyl-C(=O)-, 6-10 member aryl-C (= O)-, 5-12 membered heteroaryl-C(=O)-, C 1-6 alkyl-SO 2 -, halogenated C 1-6 alkyl-SO 2 -, 6-10 membered aryl- SO 2 - and 5-12 membered heteroaryl-SO 2 -; the 6-10 membered aryl group and the 5-12 membered heteroaryl group are optionally H (hydrogen), D (oxime), halogen or C 1 -6 alkyl substituted; n 2 is 0, 1, 2 or 3;

More preferably, R 2 is selected from the group consisting of H (hydrogen), C 1-6 alkyl and -NR c R d ; R c and R d are each independently selected from H (hydrogen), C 1-6 alkyl, C 1 -6- alkyl-C(=O)-, halogenated C 1-6 alkyl-C(=O)-, 6-10 membered aryl-C(=O)-, C 1-6 alkyl-SO 2- , halo C 1-6 alkyl-SO 2 - and 6-10 membered aryl-SO 2 -; the 6-10 membered aryl group optionally being H (hydrogen) or C 1-6 alkyl Substituted; n 2 is 0, 1, 2 or 3;

More preferably, R 2 is selected from the group consisting of H (hydrogen), C 1-6 alkyl and -NR c R d ; R c and R d are each independently selected from H (hydrogen), C 1-6 alkyl, CH 3 C(=O)-, CF 3 C(=O)-, benzoyl, CH 3 SO 2 -, CF 3 SO 2 - and p-toluenesulfonyl; n 2 is 1 or 2;

Most preferably, R 2 is selected from the group consisting of H (hydrogen), C 1-6 alkyl and -NR c R d ; R c and R d are each independently selected from H (hydrogen), methyl, isopropyl, CH 3 C(=O)-, benzoyl, CH 3 SO 2 - and p-toluenesulfonyl; n 2 is 2.
A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein R is independently present at each occurrence Selected from H (hydrogen), D (氘), C 1-6 alkyl, halogenated C 1-6 alkyl (eg -CF 3 ), C 1-6 alkoxy, and halogenated C 1-6 alkane An oxy group (for example -OCF 3 ); n is 1, 2 or 3;

Preferably, R is each independently selected from H (hydrogen), C 1-6 alkyl, and halo C 1-6 alkyl (eg, -CF 3 ); n is 1 or 2;

More preferably, R is each independently selected from H (hydrogen), methyl and -CF 3 at each occurrence; n is 1 or 2.
The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein AA is selected from

The above group is linked to the -L 1 - group by one of the two positions labeled 1 or 2, and is passed through another position
Linking, preferably, the above group is attached to the -L 1 - group via the position of the 1 label, and the position of the label is 2
connection;

More preferably, AA is selected from

The above group is attached to the -L1- group by one of the two positions marked by 1 or 2, and passes through another position
Linking, preferably, the above group is attached to the -L 1 - group via the position of the 1 label, and the position of the label is 2
Connected; and

Most preferably, AA is selected from

The above group is attached to the -L1- group by one of the two positions marked by 1 or 2, and passes through another position
Linking, preferably, the above group is attached to the -L1- group through the position of the 1 label, and the position of the label is 2
connection.
The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein L 1 is selected from
(Such as
),

The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L 2 by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L 2 is attached; R 3 , R 4 , R 5 and R 6 are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl, cyano substituted C 1-6 alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl; y1 and y2 are each independently 0, 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10;

Preferably, L 1 is selected from
(Such as
),
The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L 2 by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L 2 is attached; R 3 , R 4 , R 5 and R 6 are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C 1-6 alkyl, halogenated C 1-6 alkyl and cyano substituted C 1-6 alkyl; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

More preferably, L 1 is selected from
(Such as
),
The above group is attached to AA by one of the two positions marked by 1 or 2, and is linked to L 2 by another position, preferably, the above group is linked to AA by the position of 1 mark, and the position of the mark by 2 L 2 is attached; R 3 , R 4 , R 5 and R 6 are each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, cyano, C 1-6 alkyl, halogenated a C 1-6 alkyl group and a cyano substituted C 1-6 alkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

Most preferably, L 1 is selected from
It is linked to AA by one of the two positions labeled 1 or 2 and to L 2 by another position, preferably the above group is linked to AA by the position of 1 mark, and the position of L 2 is passed by 2 connection.
The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein L 2 is absent or
One of its two positions marks to L 1 via 1 or 2, and L 3 is connected by a further position, preferably it is connected to L 1 via a marked location, and by the marked position 2 and L 3 Connected, Z 1 is selected from O and -NH-, Z 2 is absent or selected from O and -NH-, and m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, L 2 is absent or is
One of its two positions marks to L 1 via 1 or 2, and L 3 is connected by a further position, preferably it is connected to L 1 via a marked location, and by the marked position 2 and L 3 Connection, Z 1 is -NH-, Z 2 is absent or -NH-, and m1 and m2 are each independently 0, 1 , 2 , 3, 4, 5, 6, 7, 8, 9, or 10;

More preferably, L 2 is absent or selected from

One of its two positions marks to L 1 via 1 or 2, and L 3 is connected by a further position, preferably it is connected to L 1 via a marked location, and by the marked position 2 and L 3 connection.
A compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein L 3 is absent or selected from - NR 7 - and the following groups optionally substituted by one or more R 7 : C 3-8 cycloalkylene, phenylene, naphthylene, 5-12 membered heteroarylene, 3-8 member a heterocyclylene group, a 6-12 membered subbridged heterocyclic group, a 6-12 membered siroheterocyclyl group, and a 6-12 membered fused heterocyclic group. When the above group contains a nitrogen atom, the nitrogen atom is optional. The quaternized; R 7 is each independently selected from H (hydrogen), D (oxime), halogen, carboxyl, sulfonate, C 1-4 alkyl, -CF 3 , C 1 -4 alkoxy, C 2-6 alkenyl and C 2-6 alkynyl;

Preferably, L 3 is absent or is selected from -NR 7 - and the following groups optionally substituted by one or more R 7 :

The counterion, R 7 is each independently selected from H (hydrogen), D (oxime), methyl and -CF 3 at each occurrence, and the above groups are linked to L 2 by one of two positions labeled 1 or 2. And connected to L 4 through another position, preferably, the above group is linked to L 2 through the position of 1 mark, and is connected to L 4 through the position of 2 mark;

More preferably, L 3 is absent or selected from:

E is a counterion, each of R 7 being independently selected from H (hydrogen), D (oxime), methyl and -CF 3 at each occurrence, and the above group is one of two positions labeled by 1 or 2 and L 2 connected, and connected to L 4 through another position, preferably, the above group is connected to L 2 through the position of 1 mark, and is connected to L 4 through the position of 2 mark;

Most preferably, L 3 is absent or selected from
E is a counter ion, preferably a halogen anion, more preferably a chloride ion, a bromide ion or an iodide ion, the above group being linked to L 2 by one of two positions labeled 1 or 2, and connected to L 4 through another position Preferably, the above group is attached to L 2 through the position of the 1 mark and to the L 4 through the position of the 2 mark.
A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein L 4 is absent or selected from C 1-6 alkylene, -NR 9 -C 1-6 alkylene-, -C 1-6 alkylene-NR 9 - and -NR 9 -; R 9 is independently selected from H (hydrogen), D (氘), C 1-6 alkyl and C 3-6 cycloalkyl; said C 1-6 alkyl or C 1 6 alkylene optionally being one or more H (hydrogen), D (氘) or halogen substitution;

Preferably, L 4 is absent or selected from the group consisting of -CH 2 CH 2 -, -NHCH 2 -, -CH 2 NH- and -NH-.
The compound of any one of claims 1-9, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein L 5 is selected from

and

Preferably, L 5 is selected from
The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein the compound is selected from the group consisting of:

as well as

Preferably, the compound is selected from the group consisting of
a conjugate of formula (II) or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof,

Wherein, A is a group obtained by removing γ thiol groups in the target or a group obtained by reducing a disulfide bond in the target and then removing γ thiol groups;

L 5 ' is selected from

It is linked to L 4 by one of the two positions labeled 1 or 2 and to the thiol group of the target by another position; preferably it is linked to L 4 by the position of the 1 mark and passes the position of the 2 mark Connected to the thiol group of the target; r is 0, 1, 2, 3, 4 or 5; γ is an integer from 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, γ is an integer from 4 to 8, such as γ is 4, 5, 6, 7, or 8;

The remaining groups are as defined in any of claims 1-11.
The conjugate of claim 12, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein

L 5 ' is selected from

It is linked to L 4 by one of the two positions labeled 1 or 2 and to the thiol group of the target by another position; preferably it is linked to L 4 by the position of the 1 mark and passes the position of the 2 mark Attached to the thiol group of the target;

Preferably, L 5 ' is selected from
It is linked to L 4 by one of the two positions labeled 1 or 2 and to the thiol group of the target by another position; preferably it is linked to L 4 by the position of the 1 mark and passes the position of the 2 mark Attached to the thiol group of the target.
The conjugate of claim 12 or 13 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein the target is selected from the group consisting of small molecules Body, protein, peptide and non-protein agents (such as sugar, RNA or DNA);

Preferably, the target is a substance that targets the following targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEA CAM5, mucin (eg mucin) 1 and mucin 16), CD138, CD20, CD19, CD30, SLTRK6, connexin 4, tissue factor, endothelin receptor, STEAP1, SLC39A6, guanylate cyclase C, PSMA, CCD79b, CD22, sodium phosphate synergy Transporter 2B, GPNMB, trophoblastic glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCAhlg, ETBR, MSG783, STEAP2 , TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, short proteoglycan, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1 IRTA2, TENB2, integrin α5β6, integrin α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Sealing protein 18.2, BMPR1B, Tyro7, c-Met, ApoE, CD1 lc, CD40, CD45 (PTP RC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2 CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF -1R, RNF43 and NaPi2b;

Preferably, the target is selected from the group consisting of small molecule ligands such as folic acid derivatives, glutamate urea derivatives, somatostatin derivatives, aryl sulfonamide derivatives (eg, carbonic anhydrase IX inhibitors), a combination of two aliphatic anthraquinone polyenes, a cyanine dye, and IR-783 or a derivative thereof; an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (eg, scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, pro-antibody, Bispecific antibody or multispecific antibody; RGD peptide that recognizes cell surface integrin receptor; growth factor that recognizes cell surface growth factor receptor such as EGF, PDGF or VEGF; recognizes functional cell surface plasminogen activation a peptide of factor, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, DR5 ligand and Trop-2 ligand;

More preferably, the target is a monoclonal antibody against Trop-2 or a monoclonal antibody against Her2, such as Sacituzumab, trastuzumab or pertuzumab.
The conjugate according to any one of claims 12 to 14, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, wherein the conjugate is selected from:

Wherein, A is a group obtained by removing γ sulfhydryl groups in Sacituzumab, trastuzumab or pertuzumab or a disulfide bond in Sacituzumab, trastuzumab or pertuzumab is reduced a group obtained by removing γ thiol groups, γ is an integer of 1 to 10, for example, γ is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

Preferably, the conjugate is selected from the group consisting of:

Wherein, A1 is a group obtained by removing γ thiol groups in Sacituzumab or a group obtained by reducing γ thiol groups after reduction of a disulfide bond in Sacituzumab, and γ is an integer of 4 to 8, for example, γ is 4. 5, 6, 7 or 8.
A pharmaceutical composition comprising one or more (preferably two or more) of the conjugates of any one of claims 12-15, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof Or any crystalline form or racemate thereof, and optionally one or more pharmaceutically acceptable carriers, and the pharmaceutical composition optionally further comprises one or more other anticancer agents such as chemotherapy And/or an antibody, wherein the pharmaceutical composition has an average DAR value of an integer or fraction of from 1 to 10, such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 or 10.0, preferably an integer or fraction of 4 to 8, such as 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9 or 8.0.
The conjugate of any one of claims 12-15, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof, for use in the prevention or treatment of a cancer disease Use in medicine;

Preferably, the cancer disease is selected from the group consisting of breast cancer (eg, triple negative breast cancer), gastric cancer, esophageal cancer (eg, esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, salivary gland cancer, lung cancer (eg, small cell lung cancer and Non-small cell lung cancer), squamous cell carcinoma, bladder cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, colon cancer, entity Tumor, non-Hodgkin's lymphoma, central nervous system tumor (eg glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer;

More preferably, the cancer disease is selected from the group consisting of breast cancer (eg, triple negative breast cancer) and gastric cancer;

Further preferably, the cancer disease is selected from the group consisting of gastric cancer.
Process for the preparation of a compound of formula (I') according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof:

among them:

R d ' is R d or PG 1 ,

PG 1 and PG 2 are each independently an amino protecting group, preferably Boc or MMT,

The remaining groups are as defined in any one of claims 1-11,

The method includes the following steps:

Step 1: reacting the compound (Ia)-d with the compound (Ia)-e in the presence of a metal catalyst to obtain a compound of the formula (Ia), and

Step 2: reacting the compound of the formula (Ia) under the condition of removing the amino protecting group to obtain the compound of the formula (I') or a pharmaceutically acceptable salt thereof;

Preferably, the metal catalyst is a monovalent copper salt such as a cuprous halide (such as cuprous chloride, cuprous bromide or cuprous iodide); or a divalent copper salt/reducing agent such as copper sulfate/sodium ascorbate ;

Preferably, said step 1 of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, in the first step of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-d to the metal catalyst is 1: (1-15), preferably 1: (1-10), Most preferably 1: (1-5);

Preferably, said step 1 of the process for the preparation of a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol) and any combination thereof;

Preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably the conditions in which trifluoroacetic acid is present;

Preferably, said step two of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, said step 2 of the process for the preparation of a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
Process for the preparation of a compound of formula (I') according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof:

among them:

R d " is R d or hydrogen,

R d ' is R d or PG 1 ,

PG 1 and PG 2 are each independently an amino protecting group, preferably Boc or MMT,

The remaining groups are as defined in any one of claims 1-11,

The method includes the following steps:

Step 1: obtaining a compound of (Ia)-b by reacting the compound (Ia)-a in the presence of a base, provided that when both R d " and R d ' are R d , the reaction of the first step is not required;

Step 2: reacting the compound (Ia)-b with the compound (Ia)-c in the presence of a condensation reagent and a base to obtain a compound of (Ia)-d;

Step 3: reacting the compound (Ia)-d with the compound (Ia)-e in the presence of a metal catalyst to obtain a compound of the formula (Ia), and

Step 4: reacting the compound of the formula (Ia) under the condition of removing the amino protecting group to obtain the compound of the formula (I') or a pharmaceutically acceptable salt thereof;

Preferably, the base for the preparation of the compound of the formula (I') is selected from the group consisting of an organic base and an inorganic base such as triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, sodium hydroxide or potassium hydroxide. , preferably triethylamine, diisopropylethylamine;

Preferably, said step 1 of the process for preparing a compound of formula (I') is carried out at a temperature of from 0 ° C to 40 ° C, preferably from 10 ° C to 30 ° C;

Preferably, the first step of the process for preparing the compound of the formula (I') is carried out in an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methyl Pyrrolidone, a saturated hydrocarbon (such as cyclohexane or hexane), a halogenated hydrocarbon (such as dichloromethane, chloroform or 1,2-dichloroethane), a nitrile (such as acetonitrile), and any combination thereof, preferably two Methyl chloride

Preferably, the condensation reagent of the second step of the method for preparing the compound of the formula (I') is selected from the group consisting of carbonyl diimidazole, triphosgene, preferably triphosgene;

Preferably, the base of the second step of the process for preparing the compound of the formula (I') is selected from the group consisting of an organic base and an inorganic base such as triethylamine, diisopropylethylamine, p-dimethylaminopyridine, pyridine, 2,4,6. -trimethylpyridine or the like, preferably p-dimethylaminopyridine;

Preferably, in the step 2 of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-b to (Ia)-c is 1: (0.3-2), preferably 1: (0.8-1.5);

Preferably, in the step 2 of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-b to the condensing agent and the base is 1: (0.3-1): (2-10), preferably 1: ( 0.4-0.7): (3-6);

Preferably, said step 2 of the process for preparing a compound of formula (I') is carried out in an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methyl Pyrrolidone, a saturated hydrocarbon (such as cyclohexane or hexane), a halogenated hydrocarbon (such as dichloromethane, chloroform or 1,2-dichloroethane), a nitrile (such as acetonitrile), and any combination thereof, preferably two Methyl chloride

Preferably, the metal catalyst of the third step of the method for preparing the compound of the formula (I') is a monovalent copper salt, such as a cuprous halide (such as cuprous chloride, cuprous bromide or cuprous iodide); or a divalent Copper salt/reducing agent, such as copper sulfate/sodium ascorbate;

Preferably, the step 3 of the method for preparing the compound of the formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, in the step (3) of the method for preparing the compound of the formula (I'), the molar ratio of the compound (Ia)-d to the metal catalyst is 1: (1-15), preferably 1: (1-10), most preferably 1 :(1-5);

Preferably, the method of preparing the compound of the formula (I') is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-A Pyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxyethane), a nitrile (such as acetonitrile), an alcohol (such as methanol, ethanol, isopropanol or tert-butanol) and any combination thereof;

Preferably, the method for preparing the compound of the formula (I') is carried out in the fourth step, wherein the conditions for removing the amino protecting group are acidic conditions, more preferably the conditions in which trifluoroacetic acid is present;

Preferably, said step 4 of the process for preparing a compound of formula (I') is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, said step 4 of the process for preparing a compound of formula (I') is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
Process for the preparation of a compound of formula (I") according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof:

among them:

R d ' is R d or PG 1 ,

R 10 is absent or is C 1-6 alkylene, a C 1-6 alkylene group optionally substituted by one or more of H (hydrogen), D (deuterium) or halogen,

PG 1 and PG 2 are each independently an amino protecting group, preferably Boc or MMT,

LG is a leaving group, preferably a hydroxyl group or a succinimide-N-oxy group,

The remaining groups are as defined in any one of claims 1-11,

The method includes the following steps:

Step 1: reacting compound (Ib)-a with compound (Ib)-b in the presence of a base, optionally in the presence of a condensing reagent, to obtain a compound of formula (Ib), and

Step 2: reacting the compound of the formula (Ib) under the condition that the amino protecting group is removed to obtain a compound of the formula (I") or a pharmaceutically acceptable salt thereof;

Preferably, the base is an organic base or an inorganic base; the organic base is selected from the group consisting of triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from the group consisting of NaH, NaOH, Na 2 CO 3 and K 2 CO 3 ;

Preferably, the base is DIPEA;

Preferably, the condensation reagent is selected from the group consisting of phosgene, solid phosgene,

HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP and PyBOP;

Preferably, said step 1 of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, in the step (1) of the method for preparing the compound of the formula (I"), the molar ratio of the compound (Ib)-a to the base is 1: (0.5-4), preferably 1: (0.8-3), more Preferably 1: (1-2);

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol) and any combination thereof;

Preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably the conditions in which trifluoroacetic acid is present;

Preferably, said step two of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, said step 2 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
Process for the preparation of a compound of formula (I") according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystal form or racemate thereof:

among them:

R d ' is optionally R d or PG 1 ,

R 10 is absent or is C 1-6 alkylene, a C 1-6 alkylene group optionally substituted by one or more of H (hydrogen), D (deuterium) or halogen,

PG 1 and PG 2 are each independently an amino protecting group, preferably Boc or MMT,

LG is a leaving group, preferably a hydroxyl group or a succinimide-N-oxy group,

The remaining groups are as defined in any one of claims 1-11,

The method includes the following steps:

Step 1: in the presence of a reducing agent, the compound (Ia)-d is reacted to obtain the compound (Ib)-a;

Step 2: reacting compound (Ib)-a with compound (Ib)-b in the presence of a base, optionally in the presence of a condensation reagent, to give a compound of formula (Ib), and

Step 3: reacting the compound of the formula (Ib) under the condition of removing the amino protecting group to obtain a compound of the formula (I") or a pharmaceutically acceptable salt thereof;

Preferably, the reducing agent is lithium aluminum hydride, palladium carbon, palladium hydroxide, platinum dioxide, triphenylphosphine, preferably platinum dioxide, triphenylphosphine;

Preferably, the base is an organic base or an inorganic base; the organic base is selected from the group consisting of triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from the group consisting of NaH, NaOH, Na 2 CO 3 and K 2 CO 3 ;

Preferably, the base is DIPEA;

Preferably, the condensation reagent is selected from the group consisting of phosgene, solid phosgene,

HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP and PyBOP;

Preferably, said step 1 of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 ° C to 50 ° C, preferably from 10 ° C to 30 ° C;

Preferably, said step 1 of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out in an organic solvent; said organic solvent being selected from ethers (for example tetrahydrofuran, diethyl ether, dioxane or 1,2-dimethoxy) Ethane), an alcohol (such as methanol, ethanol, isopropanol or tert-butanol), ethyl acetate and any combination thereof, preferably tetrahydrofuran;

Preferably, in the step (1) of the method for preparing the compound of the formula (I"), the molar ratio of the compound (Ib)-a to the base is 1: (0.5-4), preferably 1: (0.8-3), more Preferably 1: (1-2);

Preferably, said step 1 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol) and any combination thereof;

Preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably the conditions in which trifluoroacetic acid is present;

Preferably, said step two of the process for preparing a compound of formula (I") is carried out at a temperature of from -10 ° C to 60 ° C, preferably from 0 ° C to 35 ° C, more preferably from 0 ° C to 25 ° C;

Preferably, said step 2 of the process for the preparation of a compound of formula (I") is carried out in water and/or an organic solvent; said organic solvent being selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, saturated hydrocarbons (such as cyclohexane or hexane), halogenated hydrocarbons (such as dichloromethane, chloroform or 1,2-dichloroethane), ethers (such as tetrahydrofuran, diethyl ether, two Oxane or 1,2-dimethoxyethane), nitriles (such as acetonitrile), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
A process for the preparation of a conjugate of formula (II) according to any one of claims 12-15, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof,

The method comprises coupling a compound of formula (I) to a target,

Wherein each group is as defined in any one of claims 1-15,

Preferably, the method comprises mixing a compound of formula (I) with a target; optionally reducing a disulfide bond in the target prior to mixing;

Preferably, the disulfide bond reduction is carried out by using a reducing agent; the reducing agent is preferably TCEP;

Preferably, the method comprises: mixing a solution comprising a target with a compound of formula (I);

Preferably, the molar ratio of the target to the compound of formula (I) is 1: (1-20);

Preferably, the method is carried out at a temperature of from 0 ° C to 50 ° C, preferably from 15 ° C to 25 ° C;

Preferably, the method is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide and N- Methylpyrrolidone;

Preferably, the method further comprises purifying by one or more chromatographic methods selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, and affinity chromatography.