CN111542324A

CN111542324A - Cytotoxic agents and conjugates thereof, methods of making and uses thereof

Info

Publication number: CN111542324A
Application number: CN201980006396.1A
Authority: CN
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: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2018-02-11
Filing date: 2019-01-25
Publication date: 2020-08-14
Anticipated expiration: 2039-01-25
Also published as: CN111542324B; WO2019154120A1

Abstract

A novel cytotoxic agent which is a camptothecin derivative, a conjugate thereof with a targeting agent, a preparation method of the cytotoxic agent and the conjugate, and the use of the conjugate in preventing and/or treating proliferative diseases (including but not limited to cancer diseases) related to abnormal cell activity.

Description

Cytotoxic agents and conjugates thereof, methods of making and uses thereof

Technical Field

The invention belongs to the technical field of medicines. In particular, the present invention relates to novel cytotoxic agents (which are camptothecin derivatives), conjugates thereof with a targeting agent, methods of making said cytotoxic agents and conjugates, and uses of said conjugates in the prevention and/or treatment of proliferative diseases associated with abnormal cellular activity, including but not limited to cancer diseases.

Background

Chemotherapy was once the standard therapy for cancer, but highly lethal bioactive molecules can miskill normal cells, causing serious side effects. The targeting antitumor drug has both targeting property and antitumor activity, and has become a hotspot in the current tumor research field. Since the 20 th century, the use of biomacromolecule drugs (e.g., therapeutic antibodies or antibody fragments) for the targeted treatment of tumors has made breakthrough. However, although the biomacromolecule drug has strong targeting property, the treatment effect on solid tumors is limited; the bioactive molecules have high killing effect on cancer cells, but lack targeting property, often injure normal cells by mistake and cause serious toxic and side effects.

In recent years, it has been discovered that therapeutic antibodies can be linked to biologically active molecules to form antibody-drug conjugates (ADCs). ADC combines the targeting effect of the antibody and the high activity of bioactive molecules to form a biological missile. The antibody directs ADC binding 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 property on the tumor cell related target, the application value of the antibody is reflected in the aspect of treatment, and the antibody also becomes an ideal carrier for targeted delivery of the drug, so that the side effect of the drug is reduced.

Typically, ADC drugs consist of an antibody, a biologically active molecule, and a linker. The biologically active molecule is covalently coupled to the antibody through a linker. The antibody (such as a monoclonal antibody) can specifically recognize a specific target on the surface of the cancer cell, and further can guide the ADC to the surface of the cancer cell, so that the ADC enters the cancer cell through an endocytosis effect. Bioactive molecules can be released in cells, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

The design principle of Small Molecule Drug Conjugates (SMDCs) and antibody-drug conjugates (ADCs) is the same, i.e., biologically active molecules are chemically coupled to small molecule ligands that selectively bind to tumor cell surface receptors, thereby improving the targeting of effector molecules (biologically active molecules) to tumor cells. The chemical structure of SMDCs is almost identical to ADCs, but small molecule ligands are used instead of antibodies. At present, no SMDC is on the market.

Currently, there are four types of ADCs on the market: mylotarg (Gemtuzumab Oxogamicin, Gemtuzumab Oxazolemicin), Adcetris (Brentuximab Vedotin, CD30 monoclonal antibody-MMAE), Kadcyla (Trastuzumab Emtansine, Trastuzumab-maytansine alkaloid), and Besponsa (Inotuzumab Ozogamicin, CD22 monoclonal antibody-calicheamicin).

Summary of The Invention

Through intensive research, the inventors of the present invention have unexpectedly found a conjugate comprising a targeting moiety and a cytotoxic agent moiety (camptothecin derivative) having an antitumor effect, which has a good inhibitory ability against tumor cells.

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

wherein:

R₁each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, nitro, -NR_aR_b、C_1-6Alkyl, halo C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy, halo C_1-6Alkoxy (e.g., -OCF)₃) And C_3-6A cycloalkyl group; r_aAnd R_bEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl) and-C_1-6Alkylene- (5-12 membered heteroaryl); n is₁Is 1, 2, 3 or 4;

R₂selected from H (hydrogen), D (deuterium), C_1-6Alkyl, -NR_cR_dAnd a 3-7 membered nitrogen-containing heterocyclic group; r_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl), -C_1-6Alkylene- (5-12 membered heteroaryl), C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, 6-10 membered aryl-C_1-6alkylene-C (═ O) -, 5-12 membered heteroaryl-C_1-6alkylene-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl substitution; n is₂Is 0, 1, 2, 3 or 4;

r is independently selected for each occurrence from H (hydrogen), D (deuterium), halogen, nitro, cyano, C_1-6Alkyl, halo C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy, halo C_1-6Alkoxy (e.g., -OCF)₃)、C_3-6Cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is 1, 2, 3 or 4;

AA is selected from amino acid groups and groups comprising peptides of 2-6 amino acids;

L₁is selected from- (CH)₂)_t1-、

(e.g. using

)、

And

the above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy and C_3-6Cycloalkyl, or R₃And R₄、R₅And R₆Or R₃And R₅Together with the atoms/groups to which they are attached form a 3-8 membered ring; t1, t2, y₁And y₂Each independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L₂is absent or is

Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁Selected from O, S and-NH-, Z₂Absent or selected from O, S and-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

L₃is absent or selected from-NR₇-and renOptionally substituted by one or more R₇Substituted of the following groups: c_3-8Cycloalkylene, 6-10 membered arylene (e.g., phenylene or naphthylene), 5-12 membered heteroarylene, 3-8 membered heterocyclylene, 6-12 membered bridged heterocyclyl, 6-12 membered spiroheterocyclyl, 6-12 membered fused heterocyclyl, -NR₈-C_3-8cycloalkylene-and-C_3-8cycloalkylene-NR₈When the above groups contain a nitrogen atom, said nitrogen atom is optionally quaternized; r₇Each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, ═ O, cyano, carboxyl, sulfonic acid, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_2-6Alkenyl and C_2-6Alkynyl radical, R₈Selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-6An alkoxy group;

L₄is absent or selected from C_1-6Alkylene, -NR₉-C_1-6Alkylene-, -C_1-6alkylene-NR₉-、-NR₉And by one or more R₉A substituted 3-7 membered nitrogen containing heterocyclylene group; r₉Independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl (e.g., phenyl or naphthyl) and 5-12 membered heteroaryl; said C is_1-6Alkyl or C_1-6Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium), or halogen; and is

L₅Is 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 after gamma sulfydryl is removed from the target or a group obtained after gamma sulfydryl is removed after a disulfide bond in the target is reduced;

L₅' is selected from

Which is marked by one of the two positions 1 or 2 and L₄Attached to the thiol group of the target via another position; preferably, it is marked by 1 position and L₄Connecting and connecting with the sulfhydryl group of the target through the position marked by 2; r is 0, 1, 2, 3, 4 or 5;

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

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, hydrate, isomer, or any crystalline form or racemate thereof, optionally together with one or more pharmaceutically acceptable carriers, and optionally further comprising one or more other anticancer agents 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, e.g. 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, 6.7, 6.7.7, 6.7, 6, 6.7, 7.7, 6.7, 7, 7.7, 6, 7.7, 7, 6.7, 7, 6.0, 6.7, 7, 7.7, 6.0, 6, 6.7, 7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 or 10.0, preferably an integer or decimal from 4 to 8, for example 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 the use of a conjugate of formula (II) of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystal form or racemate thereof, for the manufacture of a medicament for the prevention or treatment of a cancer disease.

A fifth aspect of the present invention provides a conjugate of formula (II) of the present invention or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, for use in the prevention or treatment of a cancer disease.

A sixth aspect of the present invention provides a method for preventing or treating a cancer disease, the method comprising administering to a subject in need thereof an effective amount of a conjugate of formula (II) of the present invention or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof.

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

wherein:

R_dis "R_dOr a hydrogen atom,

R_d' is R_dOr PG₁，

PG₁And PG₂Each independently an amino protecting group, preferably Boc or MMT,

the remaining groups are as defined above,

the method comprises the following steps:

the method comprises the following steps: functionalization or protection (e.g. acetylation, mesylation, Boc protection) of the nitrogen atom in the presence of a base, allowing the compound (Ia) -a to react to give the compound (Ia) -b, with the proviso that when R is_d"and R_d' are both R_dIn the process, the reaction in the step one is not needed;

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

step three: reacting the compounds (Ia) -d with the compounds (Ia) -e in the presence of a metal catalyst to give the compounds of the formula (Ia), and

step four: reacting the compound of formula (Ia) under conditions to remove the amino protecting group to give the compound of formula (I') or a pharmaceutically acceptable salt thereof.

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

wherein:

R_d' is R_dOr PG₁，

R₁₀Is absent or is C_1-6Alkylene radical of the formula C_1-6Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium) or halogen,

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

the remaining groups are as defined above,

the method comprises the following steps:

the method comprises the following steps: reacting the compounds (Ia) to (d) in the presence of a reducing agent to obtain compounds (Ib) to a;

step two: reacting compound (Ib) -a with compound (Ib) -b in the presence of a base, optionally in the presence of a condensing agent, to give a compound of formula (Ib), and

step three: reacting the compound of formula (Ib) under conditions to remove the amino protecting group to give the compound of 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, or any crystalline form or racemate thereof,

the method comprising coupling a compound of formula (I) to a target,

wherein each group is as defined above.

Drawings

FIG. 1 II-1-A light chain deconvolution plot.

FIG. 2 II-1-A heavy chain deconvolution plot.

FIG. 3 II-5-A light chain deconvolution plot.

FIG. 4 II-5-A heavy chain deconvolution plot.

FIG. 5 II-6-A light chain deconvolution plot.

FIG. 6 II-6-A heavy chain deconvolution plot.

FIG. 7 II-7-A light chain deconvolution plot.

FIG. 8 II-7-A heavy chain deconvolution plot.

FIG. 9 II-8-A light chain deconvolution plot.

FIG. 10 II-8-A heavy chain deconvolution plot.

FIG. 11 II-9-A light chain deconvolution plot.

FIG. 12 II-9-A heavy chain deconvolution plot.

FIG. 13 II-10-A light chain deconvolution plot.

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

FIG. 15 II-11-A light chain deconvolution plot.

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

FIG. 17 II-12-A light chain deconvolution plot.

FIG. 18 II-12-A heavy chain deconvolution plot.

FIG. 19 is a deconvolution diagram of the light chain II-13-A.

FIG. 20 II-13-A heavy chain deconvolution plot.

FIG. 21 II-15-A light chain deconvolution plot.

FIG. 22 II-15-A heavy chain deconvolution plot.

FIG. 23 SEC chromatogram of II-1-A.

FIG. 24 SEC chromatogram of II-5-A.

FIG. 25 SEC chromatograms of II-7-A.

FIG. 26 SEC chromatogram of II-8-A.

FIG. 27 II-10-A SEC chromatograms.

FIG. 28 SEC chromatograms of II-14-A.

FIG. 29 cytotoxic agent release profile of II-1-A, II-7-A and II-8-A in cynomolgus monkey plasma.

FIG. 30 release of cytotoxic agents from rat plasma in II-1-A, II-7-A and II-8-A.

FIG. 31 release of cytotoxic agents from II-1-A, II-7-A and II-8-A in 5% Bovine Serum Albumin (BSA) PBS buffer.

Detailed Description

Definition of terms

In the present invention, unless otherwise specified, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present invention, the term "conjugate" refers to 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 via a linker. The linker is capable of cleavage in a specific environment (e.g., an intracellular low pH environment) or under a specific action (e.g., the action of a lysosomal protease), thereby separating 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 directly attached to the target by a covalent bond that is capable of breaking under a particular environment or action, thereby separating the cytotoxic agent from the target.

In the present invention, the term "cytotoxic agent" refers to a substance capable of having a deleterious effect on the growth or proliferation of cells, and also refers to a substance that inhibits or interferes with the function of cells and/or causes cell death or destruction.

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

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

In the present invention, the term "target" refers to a substance capable of specifically binding to a target (or a portion of a target) on the surface of a cell. The conjugate can be delivered to a specific cell population by interaction of the targeting 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" may be used interchangeably.

In the present invention, the term "antibody" is to be interpreted in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, so long as they possess the desired biological activity. Herein, "antibody" and "immunoglobulin" may be used interchangeably.

In the present invention, the term "monoclonal antibody" means that the antibody is derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations which may be present in minor amounts. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies in contrast thereto comprise different antibodies directed against different determinants (epitopes). In addition to specificity, monoclonal antibodies also have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" used herein indicates that the antibody is characterized as being from a substantially homogeneous population of antibodies, and is not to be construed as requiring production by a particular method.

In some embodiments of the invention, monoclonal antibodies also specifically include chimeric antibodies, i.e., antibodies in which a portion of the heavy and/or light chain is identical to or homologous to an antibody of one class, subclass, or subclass, and the remainder is identical to or homologous to an antibody of another class, subclass, or subclass, so long as they possess the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al, 1984, PNAS, 81: 6851-. Chimeric antibodies useful in the invention include primatized antibodies comprising variable region antigen binding sequences from a non-human primate (e.g., an ancient monkey or chimpanzee, etc.) and human constant region sequences.

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

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

The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies having three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies having four different antigen-binding specificities.

The term "whole antibody" refers to an antibody comprising antigen-binding variable regions and light chain constant regions (CL), heavy chain constant regions (CH1, CH2, and CH 3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. The intact antibody is preferably an intact antibody having one or more effector functions.

The term "pro-anti (pro body)" is a modified antibody, comprising an antibody or an antibody fragment, capable of binding specifically to its target, capable of being coupled to a masking group, wherein the masking group means that the cleavage constant for the binding capacity of the antibody or antibody fragment to its target is at least 100 times, 1000 times or 10000 times greater than the cleavage constant for the binding capacity of the antibody or antibody fragment to its target without the coupling of the masking group.

In the present invention, a "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that contains minimal non-human immunoglobulin sequences. Most humanized antibodies are recipient human immunoglobulins in which residues from the hypervariable region are replaced with residues from a non-human (e.g., mouse, rat, rabbit or non-human primate) hypervariable region (donor antibody) having the desired specificity, affinity, and function. In some embodiments, Framework Region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Furthermore, humanized antibodies may also comprise residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further optimize the performance of the antibody. Humanized antibodies typically comprise at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops (hypervariable loops) correspond to those of a non-human immunoglobulin and the FRs are fully or substantially fully sequences of a human immunoglobulin. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details see, e.g., 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 classified into different "classes" according to the amino acid sequence of the heavy chain constant region. The main five classes are IgA, IgD, IgE, IgG and IgM, of which several may also be divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions of the different classes of antibodies are referred to as α, γ and μ, respectively. The different subunit structures and three-dimensional configurations of immunoglobulins are well known in the art.

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

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 mouse, hamster, rat and human cells (see, e.g., Kohler et al, 1975, Nature, 256: 495), or by recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567), or isolated from phage antibody libraries (see, e.g., Clackson et al, 1991, Nature, 352: 624-. Monoclonal antibodies useful in the present invention include, but are not limited to: anti-Trop-2 monoclonal antibodies, such as, for example, Sacituzumab (i.e., isatuzumab or hRS7 antibody), or anti-Her 2 monoclonal antibodies, such as, for example, trastuzumab or Pertuzumab (Pertuzumab).

In some embodiments of the invention, the target anti-Trop-2 antibody is RS7, described in U.S. patent No. 7,517,964; and hRS7 (i.e., Sacituzumab of the invention) as described in US 2012/0237518. The anti-Trop-2 antibody which can be used in the invention can also be obtained by screening through a method of designing, constructing and constructing an antibody library displaying the antibody by using a carrier disclosed in CN 103476941A.

In the present invention, Trop-2 or Trop2 refers to human trophoblast cell-surface antigens-2 (human trophoblast cell-surface antigens 2), also known as tactd 2, M1S1, GA733-1, EGP-1, which are cell surface receptors expressed by many human tumor (e.g., breast, colorectal, lung, pancreatic, ovarian, prostate, or cervical cancer) cells. In some embodiments, the compounds or conjugates of the invention are capable of inhibiting or killing a cell that expresses a TROP2 receptor, such as breast, colorectal, lung, pancreatic, ovarian, prostate, or cervical cancer.

In some embodiments of the invention, the target is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her2, the amino acid sequence of which is known to those skilled in the art, and schematic sequences of which can be found, for example, in CN 103319599. Trastuzumab heavy chain terminal Lys is easily deleted, but this deletion does not affect biological activity, see Dick, l.w. et al, biotechnol.bioeng., 100: 1132-1143. Exemplary heavy and light chain sequences of pertuzumab are set forth in SEQ ID nos. 16 and 15 of US 7560111.

In the present invention, ErbB2 and Her2 are used interchangeably and both represent the native sequence of the human Her2 protein (Genebank accession number X03363, see, e.g., Semba et al, 1985, PNAS, 82: 6497-. ErbB2 denotes the gene encoding human Her2 and neu denotes the gene encoding rat p185 neu. In some embodiments, a compound or conjugate of the invention is capable of inhibiting or killing a cell that expresses an ErbB2 receptor, such as a breast cancer cell, ovarian cancer cell, gastric cancer cell, endometrial cancer cell, salivary gland cancer cell, lung cancer cell, kidney cancer cell, colon cancer cell, thyroid cancer cell, pancreatic cancer cell, bladder cancer cell, or liver cancer cell.

In the present invention, the term "amino acid" includes natural amino acids and unnatural amino acids. The natural amino acids and the unnatural amino acids 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, D-isoleucine, L-aspartic acid, D-aspartic acid, L-asparagine, D-asparagine, L-glutamic acid, D-glutamic acid, L-glutamine, D-glutamine, L-arginine, D-arginine, L-histidine, L-tyrosine, D-tyrosine, L-methionine, L-leucine, L-arginine, L-histidine, L-tyrosine, L-methionine, L-tyrosine, L-arginine, L-methionine, L-isoleucine, L-leucine, L-, D-methionine, and the like.

In the present invention, the term "C_1-6Alkyl "denotes straight or branched alkyl having 1 to 6 carbon atoms, including for example" C_1-4Alkyl group "," C_1-3Alkyl "and the like, specific examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, and 1, 2-dimethylpropyl, and the like. The term "C_1-6Alkylene "is a corresponding divalent radical, including, for example," C_1-4Alkylene group "," C_1-3Alkylene "and the like, specific examples include, but are not limited to: methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like.

In the present invention, the term "C_2-10The "alkenyl group" means a straight-chain, branched or cyclic alkenyl group having 2 to 10 carbon atoms and containing at least one double bond, and includes, for example, "C_2-6Alkenyl group "," C₂₄Alkenyl groups "and the like. Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1, 3-pentadienyl, 1, 4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1, 4-hexadienyl, cyclopentenyl, 1, 3-cyclopentadienyl, cyclohexenyl, 1, 4-cyclohexadienyl and the like. The term "C_2-10Alkenylene "is a corresponding divalent radical, including, for example," C_2-6Alkenylene group and C_2-4Alkenylene "and the like. Examples include, but are not limited to: vinylene, propenylene, butenylene, pentenylene, hexenylene, cyclopentenylene, cyclohexenylene, and the like.

In the present invention, the term "C_2-10Alkynyl "refers to a straight or branched chain alkynyl group containing at least one triple bond and having 2 to 10 carbon atoms, including, for example," C_2-6Alkynyl group "," C_2-4Alkynyl "and the like. Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl and the like. The term "C_2-10Alkynylene "is a corresponding divalent radical, including, for example," C_2-6Alkynylene group and C_2-4Alkynylene "and the like. Examples include, but are not limited to: ethynylene, propynyl, butynyl, pentynyl, hexynyl and the like.

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

In the present invention, the term "3-to 10-membered cycloalkyl" or "C_3-10Cycloalkyl "refers to a saturated cyclic alkyl group containing 3 to 10 carbon atoms, including, for example," C_3-8Cycloalkyl group "," C_3-6Cycloalkyl group "," C_4-6Cycloalkyl group "," C_5-7Cycloalkyl radicals "or" C_5-6Cycloalkyl groups "and the like. Specific examples include, but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, and the like. The term "3-10 membered cycloalkylene" or "C_3-10Cycloalkylene "is a corresponding divalent radical, including, for example," C_3-8Cycloalkylene group "," C_3-6Cycloalkylene group "," C_4-6Cycloalkylene group "," C_5-7Cycloalkylene "or" C_5-6Cycloalkylene "and the like. Specific examples include, but are not limited to: cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene, and the like.

In the present invention, the term "3-8 membered heterocyclic group" means a cyclic group containing 3 to 8 ring atoms, at least one of which is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. "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 oxygen-containing heterocyclic group", "4-7 membered heterocyclic group", "4-6 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered nitrogen-containing heterocyclic group", preferably includes, but is not limited to, oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and homopiperazinyl groups and the like. The term "3-to 8-membered heterocyclylene" is a corresponding divalent group, and includes, for example, "3-to 8-membered nitrogen-containing heterocyclylene", "3-to 7-membered nitrogen-containing heterocyclylene", "3-to 8-membered oxygen-containing heterocyclylene", "3-to 6-membered oxygen-containing heterocyclylene", "4-to 7-membered heterocyclylene", "4-to 6-membered heterocyclylene", "5-to 7-membered heterocyclylene", "5-to 6-membered nitrogen-containing heterocyclylene", and preferably includes, but is not limited to, oxiranylene, oxocyclobutaneylene, pyrrolidinylene, tetrahydrofurylene, piperidylene, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like.

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

and the like. The term "6-12-membered spirocyclic ylidene" is a corresponding divalent group and includes, for example, "6-11-membered spirocyclic ylidene", "6-10-membered spirocyclic ylidene", "7-9-membered spirocyclic ylidene", "7-8-membered spirocyclic ylidene", "9-10-membered spirocyclic ylidene", and "8-10-membered spirocyclic ylidene", etc.

As used herein, the term "6-12 membered bridged cyclic group" refers to a cyclic structure containing 6 to 12 ring carbon atoms formed by two or more cyclic structures sharing two non-adjacent carbon atoms with each other. Optionally, the carbon atom in the cyclic structure may be oxo. "6-12-membered bridge ring group" includes, for example, "6-11-membered bridge ring group", "6-10-membered bridge ring group", "7-9-membered bridge ring group", "7-8-membered bridge ring group", "9-10-membered bridge ring group" and "8-10-membered bridge ring group", etc. Specific examples include, but are not limited to:

and the like. The term "6-12 membered bridged ring group" is a corresponding divalent group, and includes, for example, "6-11 membered bridged ring group", "6-10 membered bridged ring group", "7-9 membered bridged ring group", "7-8 membered bridged ring group", "9-10 membered bridged ring group" and "8-10 membered bridged ring group", etc.

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

and the like. 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", "6-8-membered fused ring group", "10-12-membered fused ring group" and "7-10-membered fused ring group".

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

and the like. The term "6-to 12-membered spirocyclic group" is a corresponding divalent group, and includes, for example, "6-to 12-membered nitrogen-containing spirocyclic group", "8-to 11-membered nitrogen-containing spirocyclic group", "6-to 11-membered spirocyclic group", "6-to 10-membered spirocyclic group", "7-to 11-membered spirocyclic group", "7-to 10-membered spirocyclic group", "7-to 9-membered spirocyclic group", "7-to 8-membered spirocyclic group", "9-to 10-membered spirocyclic group" and "8-to 10-membered spirocyclic group", etc.

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

and the like. The term "6-to 12-membered bridged heterocyclic group" is a corresponding divalent group, and includes, for example, "6-to 12-membered nitrogen-containing bridged heterocyclic group", "8-to 11-membered nitrogen-containing bridged heterocyclic group", "8-membered nitrogen-containing bridged heterocyclic group", "6-to 11-membered bridged heterocyclic group", "6-to 9-membered bridged heterocyclic group", "6-to 10-membered bridged heterocyclic group", "7-to 9-membered bridged heterocyclic groupHeterocyclyl group "," 7-to 8-membered bridged heterocyclyl group "," 9-to 10-membered bridged heterocyclyl group ", and" 8-to 10-membered bridged heterocyclyl 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 cyclic structure containing 6 to 12 ring atoms (at least one of which is a hetero atom such as a nitrogen atom, an oxygen atom or a sulfur atom) formed by two or more cyclic structures sharing two adjacent atoms with each other. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be 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", "7-10-membered fused heterocyclic group", "8-10-membered nitrogen-containing fused heterocyclic group", "9-10-membered nitrogen-containing fused heterocyclic group" and "6-12-membered oxygen-containing fused heterocyclic group", etc. 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, 6 a-tetrahydro-1H-furo [3, 4-d ] imidazolyl, 4, 6-dihydro-1H-thieno [3, 4-d ] imidazolyl, 4, 6-dihydro-1H-pyrrolo [3, 4-d ] imidazolyl, benzimidazolyl, octahydro-benzo [ d ] imidazolyl, decahydroquinolinyl, hexahydrothienoimidazolyl, benzimidazolyl, octahydro-benzo [ d ] imidazolyl, and the like, Hexahydrofuroimidazolyl, 4, 5, 6, 7-tetrahydro-1H-benzo [ d ] imidazolyl, octahydrocyclopenta [ c ] pyrrolyl, indolinyl, dihydroisoindolyl, benzoxazolinyl, benzothiazolinyl, 1, 2, 3, 4-tetrahydroisoquinolinyl, 1, 2, 3, 4-tetrahydroquinolinyl, 4H-1, 3-benzoxazinyl and the like. The term "6-to 12-membered fused heterocyclic group" is a corresponding divalent group, and includes, for example, "6-to 12-membered nitrogen-containing fused heterocyclic group", "6-to 11-membered fused heterocyclic group", "6-to 10-membered fused heterocyclic group", "7-to 10-membered fused heterocyclic group", "8-to 11-membered nitrogen-containing fused heterocyclic group", "8-to 10-membered nitrogen-containing fused heterocyclic group", "8-membered nitrogen-containing fused heterocyclic group", "9-to 10-membered nitrogen-containing fused heterocyclic group" and "6-to 12-membered oxygen-containing fused heterocyclic group", etc.

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

In the present invention, the term "heteroaryl" refers to a cyclic group having aromaticity, wherein at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. Specific examples include, but are not limited to, 5-to 10-membered heteroaryl, 5-to 10-membered nitrogen-containing heteroaryl, 6-to 10-membered oxygen-containing heteroaryl, 6-to 8-membered nitrogen-containing heteroaryl, and 5-to 8-membered oxygen-containing heteroaryl, etc., 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, 3, 4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1, 4-dioxadienyl, 2H-1, 2-oxazinyl, 4H-1, 2-oxazinyl, 6H-1, 2-oxazinyl, 4H-1, 3-oxazinyl, 6H-1, 3-oxazinyl, 4H-1, 4-oxazinyl, pyridazinyl, pyrazinyl, 1, 2, 3-triazinyl, 1, 3, 5-triazinyl, 1, 2, 4, 5-tetrazinyl, azepinyltrienyl, 1, 3-diazacycloheptyltrienyl, and azepinyltrienyl, 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 nitrogen-containing heteroarylene, 5-6-membered nitrogen-containing heteroarylene, 6-10-membered oxygen-containing heteroarylene, 6-8-membered nitrogen-containing heteroarylene, and 5-8-membered oxygen-containing heteroarylene, etc., such as furanylene, thiophenylene, pyrrolylene, thiazolyl, oxazolylene, imidazolyl, pyrazolyl, triazolylene, pyridyl, pyrimidylene, oxazinylene, pyridazinylene, pyrazinylene, triazinylene, and tetrazinylene.

In the present invention, the term "counter ion" refers to an ion accompanying an ionic substance to maintain electrical neutrality, for example, in sodium chloride, the sodium cation is the counter ion of the chloride anion, and vice versa.

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

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

As used herein, the term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the current circumstances is not exceeded and that the substitution forms a stable compound/conjugate. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds/conjugates.

If a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced individually and/or together with an independently selected optional substituent. If the nitrogen of a substituent is described as being optionally substituted with one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogen is present) may each be replaced with an independently selected optional substituent.

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 as 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.

As used herein, unless otherwise indicated, the point of attachment of a substituent may be from any suitable position of the substituent.

When a bond of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom 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 (e.g., methanol, ethanol, propanol, acetonitrile, etc.) or the like. For example, the compounds/conjugates of the invention may form ethanolates with ethanol.

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

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

As used herein

Chemical bonds of the compounds/conjugates of the invention are depicted. Use of

To depict the bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) at that carbon atom are included. Use of

The presence of the stereoisomers shown is intended to be indicated by the depiction of the bonds to asymmetric carbon atoms. When present in a racemic mixture, use

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

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

Pharmaceutically acceptable salts of the compounds/conjugates of the present invention include 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 Stahl and Wermuth, "Handbook of Pharmaceutical Salts: properties, Selection, and Use "(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 present invention are metabolites of the compounds/conjugates of the invention, i.e., substances formed in vivo upon administration of the compounds/conjugates of the invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound/conjugate. Accordingly, the present invention includes metabolites of the compounds/conjugates of the present invention, including compounds/conjugates made by the method of contacting a compound/conjugate of the present invention with a mammal for a time sufficient to produce a metabolite thereof.

As used herein, "DAR" refers to the drug/antibody ratio (drug antibody ratio). Mean DAR values refer to the mean value of the DAR of each conjugate in the pharmaceutical composition.

Conjugates

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

wherein:

R₁each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, nitro, -NR_aR_b、C_1-6Alkyl, halo C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy, halo C_1-6Alkoxy (e.g., -OCF)₃) And C_3-6A cycloalkyl group; r_aAnd R_bEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl) and-C_1-6Alkylene- (5-12 membered heteroaryl); n is₁1.2, 3 or 4;

L₁is selected from- (CH)₂)_t1-、

(e.g. using

)、

L₂is absent or is

L₃is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups: c_3-8Cycloalkylene, 6-10 membered arylene (e.g., phenylene or naphthylene), 5-12 membered heteroarylene, 3-8 membered heterocyclylene, 6-12 membered bridged heterocyclyl, 6-12 membered spiroheterocyclyl, 6-12 membered fused heterocyclyl, -NR₈-C_3-8cycloalkylene-and-C_3-8cycloalkylene-NR₈When the above groups contain a nitrogen atom, said nitrogen atom is optionally quaternized; r₇Each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, ═ O, cyano, carboxyl, sulfonic acid, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_2-6Alkenyl and C_2-6Alkynyl radical, R₈Selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-6An alkoxy group;

L₅Is selected from

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

In a preferred embodiment, R₁Each occurrence independently selected from H (hydrogen), D (deuterium), F, C1, nitro, -NR_aR_b、C_1-6Alkyl, -CF₃、-OCH₃and-OCF₃；R_aAnd R_bEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl) and-C_1-6Alkylene- (5-12 membered heteroaryl); n is₁Is 1, 2 or 3.

Preferably, R₁Each occurrence is independently selected from H (hydrogen), F, nitro, -NR_aR_b、C_1-4Alkyl, -CF₃、-OCH₃and-OCF₃；R_aAnd R_bEach independently selected from H (hydrogen), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is₁Is 1 or 2.

More preferably, R₁Independently at each occurrence, selected from H (hydrogen), nitro, -NH₂、-N(CH₃)₂and-OCH₃；n₁Is 1 or 2.

In a preferred embodiment, R₂Selected from H (hydrogen), D (deuterium), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl), -C_1-6Alkylene- (5-12 membered heteroaryl), C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl radical takingGeneration; n is₂Is 0, 1, 2, 3 or 4.

Preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1- ₆Alkyl radical, C_3-6Cycloalkyl radical, C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl substitution; n is₂Is 0, 1, 2 or 3.

More preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), C_1-6Alkyl radical, C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂And 6-to 10-membered aryl-SO₂-; said 6-to 10-membered aryl group being optionally substituted by H (hydrogen) or C_1-6Alkyl substitution; n is₂Is 0, 1, 2 or 3.

More preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), C_1-6Alkyl radical, CH₃C(＝O)-、CF₃C (═ O) -, benzoyl, CH₃SO₂-、CF₃SO₂-and p-toluenesulfonyl; n is₂Is 1 or 2.

Most preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), methyl, isopropyl, CH₃C (═ O) -, benzoyl, CH₃SO₂-and p-toluenesulfonyl; n is₂Is 2.

In a preferred embodiment, R is present at each occurrenceEach occurrence is independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl, halo C_1- ₆Alkyl (e.g. -CF)₃)、C_1-6Alkoxy and halo C_1-6Alkoxy (e.g., -OCF)₃) (ii) a n is 1, 2 or 3.

Preferably, R is independently selected for each occurrence from H (hydrogen), C_1-6Alkyl and halo C_1-6Alkyl (e.g. -CF)₃) (ii) a n is 1 or 2.

More preferably, R is independently selected for each occurrence from H (hydrogen), methyl and-CF₃(ii) a n is 1 or 2.

In a preferred embodiment, AA is selected from

The above groups being linked to the-L1-group via one of the two positions marked 1 or 2 and via the other position

Attached, preferably, the above group is via the position marked with 1 and-L₁-the group is linked and is linked via the 2-labelled position

And (4) connecting.

More preferably, AA is selected from

Attached, preferably, the above group is attached to the-L1-group via the 1-labeled position and via the 2-labeled position

And (4) connecting.

Most preferably, AA is selected from

And

And (4) connecting.

In a preferred embodiment, L₁Is selected from

(e.g. using

)、

The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy and C_3-6A cycloalkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Preferably, L₁Is selected from

(e.g. using

)、

The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl and cyano-substituted C_1-6An alkyl group; y1 and y2 are each independently 0, 1, 2, 3,4.5, 6, 7, 8, 9 or 10.

More preferably, L₁Is selected from

(e.g. using

)、

The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl and cyano-substituted C_1-6An alkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Most preferably, L₁Is selected from

It is linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂And (4) connecting.

In a preferred embodiment, L₂Is absent or is

Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁Selected from O and-NH-, Z₂Absent or selected from O and-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Preferably, L₂Is absent or is

Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁is-NH-, Z₂Absent or-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

More preferably, L₂Is absent or selected from

Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃And (4) connecting.

In a preferred embodiment, L₃Is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups: c_3-8Cycloalkylene, phenylene, naphthylene, 5-12 membered heteroarylene, 3-8 membered heterocyclene, 6-12 membered bridged heterocyclene, 6-12 membered spiroheterocyclene, and 6-12 membered fused heterocyclene, when the above groups contain a nitrogen atom, the nitrogen atom is optionally quaternized; r₇Each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid, C_1-4Alkyl, -CF₃、C_1-4Alkoxy radical, C_2-6Alkenyl and C_2-6Alkynyl.

Preferably, L₃Is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups:

e is a counterion, R₇Each independently at each occurrence is selected from H (hydrogen), D (deuterium), methyl and-CF₃One of the two positions of the above radicals marked by 1 or 2 is linked to L₂Is connected to L via another position₄Linking, preferably, the above group via the 1-labelled position with L₂Is connected and passes the position marked by 2 and L₄And (4) connecting.

More preferably, L₃Absent or selected from:

Most preferably, L₃Is absent or selected from

E is a counterion, preferably a halide anion, more preferably chloride, bromide or iodide, in one of the two positions of which the above radical is marked by 1 or 2, with L₂Is connected to L via another position₄Linking, preferably, the above group via the 1-labelled position with L₂Is connected and passes the position marked by 2 and L₄And (4) connecting.

In a preferred embodiment, L₄Is absent or selected from C_1-6Alkylene, -NR₉-C_1-6Alkylene-, -C_1-6alkylene-NR₉-and-NR₉-；R₉Independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl and C_3-6A cycloalkyl group; said C is_1-6Alkyl or C_1-6Alkylene groups are optionally substituted with one or more H (hydrogen), D (deuterium), or halogen.

Preferably, L₄Is absent or selected from-CH₂CH₂-、-NHCH₂-、-CH₂NH-and-NH-.

In a preferred embodiment, L₅Is selected from

Preferably, L₅Is selected from

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

in a preferred embodiment, the compound of formula (I) is selected from:

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

L₅' is selected from

γ is an integer from 1 to 10, e.g., 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 is

The remaining groups are as defined above.

In a preferred embodiment, L₅' is selected from

Which is marked by one of the two positions 1 or 2 and L₄Attached to the thiol group of the target via another position; preferably, it is marked by 1 position and L₄Attached to the thiol group of the target via the 2-labeled position.

Preferably, L₅' is selected from

In preferred embodiments, the target is selected from the group consisting of small molecule ligands, proteins, polypeptides, and non-protein agents (such as sugars, RNA, or DNA).

Preferably, the target is a substance that targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEA CAM5, mucins (such as 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 cotransporter 2B, GPNMB, trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66 33, CD33, PD-L33, DR 33, E33, FcFcRH 72P, MPF, Napi3 33, Sema 5b, PShlCA 33, PShlAP, MSG783, STEP 33, TrpM 33, CD33, CRIPPTO, CRIPTC 33, CD 6572, CD33, CD 36, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, sealprotein 18.2, BMPR1B, Tyro7, C-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, GREIL 10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEL, STAT 36 1, TGTGR 1, CTSS, PGF, VEGFA, VEGFC 1, TGFACD 1QA, TGFAC QA, TGFACT QA, TGFAAK QA, TGFAN QA, TGFAAK QA, TFAK QA, PGSAGF 72, TFAS QA, TFS 36.

Preferably, the target is selected from small molecule ligands such as folic acid derivatives, glutamic urea derivatives, somatostatin derivatives, arylsulfonamide derivatives (e.g. carbonic anhydrase IX inhibitors), polyenes linking two aliphatic indoles, cyanine dyes and IR-783 or derivatives thereof; an antibody, e.g., a monoclonal antibody or 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 (e.g., scFv), nonhuman antibody, humanized antibody, chimeric antibody, fully human antibody, preanti, bispecific antibody, or multispecific antibody; RGD peptides that recognize cell surface integrin receptors; growth factors that recognize cell surface growth factor receptors such as EGF, PDGF or VEGF; peptides capable of recognizing functional cell surface plasminogen activator, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACTD 2 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, for example, 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 thiol groups from Sacituzumab, trastuzumab or pertuzumab, or a group obtained by removing γ thiol groups from Sacituzumab, trastuzumab or pertuzumab after reduction of disulfide bonds, and 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:

wherein a1 is a group obtained by removing γ thiol groups from Sacituzumab or a group obtained by removing γ thiol groups from Sacituzumab after reduction of the disulfide bond, 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 present invention provides a pharmaceutical composition comprising one or more (preferably two or more) conjugates of formula (II) of the present invention or pharmaceutically acceptable salts, solvates, hydrates, isomers, or any crystalline forms or racemates thereof, and optionally one or more pharmaceutically acceptable carriers, and optionally further comprising one or more other anticancer drugs such as chemotherapeutic agents and/or antibodies, wherein the pharmaceutical composition has an average DAR value of 1 to 10, e.g., 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, 6.9, 6.6, 6.0, 6.7, 6.6, 6.7, 6, 7, 6.0, 6.7, 6, 6.7, 7, 6.7, 6.0, 6, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 or 10.0, preferably an integer or decimal from 4 to 8, for example 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 present invention provides a conjugate of formula (II) of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, for use in the prevention or treatment of a cancer disease.

In some embodiments, the present invention provides a method for preventing or treating a cancer disease, the method comprising administering to a subject in need thereof an effective amount of a conjugate of formula (II) of the present invention or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof.

Preferably, the cancer disease is selected from breast cancer (e.g., triple negative breast cancer), gastric cancer, esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, salivary gland cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell cancer, bladder cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, renal cancer, colon cancer, solid tumors, non-hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, and thyroid cancer.

More preferably, the cancer disease is selected from breast cancer (e.g., triple negative breast cancer) and gastric cancer.

Further preferably, the cancer disease is selected from gastric cancer.

By "pharmaceutically acceptable carrier" in the context of the present invention is meant a diluent, adjuvant, excipient, or vehicle that is administered together with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that may be employed 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 and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical compositions of the present invention may act systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

The pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route. Such as tablets, capsules, troches, hard candies, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, gels, ointments, creams, pastes, lotions, liniments, aqueous suspensions, elixirs, ophthalmic preparations, pills, implants, aerosols, powders, sprays and the like.

When administered orally, the pharmaceutical composition may be formulated into any orally acceptable dosage form including, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, oral emulsions, and the like. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents for use in capsules typically include lactose and dried corn starch. Oral suspensions are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When administered transdermally or topically, the pharmaceutical compositions may be formulated in the form of suitable ointments, lotions or liniments in which the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; lotions or liniments carriers that may be used include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutical composition can also be administered in the form of injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

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

The dosing regimen may be adjusted to provide the best 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 exigencies of the therapeutic situation. It is noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering the composition or supervising the administration of the composition.

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

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

As used herein, unless otherwise specified, the term "treating" means reversing, alleviating, inhibiting the progression of, or preventing such a disorder or condition, or one or more symptoms of such a disorder or condition, to which such term applies.

As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Preparation method

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

wherein:

R_d' is R_dOr PG₁，

the remaining groups are as defined above,

the method comprises the following steps:

the method comprises the following steps: reacting the compounds (Ia) -d with the compounds (Ia) -e in the presence of a metal catalyst to give the compounds of the formula (Ia), and

step two: reacting the compound of formula (Ia) under conditions to remove the amino protecting group to give the compound of formula (I') or a pharmaceutically acceptable salt thereof.

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

Preferably, said first step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃.

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

Preferably, said step one of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably in the presence of trifluoroacetic acid.

Preferably, said second step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃.

Preferably, said step two of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

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

wherein:

R_dis "R_dOr a hydrogen atom,

R_d' is R_dOr PG₁，

the remaining groups are as defined above,

the method comprises the following steps:

Preferably, the base of step one of the process for the preparation of the compound of formula (I') is selected from organic and inorganic bases, such as triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, preferably triethylamine, diisopropylethylamine.

Preferably, said first step of the process for the preparation of the compound of formula (I') is carried out at a temperature of from 0 ℃ to 40 ℃, preferably from 10 ℃ to 30 ℃.

Preferably, said step one of the process for the preparation of the compound of formula (I') is carried out in an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), nitriles (e.g., acetonitrile), and any combination thereof, preferably dichloromethane.

Preferably, the condensation reagent of step two of the process for the preparation of the compound of formula (I') is selected from carbonyldiimidazole, triphosgene, preferably triphosgene.

Preferably, the base of step two of the process for the preparation of the compound of formula (I') is selected from organic and inorganic bases, such as triethylamine, diisopropylethylamine, p-dimethylaminopyridine, pyridine, 2, 4, 6-trimethylpyridine and the like, preferably p-dimethylaminopyridine.

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

Preferably, in the process for preparing the compound of formula (I'), in step two, the molar ratio of the compounds (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 two of the process for the preparation of the compound of formula (Γ) is carried out in an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), nitriles (e.g., acetonitrile), and any combination thereof, preferably dichloromethane.

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

Preferably, the process for the preparation of the compound of formula (I') said step three is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃.

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

Preferably, the process for the preparation of the compound of formula (I') said step three is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the conditions for removing the amino protecting group in step four of the process for preparing the compound of formula (I') are acidic conditions, more preferably in the presence of trifluoroacetic acid.

Preferably, said step four of the process for the preparation of the compound of formula (Γ) is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃.

Preferably, said step four of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

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

wherein:

R_d' is R_dOr PG₁，

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

the remaining groups are as defined above,

the method comprises the following steps:

the method comprises the following steps: reacting compound (Ib) -a with compound (Ib) -b in the presence of a base, optionally in the presence of a condensing agent, to give a compound of formula (Ib), and

step two: reacting the compound of formula (Ib) under conditions to remove the amino protecting group to give the compound of formula (I') or a pharmaceutically acceptable salt thereof.

Preferably, the base is an organic base or is free ofOrganic alkali; the organic base is selected from triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from NaH, NaOH and Na₂CO₃And K₂CO₃。

Preferably, the base is DIPEA.

Preferably, the condensation reagent is selected from the group consisting of phosgene, phosgene solids, phosgene, and mixtures thereof,

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

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

Preferably, said step one of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, said second step of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

Preferably, the process for preparing a compound of formula (I ″), or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, is as follows:

wherein:

R_d' is R_dOr PG₁，

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

the remaining groups are as defined above,

the method comprises the following steps:

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

Preferably, the base is an organic base or an inorganic base; the organic base is selected from triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from NaH, NaOH and Na₂CO₃And K₂CO₃。

Preferably, the base is DIPEA.

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

Preferably, said first step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 50 ℃, preferably 10 ℃ to 30 ℃.

Preferably, said step one of the process for the preparation of the compound of formula (I ") is carried out in an organic solvent; the organic solvent is selected from ethers (such as tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), ethyl acetate and any combination thereof, preferably tetrahydrofuran.

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

Preferably, said step three of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃.

Preferably, said step three of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.

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

the method comprising coupling a compound of formula (I) to a target,

wherein each group is as defined above.

Preferably, the method comprises: mixing a compound of formula (I) with a target; the disulfide bonds in the targets are optionally reduced prior to mixing.

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

Preferably, the method comprises: a solution containing the target is mixed 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 of from 0 ℃ to 50 ℃, preferably from 15 ℃ to 25 ℃.

Preferably, the process is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide and N-methylpyrrolidone.

Preferably, the method further comprises purification 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 formula (I) of the invention can be efficiently coupled with a target (such as an antibody), and has high coupling efficiency, and few unconjugated targets (such as antibodies). The obtained conjugate has obvious in-vitro tumor cell growth inhibition activity and excellent anti-tumor effect.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

The structures of the compounds/conjugates described in the following examples were measured by nuclear magnetic resonance spectroscopy (¹H NMR) and/or Mass Spectrometry (MS).

Nuclear magnetic resonance spectrum (¹H NMR) was performed using a Bruker 400 MHz nuclear magnetic resonance apparatus; the solvent was determined to be deuterated methanol (CD)₃OD), deuterationChloroform (CDCl)₃) Or hexadeutero dimethyl sulfoxide (DMSO-d)₆) (ii) a The internal standard substance is Tetramethylsilane (TMS).

Abbreviations in the Nuclear Magnetic Resonance (NMR) spectroscopic data of the examples have the following meanings:

s: unimodal (singlet), d: doublet (doublt), t: triplet (triplet), q: quartet (quartz), dd: doublet (doubledoubledoublet), qd: quartet doubtet, ddd: double doublet (double doublet), ddt: double double triplet (double double triple), dddd: double double doublet (double double doublet), m: multiplet (multiplex), br: broad (broad), J: coupling constant, Hz: hertz, DMSO-d₆: hexadeuterio dimethyl sulfoxide.

All values are expressed in ppm.

Mass Spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

The preparation method of the high-performance liquid phase comprises the following steps:

the method A comprises the following steps:

a chromatographic column: waters Bridge Prep C18 OBD 5 μm 19x150mm

Mobile phase A: acetonitrile; mobile phase B: water (W)

Time [ min ]]	Mobile phase A [% ]]	Mobile phase B [% ]]	Flow rate [ mL/min]
0.00	10.0	90.0	24
16.00	90.0	10.0	24

The method B comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19x150mm

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

Time [ min ]]	Mobile phase A [% ]]	Mobile phase B [% ]]	Flow rate [ mL/min]
0.00	10.0	90.0	28
16.00	90.0	10.0	28

The method C comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19x150mm

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

The method D comprises the following steps:

a chromatographic column: waters Bridge Prep C18 OBD 5 μm 19x150mm

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

Time [ min ]]	Flow ofPhase A [% ]]	Mobile phase B [% ]]	Flow rate [ mL/min]
0.00	10.0	90.0	24
16.00	90.0	10.0	24

Abbreviations used in the present invention have the following meanings:

boc tert-butyloxycarbonyl group

BOP benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate

DCC N, N' -dicyclohexylcarbodiimide

DCM dichloromethane

DEPC Cyanophosphoric acid diethyl ester

DIC N, N' -diisopropylcarbodiimide

DIPEA N, N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMSO-d₆Hexahydro-deuterated dimethyl sulfoxide

DTT dithiothreitol

EDC 1, 2-dichloroethane

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

FA formic acid

HATU 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

HBTU benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate

MMT p-methoxy trityl

MeOH methanol

NMM N-methylmorpholine

PyAOP (3H-1, 2, 3-triazolo [4, 5-b ] pyridin-3-yloxy) tri-1-pyrrolidinophosphonium 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (isopropylamino) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-1)

The method comprises the following steps:

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

Di-tert-butyl dicarbonate (288mg, 1.32mmol) was added dropwise to a solution of compound 1-1(310mg, 0.66mmol) and triethylamine (200mg, 1.98mmol) in dichloromethane (10mL) and reacted at room temperature for 24 hours. Purification by silica gel column chromatography (DCM/MeOH ═ 10: 1) afforded the title compound, 412 mg. ESI-MS (m/z): 534.2[ M + H]⁺。

Step two:

synthesis of tert-butyl (2- ((S) -4- (((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-diazatripentadecanamido) benzyl) oxy) carbonyl) oxy) -4-ethyl-3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamate (Compound 1-3)

A solution of triphosgene (85mg, 0.29mmol) in dichloromethane (1mL) was added dropwise to a mixed solution of 4-dimethylaminopyridine (234mg, 1.92mmol) and compound 1-2(256mg, 0.48mmol) in dichloromethane (4mL) at 0 ℃ under a nitrogen atmosphere, and reacted for 1 h. (S) -2- (32-azido-5-oxo-3, 9, 12, 15, 18, 21, 24, 27, 30-nonaoxa-6-azadotriamido) -N- (4- (hydroxymethyl) phenyl) -6- (((4-methoxyphenyl) diphenylmethyl) amino) hexanamide (212.05mg, 0.2mmol) was added to the reaction mixture under nitrogen protection at 0 ℃ and reacted for 2h at 0 ℃. The reaction was purified by preparative high performance liquid chromatography (method A) to give the title compound, 75 mg. ESI-MS (m/z): 1619.8[ M + H]⁺。

Step three:

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

Cuprous bromide (18.67mg, 0.13mmol) and water (0.5mL) were added to a solution of (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide (25mg, 0.09mmol) and compound 1-3(75mg, 0.046mmol) in dimethyl sulfoxide (3mL) at room temperature for 2H. The reaction was purified by preparative high performance liquid chromatography (method A) to give the title compound, 68 mg. ESI-MS (m/z): 1893.9[ M + H]⁺。

Step four:

synthesis of Compound I-1

At room temperature, compounds 1-4(75mg, 0.04mmol) were added to trifluoroacetic acid (0.2mL), dichloromethane (0.05mL) and water (0.025mL)mL) was added to the mixed solvent, and the reaction was stirred for 5 min. The reaction was purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 21.6 mg. 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) hexanamido) methyl) -1H-1, 2, 3-triazol-1-yl) -4, 8-dioxo-6, 12, 15, 18, 21, 24, 27, 30, 33-nonaoxa-3, 9-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (isopropylamino) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6), synthesis of 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-3)

The method comprises the following steps:

(2- ((S) -4- (((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-diazatripentadecanamido) benzyl) oxy) carbonyl) oxy) -4-ethyl-3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamic acid tert-butyl ester (Compound 2-1)

Using a synthesis analogous to the third step of example 1, 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N- (prop-2-yn-1-yl) hexanamide was used instead of (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide and the title compound was purified by preparative high performance liquid chromatography (method A) to give 28 mg. ESI-MS (m/z): 1867.9[ M + H]⁺。

Step two:

preparation of Compound I-3

Using a procedure analogous to step four of example 1Synthesis procedure, substituting Compound 2-1 for Compound 1-4, gave the title compound, trifluoroacetate 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-pyrrol-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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (isopropylamino) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-4)

The method comprises the following steps:

synthesis of 1- (piperidin-4-ylmethyl) -1H-pyrrole-2, 5-dione (Compound 3-2)

Trifluoroacetic acid (1mL) was added to a solution of compound 3-1 in dichloromethane (3mL) at room temperature. The reaction was carried out at room temperature for 2 h. The reaction was concentrated to give the crude title compound, 98 mg. 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-ynoyl) piperidin-4-yl) methyl) -1H-pyrrole-2, 5-dione (Compound 3-3)

N, N-diisopropylethylamine (67mg, 0.52mmol) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (235mg, 0.62mmol) were added to a solution of pent-4-ynoic acid (55mg, 0.56mmol) and compound 3-2(100mg, 0.52mmol) in dichloromethane (5mL) at room temperature, respectively, and reacted at room temperature for 3 hours. The reaction was purified by preparative high performance liquid chromatography (method C) to give the title compound, 89 mg. ESI-MS (m/z): 275.1[ M + H]⁺。

Step three:

(2- ((S) -4- (((4- ((S) -35- (4- (3- (4- ((2, 5-dioxo-2, 5-dihydro-1H-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-diazatripentadecanamido) benzyl) oxy) carbonyl) oxy) -4-ethyl-3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamic acid tert-butyl ester (Compound 3-4)

Using a synthesis method analogous to step three of example 1, substituting compound 3-3 for (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide, the title compound was obtained, 26 mg. ESI-MS (m/z): 1893.9[ M + H]⁺。

Step four:

synthesis of Compound I-4

Using a synthesis similar to step four of example 1, compound 3-4 was substituted for compound 1-4 to give the title compound as the trifluoroacetate salt, 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-5)

The method comprises the following steps:

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

Compound 4-1 hydrochloride (150mg, 0.33mmol) was dissolved in dichloromethane (4mL) at room temperature, cooled to 0 deg.C, methanesulfonyl chloride (109.5mg, 0.96mmol) was added dropwise, and the reaction was stirred at room temperature overnight. Purification by preparative high performance liquid chromatography (method C) afforded 75mg of the title compound. ESI-MS (m/z): 512.3[ M + H]⁺。

Step two:

synthesis of 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethylsulphonylamino) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 4-3)

A solution of triphosgene (41mg, 0.14mmol) in dichloromethane (1mL) was added dropwise to a mixed solution of 4-dimethylaminopyridine (136.5mg, 1.12mmol) and compound 4-2(71.7mg, 0.14mmol) in dichloromethane (4mL) at 0 ℃ under a nitrogen atmosphere, and reacted for 1 h. (S) -2- (32-azido-5-oxo-3, 9, 12, 15, 18, 21, 24, 27, 30-nonaoxa-6-azadotriamido) -N- (4- (hydroxymethyl) phenyl) -6- (((4-methoxyphenyl) diphenylmethyl) amino) hexanamide (14.0mg, 0.14mmol) was added to the reaction mixture under nitrogen protection at 0 ℃ and reacted for 2h at 0 ℃. The reaction was purified by preparative high performance liquid chromatography (method A) to give the title compound, 43.0 mg. 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) cyclohexanecarboxamido) 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 4-4).

The compound 4-3(43mg, 0.025mmol) and (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide (14.0mg, 0.050mmol) were added to H at room temperature₂To a mixed solvent of O (0.4ml) and DMSO (1.6ml) was added cuprous bromide (10.8mg, 0.075mmol), and the reaction was stirred for 2 hours. Filtration and purification of the filtrate by preparative high performance liquid chromatography (method A) gave the title compound, 30.0 mg. ESI-MS (m/z): 1871.8[ M + H]⁺。

Step four:

synthesis of Compound I-5

Compound 4-4(30mg, 0.016mmol) was added to a mixed solvent of trifluoroacetic acid (0.2mL), dichloromethane (0.05mL) and water (0.025mL) at room temperature, and the reaction was stirred for 5 min. The reaction was purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 5.68 mg.

¹H NMR(400MHz，DMSO-d₆)10.19(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＝7.9Hz，1H)，7.82(s，1H)，7.80(t，J＝7.5Hz，1H)，7.68(s，3H)，7.61(d，J＝8.4Hz，2H)，7.33(d，J＝8.5Hz，2H)，7.06(s，1H)，7.01(s，2H)，5.54(d，J＝3.9Hz，2H)，5.46(s，2H)，5.11(q，J＝12.2Hz，2H)，4.47(t，J＝4.9Hz，3H)，4.25(d，J＝5.5Hz，2H)，4.04(d，J＝3.6Hz，2H)，4.00(d，J＝2.5Hz，2H)，3.99-3.93(m，1H)，3.78(t，J＝5.2Hz，2H)，3.50-3.43(m，32H)，3.39-3.35(m，2H)，3.28(q，J＝5.6Hz，2H)，3.23(d，J＝7.0Hz，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＝6.9Hz，6H)，0.91(t，J＝7.4Hz，3H)，0.89-0.84(m，2H).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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-6)

The method comprises the following steps:

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

Triethylamine (300mg, 3.0mmol) and acetyl chloride (100mg, 1.2mmol) were added dropwise to a solution of compound 5-1(440mg, 1.0mmol) in dichloromethane (30mL) in this order at room temperature, and the reaction was carried out at room temperature for 2 hours. The reaction was concentrated and purified by preparative high performance liquid chromatography (method C) to give the title compound, 185.2 mg. ESI-MS (m/z): 476.3[ M + H]⁺。

Step two:

synthesis of 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 5-3)

Compound 5-2(60mg, 0.13mmol) was dissolved in dry dichloromethane (2mL) at room temperature under nitrogen, cooled to 0 deg.C, added a solution of 4-dimethylaminopyridine (185mg, 1.51mmol) in dry dichloromethane (0.5mL), then triphosgene (37mg, 0.13mmol) in dry dichloromethane (0.5mL) was added dropwise slowly and the reaction was stirred at 0 deg.C for 1 hour after the addition. (S) -2- (32-azido-5-oxo-3, 9, 12, 15, 18, 21, 24, 27, 30-nonaoxa-6-azadotriamido) -N- (4- (hydroxymethyl) phenyl) -6- (((S-2-azido-5-oxo-3, 9-12, 15-azadotriamido) S-e4-methoxyphenyl) diphenylmethyl) amino) hexanamide (118mg, 0.11mmol) in dry dichloromethane (0.5mL) was reacted at 0 ℃ for 2h after the addition was completed. Preparative high performance liquid chromatography purification (method A) gave the title compound, 60 mg. 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) cyclohexanecarboxamido) 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 5-4)

Compound 5-3(20mg, 0.012mmol) and (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide (5.3mg, 0.019mmol) were dissolved in dimethyl sulfoxide (0.5mL) and water (0.1mL) at room temperature, cuprous bromide (3.67mg, 0.026mmol) was added, and the reaction was stirred for 1H. Filtration and purification of the filtrate by preparative high performance liquid chromatography (method A) gave the title compound, 18 mg. ESI-MS (m/z): 1836.5[ M + H]⁺。

Step four:

synthesis of Compound I-6

Compound 5-4(18mg, 0.011mmol) was dissolved in acetonitrile (0.4ml) and water (0.1ml) at room temperature, and a solution of trifluoroacetic acid (0.5ml) in acetonitrile (0.5ml) was added dropwise thereto and stirred at room temperature for 2 hours. The reaction was purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 14 mg.

¹H NMR(400MHz，DMSO-d₆)10.19(s，1H)，8.57(d，J＝8.5Hz，1H)，8.21(t，J＝6.4Hz，3H)，8.10(t，J＝5.6Hz，1H)，7.89(t，J＝7.9Hz，1H)，7.82(s，1H)，7.79(t，J＝7.3Hz，1H)，7.69(s，3H)，7.61(d，J＝8.6Hz，2H)，7.33(d，J＝8.6Hz，2H)，7.05(s，1H)，7.01(s，2H)，5.54(d，J＝4.0Hz，2H)，5.45(s，2H)，5.11(q，J＝12.2Hz，2H)，4.47(t，J＝5.1Hz，3H)，4.25(d，J＝5.6Hz，2H)，4.13-4.06(m，1H)，4.04(d，J＝3.6Hz，2H)，4.00(d，J＝2.6Hz，2H)，3.78(t，J＝5.3Hz，2H)，3.50-3.40(m，34H)，3.28(q，J＝6.0Hz，2H)，3.23(d，J＝7.1Hz，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＝3.1Hz，6H)，0.91(t，J＝7.3Hz，3H)，0.89-0.83(m，2H).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) hexanamido) methyl) -1H-1, 2, 3-triazol-1-yl) -4, 8-dioxo-6, 12, 15, 18, 21, 24, 27, 30, 33-nonaoxa-3, 9-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-7)

The method comprises the following steps:

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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 6-1)

Using a synthesis analogous to the third step of example 5, 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) N- (prop-2-yn-1-yl) hexanamide was used instead of (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanemethylmethaneAmide, purified by preparative high performance liquid chromatography (method A) to give 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-nonaoxa-3, 9-diazatripentadecanylamino) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate trifluoroacetate (Compound I-7)

Using a synthesis similar to step four of example 5, compound 6-1 was substituted for compound 5-4 and purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 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-pyrrol-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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-8)

The method comprises the following steps:

4- ((S) -35- (4- (3- (4- ((2, 5-dioxo-2, 5-dihydro-1H-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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 7-1)

Using a synthesis analogous to the third step of example 5, substituting 1- ((1- (pent-4-ynoyl) piperidin-4-yl) methyl) -1H-pyrrole-2, 5-dione for (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide, the title compound was purified by preparative high performance liquid chromatography (method A) to give 35 mg. ESI-MS (m/z): 1871.8[ M + H]⁺。

Step two:

preparation of Compound I-8

Using a synthetic method similar to step four of example 5, compound 7-1 was substituted for compound 5-4 and purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 9.15 mg. 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-pyrrol-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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-9)

The method comprises the following steps:

4- ((S) -35- (4- (3- (4- ((2, 5-dioxo-2, 5-dihydro-1H-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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 8-1)

Using a synthesis analogous to the third step of example 5, 1- ((1- (pent-4-ynoyl) piperidin-4-yl) methyl) -1H-pyrrole-2, 5-dione was used instead of (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide and the title compound was purified by preparative high performance liquid chromatography (method A) to give 30 mg. ESI-MS (m/z): 1835.8[ M + H]⁺。

Step two:

preparation of Compound I-9

Using a synthesis method similar to step four of example 5, compound 8-1 was substituted for compound 5-4 and purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 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-pyrrol-1-yl) methyl) cyclohexyl) -1, 30, 34-trioxo-5, 8, 11, 14, 17, 20, 23, 26, 32-nonaoxa-2, 29, 35-triaza-trinexaheptadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethylsulphonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, synthesis of 2-b ] quinolin-4-yl) carbonate (Compound I-10)

The method comprises the following steps:

synthesis of 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethylsulphonylamino) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 9-1)

Compound 4-3(30mg, 0.019mmol) was dissolved in tetrahydrofuran (6.0mL) at room temperature, N₂Platinum dioxide (5mg) was added under protection, and the mixture was replaced with hydrogen three times and reacted at room temperature for 3 hours. Insoluble material was filtered off, the filtrate was concentrated and the crude product was purified by preparative high performance liquid chromatography (method D) to give the title compound, 15 mg. ESI-MS (m/z): 650.3[ (M-273)/2+ H]⁺。

Step two:

4- ((S) -1- ((1r, 4r) -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-triazatetraheptadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 9-2)

Compound 9-1(20mg, 0.013mmol) and methyl (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexanecarboxylate (6mg, 0.019mmol) were dissolved in dichloromethane (2mL) at room temperature, N-diisopropylethylamine (3.0mg, 0.025mmol) was added dropwise, and the reaction was completed at room temperature for 30 min. The reaction solution was used directly in the next reaction.

Step three:

synthesis of Compound I-10

Trifluoroacetic acid (0.1mL) was added to a reaction solution of compound 9-2(20mg, 0.011mmol) in dichloromethane (2.0mL) and reacted at room temperature for 30 min. The reaction was concentrated and purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 4.0 mg. ESI-MS (m/z): 760.0[ M/2+ H]⁺。

Example 10

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

The method comprises the following steps:

synthesis of 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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 10-1)

Using a synthesis method similar to the one step of example 9, compound 5-3 was used instead of compound 4-3, and purification by preparative high performance liquid chromatography (method D) was performed to give the title compound, 15 mg. ESI-MS (m/z): 632.5[ (M-273)/2+ H]⁺。

Step two:

4- ((S) -1- ((1r, 4r) -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-triazatetraheptadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 10-2)

Using a synthesis method similar to the second step of example 9, compound 10-1 was used in place of compound 9-1, and the reaction mixture was used directly in the next reaction.

Step three:

synthesis of Compound I-11

Using a synthetic method similar to the third step of example 9, compound 10-2 was used in place of compound 9-2 to prepare a high performance liquidPurification by phase chromatography (method B) gave the title compound as the trifluoroacetate 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-diazatripentadecanamido) -2-methylbenzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-12)

The method comprises the following steps:

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

4-amino-2-methylbenzyl alcohol (107mg, 0.78mmol) and compound 11-1(500mg, 0.78mmol) were dissolved in dichloromethane (5mL) at room temperature, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (289mg, 1.17mmol) was added, and the reaction was stirred at 35 ℃ for 5 h. After the temperature was reduced to room temperature, diethylamine (1mL) was added to the reaction solution, and the mixture was stirred for 2 hours. The reaction was concentrated and purified by silica gel column chromatography (DCM/MeOH ═ 30/1) to afford the title compound, 300 mg.

Step two:

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

Compound 11-2(100mg, 0.19mmol) and 32-azido-5-oxo-3, 9, 12, 15, 18, 21, 24, 27, 30-nonaoxa-6-azadotriacontane-1-oic acid (103mg, 0.19mmol) were dissolved in dichloromethane (5mL) at room temperature, and 2-ethyl-2-acetate was addedOxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (69mg, 0.28mmol) was reacted at 30 ℃ with stirring for 5 h. The reaction was concentrated and purified by silica gel column chromatography (DCM/MeOH ═ 10/1) to give the title compound, 154 mg. ESI-MS (m/z): 1074.5[ M + H]⁺。

Step three:

synthesis of 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-diazatripentadecanamido) -2-methylbenzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 11-4)

Using a synthesis analogous to the second step of example 5, substituting compound 11-3 for (S) -2- (32-azido-5-oxo-3, 9, 12, 15, 18, 21, 24, 27, 30-nonaoxa-6-azadotriamido) -N- (4- (hydroxymethyl) phenyl) -6- (((4-methoxyphenyl) diphenylmethyl) amino) hexanamide, the title compound was purified by preparative high performance liquid chromatography (method A) to give 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) cyclohexanecarboxamido) 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-diazatripentadecanamido) -2-methylbenzyl ((S) -4-ethyl-11- (2- (N-isopropylacetamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 11-5)

Using a synthetic method analogous to the third step of example 5, compound 11-4 was substituted for compound 5-3 and purified by preparative high performance liquid chromatography (method A) to give the title compound, 15 mg. ESI-MS (m/z): 1824.7[ M + H]⁺。

Step five:

synthesis of Compound I-12

Compound 11-5(15mg, 0.008mmol) was dissolved in acetonitrile (0.4mL) and water (0.1mL) at room temperature, and a solution of trifluoroacetic acid (0.5mL) in acetonitrile (0.5mL) was added dropwise. Stirred at room temperature for 2 h. Purification by preparative high performance liquid chromatography (method B) gave the title compound as the trifluoroacetate salt, 8.12 mg. 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) hexanamido) methyl) -1H-1, 2, 3-triazol-1-yl) -4, 8-dioxo-6, 12, 15, 18, 21, 24, 27, 30, 33-nonaoxa-3, 9-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethylsulphonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [ 3', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound I-13)

The method comprises the following steps:

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-diazatripentadecanamido) benzyl ((S) -4-ethyl-11- (2- (N-isopropylmethanesulfonamido) ethyl) -3, 14-dioxo-3, 4, 12, 14-tetrahydro-1H-pyrano [3 ', 4': synthesis of 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbonate (Compound 12-1)

Using a synthesis analogous to the procedure three in example 4, 6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N- (prop-2-yn-1-yl) hexanamide was used instead of (1r, 4r) -4- ((2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) methyl) -N- (prop-2-yn-1-yl) cyclohexanecarboxamide and the title compound was purified by preparative high performance liquid chromatography (method A) to give 30 mg. ESI-MS (m/z): 1846.7[ M + H]⁺。

Step two:

synthesis of Compound I-13

Using a synthetic method analogous to the fourth step in example 4, compound 12-1 was substituted for compound 4-4 and purified by preparative high performance liquid chromatography (method B) to give the title compound as the trifluoroacetate salt, 19.2 mg. ESI-MS (m/z): 1574.6[ M + H]⁺。

Preparation of conjugates

Example 13 preparation of II-1-A

Taking 5mg of Sacituzumab, diluting with a diluent (20mM PB +150mM NaCl +20mM sodium edetate solution, pH 7.6) (the final concentration of sodium edetate is 5mM, and the final concentration of antibody is 10mg/mL), and mixing uniformly; then 1M Na was used₂Adjusting the pH value of the PO4 solution to 7.4, adding 10mMTCEP solution, mixing uniformly, and standing at room temperature for 30 min; adding I-1 (molar ratio of I-1 to antibody is 9: 1) dissolved in dimethyl sulfoxide into the solution system, mixing, and standing at room temperature for 2 h. And finally, replacing the buffer solution with a PBS solution with the pH value of 6.5 by adopting a G-25 gel column to obtain a product of coupling the I-1 and the antibody, which is named as II-1-A.

Example 14 preparation of II-5-A

Using a method similar to example 13, I-1 was replaced with I-3 to give a product of I-3 conjugated with an antibody, which was designated as II-5-A.

Example 15 preparation of II-6-A

Using a method similar to example 13, I-1 was replaced with I-4 to give a product of I-4 conjugated with an antibody, which was designated as II-6-A.

Example 16 preparation of II-7-A

Using a method similar to example 13, I-1 was replaced with I-5 to give a product of I-5 conjugated with an antibody, which was designated as II-7-A.

Example 17 preparation of II-8-A

Using a method similar to example 13, I-1 was replaced with I-6 to give a product of I-6 conjugated with an antibody, which was designated as II-8-A.

Example 18 preparation of II-9-A

Using a method similar to example 13, I-1 was replaced with I-7 to give a product of I-7 conjugated with an antibody, which was designated as II-9-A.

Example 19 preparation of II-10-A

Using a method similar to example 13, I-1 was replaced with I-8 to give a product of I-8 conjugated to an antibody, which was designated as II-10-A.

Example 20 preparation of II-11-A

Using a method similar to example 13, I-1 was replaced with I-9 to give a product of I-9 conjugated with an antibody, which was designated as II-11-A.

Example 21 preparation of II-12-A

Using a method similar to example 13, I-1 was replaced with I-10 to give a product of I-10 conjugated with an antibody, which was designated as II-12-A.

Example 22 preparation of II-13-A

Using a method similar to example 13, I-1 was replaced with I-11 to give a product of I-11 conjugated with an antibody, which was designated as II-13-A.

Example 23 preparation of II-14-A

Using a method similar to example 13, I-1 was replaced with I-12 to give a product of I-12 conjugated with an antibody, which was designated as II-14-A.

Example 24 preparation of II-15-A

Using a method similar to example 13, I-1 was replaced with I-13 to give a product of I-13 conjugated with an antibody, which was designated as II-15-A.

Molecular weight determination and DAR value analysis of conjugates

In the following examples, LC represents an antibody light chain, HC represents an antibody heavy chain, DAR1 represents a conjugate comprising one antibody light or heavy chain and one cytotoxic agent, DAR2 represents a conjugate comprising one antibody light or heavy chain and two cytotoxic agents, 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 conjugation

Sample treatment: diluting a sample to be detected to 1mg/mL, adding 1M DTT (dithiothreitol) until the final concentration of DTT is 20mmol/L, uniformly mixing, and carrying out water bath at 37 ℃ for 30 min.

The chromatographic measurement conditions were as follows:

liquid chromatography column: ACQUITU

Protein BEH C18 1.7μm，2.1mm×100mm

Mobile phase A: 0.1% Formic Acid (FA)/98% H₂O/2% Acetonitrile (ACN); mobile phase B: 0.1% FA/2% H₂O/98％ACN

Flow rate: 0.25 mL/min; temperature of the sample chamber: 8 ℃; column temperature: 60 ℃; sample introduction amount: 1 μ g

Elution conditions:

time (minutes)	2	20	22	25	26	30
Mobile phase B (% by volume)	20	40	90	90	20	20

Mass spectrometry conditions:

the mass spectrum model is as follows: triple TOF 5600+

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

The theoretical molecular weights of the light and heavy chains of conjugate II-1-A formed after conjugation of Compound I-1 to an antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following Table:

mass spectrum deconvolution results as shown in fig. 1 and 2, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.0 by coupling 0 to 2 cytotoxic agents to the light chain (22%, 72%, 6% for LC, DAR1 and DAR2 ratios, respectively) and 1 to 3 cytotoxic agents to the heavy chain (49%, 27%, and 24% for DAR1, DAR2 and DAR3 ratios, respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-5-A formed after conjugation of Compound I-3 to an antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 3 and 4, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of antibody to cytotoxic agent (average DAR value) was calculated to be 5.4 by coupling 0 to 1 cytotoxic agent to the light chain (18% and 82% for LC and DAR1 ratios, respectively) and 1 to 3 cytotoxic agents to the heavy chain (46%, 20% and 34% for DAR1, DAR2 and DAR3 ratios, respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-6-A formed after conjugation of Compound I-4 to the antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 5 and 6, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of antibody to cytotoxic agent (average DAR value) was calculated to be 6.0 by coupling 0 to 1 cytotoxic agent to the light chain (16% and 84% for LC and DAR1 ratios, respectively) and 1 to 3 cytotoxic agents to the heavy chain (31%, 23% and 46% for DAR1, DAR2 and DAR3 ratios, respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-7-A formed after conjugation of Compound I-5 to the antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 7 and 8, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.2 by coupling 0 to 1 cytotoxic agent to the light chain (24% and 76% for LC and DAR1 ratios, respectively) and 0 to 4 cytotoxic agents to the heavy chain (6%, 43%, 20%, 25% and 6% for HC, DAR1, DAR2, DAR3 and DAR4 ratios, respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-8-A formed after conjugation of Compound I-6 to the antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 9 and 10, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.5 by coupling 0 to 1 cytotoxic agent to the light chain (19% and 81% for LC and DAR1 respectively) and 0 to 4 cytotoxic agents to the heavy chain (6%, 37%, 18%, 32% and 7% for HC, DAR1, DAR2, DAR3 and DAR4 respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-9-A formed after conjugation of Compound I-7 to an antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 11 and 12, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of antibody to cytotoxic agent (average DAR value) was calculated to be 5.3 by coupling 0 to 1 cytotoxic agent to the light chain (19% and 81% for LC and DAR1 ratios, respectively) and 0 to 3 cytotoxic agents to the heavy chain (3%, 45%, 18% and 34% for HC, DAR1, DAR2 and DAR3 ratios, respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-10-A formed after conjugation of Compound I-8 to the antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 13 and 14, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.3 by coupling 0 to 1 cytotoxic agent to the light chain (20% and 80% for LC and DAR1 respectively) and 0 to 4 cytotoxic agents to the heavy chain (4%, 43%, 20%, 29% and 4% for HC, DAR1, DAR2, DAR3 and DAR4 respectively).

The theoretical molecular weights and the measured molecular weights (calculated as the predominant saccharide form G0F) of the light and heavy chains of conjugate II-11-A formed after conjugation of Compound I-9 to the antibody are shown in the following table:

mass spectrum deconvolution results as shown in fig. 15 and 16, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.3 by coupling 0 to 1 cytotoxic agent to the light chain (19% and 81% for LC and DAR1 respectively) and 0 to 4 cytotoxic agents to the heavy chain (5%, 44%, 17%, 31% and 3% for HC, DAR1, DAR2, DAR3 and DAR4 respectively).

The theoretical molecular weights of the light and heavy chains of conjugate II-12-A formed after conjugation of Compound I-10 to an antibody, as well as the measured molecular weights (calculated as the predominant saccharide form G0F) are given in the following table:

mass spectrum deconvolution results as shown in fig. 17 and 18, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of the antibody to the cytotoxic agent (average DAR value) was calculated to be 5.2 by coupling 0 to 1 cytotoxic agent to the light chain (26% and 74% for LC and DAR1 respectively) and 0 to 4 cytotoxic agents to the heavy chain (6%, 41%, 21%, 23% and 9% for HC, DAR1, DAR2, DAR3 and DAR4 respectively).

The theoretical molecular weights and the measured molecular weights (calculated as the predominant saccharide form G0F) of the light and heavy chains of conjugate II-13-A formed after conjugation of Compound I-11 to the antibody are shown in the following table:

mass spectrum deconvolution results as shown in fig. 19 and 20, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of antibody to cytotoxic agent (average DAR value) was calculated to be 5.2 by coupling 0 to 1 cytotoxic agent to the light chain (19% and 81% for LC and DAR1 ratios, respectively) and 0 to 3 cytotoxic agents to the heavy chain (3%, 47%, 18% and 32% for HC, DAR1, DAR2 and DAR3 ratios, respectively).

The theoretical molecular weights and the measured molecular weights (calculated as the predominant saccharide form G0F) of the light and heavy chains of conjugate II-15-A formed after conjugation of Compound I-13 to the antibody are shown in the following table:

mass spectrum deconvolution results as shown in fig. 21 and 22, DAR was calculated from the mass spectrum deconvoluted signal intensity ratio: the coupling ratio of antibody to cytotoxic agent (average DAR value) was calculated to be 4.7 by coupling 0 to 1 cytotoxic agent to the light chain (28% and 72% for LC and DAR1 ratios, respectively) and 0 to 4 cytotoxic agents to the heavy chain (9%, 48%, 19%, 18%, and 6% for HC, DAR1, DAR2, DAR3, and DAR4 ratios, respectively).

Size Exclusion Chromatography (SEC) analysis

Example 26 coupling reactions were monitored by SEC-HPLC for SEC detection.

Chromatographic conditions are as follows:

liquid chromatography column: TOSOH TSKgel G3000 SWXL 7.8X 300mm

Mobile phase: 25mmol/L Na₂HPO₄，25mmol/L NaH₂PO₄，300mmol/L NaCl，pH6.5

Flow rate: 0.5 mL/min; detection wavelength: 280 nm; column temperature: room temperature; temperature of the sample chamber: 8 deg.C

Sample introduction amount: 30 mu g of the solution; isocratic operation for 30min

From the SEC retention time and the peak area ratio (FIG. 23), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-1-A obtained by coupling I-1 to the antibody, and the intact structure of the antibody was still maintained.

From SEC retention time and peak area ratio (FIG. 24), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-5-A obtained by coupling I-3 to the antibody, still maintained the intact structure of the antibody.

From SEC retention time and peak area ratio (FIG. 25), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-7-A obtained by coupling I-5 to the antibody, still maintained the intact structure of the antibody.

From SEC retention time and peak area ratio (FIG. 26), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-8-A obtained by coupling I-6 to the antibody, still maintained the intact structure of the antibody.

From SEC retention time and peak area ratio (FIG. 27), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-10-A obtained by coupling I-8 to the antibody, still maintained the intact structure of the antibody.

From SEC retention time and peak area ratio (FIG. 28), it was confirmed that the molecular weight of the main coupling product was about 150kD, i.e., II-14-A obtained by coupling I-12 to the antibody, still maintained the intact structure of the antibody.

Size Exclusion Chromatography (SEC) analysis shows that after the antibody is coupled with a cytotoxic agent, the light chain and the heavy chain of the antibody are still combined in a mode of hydrophobic acting force and the like, the complete structure of the antibody is maintained, and the activity of the antibody is well maintained.

Biological assay

EXAMPLE 27 in vitro potency assay of conjugate

The assay evaluates the inhibitory effect of the conjugates on cell proliferation by co-culturing the cytotoxic agents of the present invention (compound 1-1 in example 1, compound 4-2 in example 4, and compound 5-2 in example 5) and the antibody-drug conjugates, respectively, with tumor cells and detecting the activity of dehydrogenase in mitochondria. The testing steps are as follows: the conjugate was diluted with the corresponding assay medium (RPMI1640 with 2% FBS) and cytotoxic agent as control, starting at 1000nM, diluted 4-fold, and diluted 12 concentration gradients. Breast cancer cells HCC1806(Trop-2 positive cell line, tokyo bobo) were digested by conventional methods using pancreatin, tumor cells were collected and resuspended in the corresponding detection medium (RPMI1640 with 2% FBS). Adding the diluted conjugate and cytotoxic agent into the solution respectivelyCells were added to 96-well plates. After 3 days of cell culture, 20. mu.L of CCK8 reagent (RHINO BIO) was added to each well, reacted for 2 hours, and the activity of dehydrogenase in the mitochondria was examined using a microplate reader (manufacturer: Molecular Devices, type: SpectraMax M2, detection wavelength 450nm), from which EC of cytotoxic agent and conjugate for growth inhibition of breast cancer HCC1806 was calculated₅₀The value is obtained. The results of the conjugate on the growth inhibition of 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).

In addition, EC of Compound 1-1 in example 1, Compound 4-2 in example 4, and Compound 5-2 in example 5 for growth inhibition of Breast cancer cell HCC1806₅₀The values were 4.07nM, 7.56nM and 8.72nM, respectively, of the EC of the corresponding antibody-drug conjugates II-1-A, II-7-A and II-8-A₅₀Values of 1.54nM, 1.63nM and 1.15nM, respectively, are significantly lower than the EC of the corresponding cytotoxic agent₅₀The value is obtained. This further illustrates that the activity of the cytotoxic agent in inhibiting tumor is significantly enhanced due to the improved targeting property after the cytotoxic agent is coupled with the antibody.

Example 28 conjugate plasma stability test

The experimental method comprises the following steps: respectively preparing II-1-A, II-7-A and II-8-A samples by using rat plasma, cynomolgus monkey plasma and 0.5% Bovine Serum Albumin (BSA) PBS buffer solution, placing the samples in a biochemical incubator at 37 ℃, respectively incubating for 0h, 1h, 4h, 24h, 48h and 72h, then taking out the samples, placing the samples in a storage at-80 ℃, and detecting the release condition of a cytotoxic agent in the conjugate by adopting LC/MS (liquid chromatography/mass spectrometry) to determine the plasma stability of the conjugate. The cytotoxic agents corresponding to II-1-A, II-7-A and II-8-A were Compound 1-1 in example 1, Compound 4-2 in example 4 and Compound 5-2 in example 5, respectively.

As shown in FIGS. 29 to 31, both II-1-A, II-7-A and II-8-A showed good plasma stability in cynomolgus monkey plasma, rat plasma and 0.5% BSA PBS buffer.

Example 29 in vivo potency assay of conjugates

The experimental method comprises the following steps: NCI-N87 cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, and NCI-N87 cells in the exponential growth phase were collected. Resuspended to appropriate concentration with PBS, and inoculated subcutaneously at the right scapular region of Balb/c nude mice. Tumor growth volume after inoculation is about 100-³When the tumor is removed, the volume of the tumor is too small (less than 100 mm)³) Or too large (greater than 200 mm)³) The remaining mice were randomly grouped (8 mice/group) and administered conjugate or solvent control (saline), tail vein injection, 2 times weekly for a total of 3 weeks. Tumor diameters were measured 2 times a week for 3 weeks after administration with a vernier caliper, and tumor volumes were calculated according to the following calculation formula: v is 0.5a²× b, wherein a and b represent the major and minor diameters of the tumor, respectively.

Tumor growth inhibition ratio TGI (%) was calculated using the following formula for evaluating the tumor-inhibiting efficacy of the antibody-drug conjugate:

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

Wherein, VT is not: mean tumor volume at the end of the experiment in the conjugate treatment group,

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

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

VC is started: mean tumor volume at the beginning of vehicle control group dosing.

The results of the experiment are shown in table 2 below:

TABLE 2 results of in vivo drug efficacy testing of conjugates

As can be seen from Table 2, the conjugates of the present invention (e.g., II-8-A and II-10-A) have significant anti-tumor effects, and the P-value results indicate that the conjugates exhibit a significant trend in anti-tumor efficacy compared to the saline group.

In addition, no animal death or weight loss occurred in the conjugate-treated groups during the evaluation period, indicating that the conjugates of the present invention (e.g., II-8-A and II-10-A) not only have significant antitumor effects but also have excellent safety.

While the invention has been illustrated by the foregoing specific embodiments, it should be understood that it is not to be construed as being limited thereby. The present invention covers the general aspects previously disclosed, and various modifications or changes in detail thereof may be made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, the description is to be regarded as illustrative in nature, and not as restrictive.

Claims

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

wherein:

R₁each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, nitro, -NR_aR_b、C_1-6Alkyl, halo C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy, halo C_1-6Alkoxy (e.g., -OCF)₃) And C_3-6A cycloalkyl group; r_aAnd R_bEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl) and-C_1-6Alkylene- (5-12 membered heteroaryl); n is₁Is 1, 2, 3 or 4;

R₂selected from H (hydrogen), D (deuterium), C_1-6Alkyl, -NR_cR_dAnd a 3-7 membered nitrogen-containing heterocyclic group; r_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl), -C_1-6Alkylene- (5-12 membered heteroaryl), C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, 6-10 membered aryl-C_1-6alkylene-C (═ O) -, 5-12 membered heteroaryl-C_1-6alkylene-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl substitution; n is₂Is 0, 1, 2, 3 or 4;

r is independently selected for each occurrence from H (hydrogen), D (deuterium), halogen, nitro, cyano, C_1-6Alkyl, halo C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy, halo C_1-6Alkoxy (e.g., -OCF)₃)、C_3-6Cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is 1, 2, 3 or 4;

AA is selected from amino acid groups and groups comprising peptides of 2-6 amino acids;

L₁is selected from- (CH)₂)_t1-、

(e.g. using
)、

The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy and C_3-6Cycloalkyl, or R₃And R₄、R₅And R₆Or R₃And R₅Together with the atoms/groups to which they are attached form a 3-8 membered ring; t1, t2, y₁And y₂Each independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

L₂is absent or is
Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁Selected from O, S and-NH-, Z₂Absent or selected from O, S and-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

L₃is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups: c_3-8Cycloalkylene, 6-10 membered arylene (e.g., phenylene or naphthylene), 5-12 membered heteroarylene, 3-8 membered heterocyclylene, 6-12 membered bridged heterocyclyl, 6-12 membered spiroheterocyclyl, 6-12 membered fused heterocyclyl, -NR₈-C_3-8cycloalkylene-and-C_3-8cycloalkylene-NR₈When the above groups contain a nitrogen atom, said nitrogen atom is optionally quaternized; r₇Each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, ═ O, cyano, carboxyl, sulfonic acid, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy radical, C_2-6Alkenyl and C_2-6Alkynyl radical, R₈Selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-6An alkoxy group;

L₄is absent or selected from C_1-6Alkylene, -NR₉-C_1-6Alkylene-, -C_1-6alkylene-NR₉-、-NR₉And by one or more R₉A substituted 3-7 membered nitrogen containing heterocyclylene group; r₉Independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl (e.g., phenyl or naphthyl) and 5-12 membered heteroaryl; said C is_1-6Alkyl or C_1-6Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium), or halogen; and is

L₅Is 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 R is₁Each occurrence independently selected from H (hydrogen), D (deuterium), F, Cl, nitro, -NR_aR_b、C_1-6Alkyl, -CF₃、-OCH₃and-OCF₃；R_aAnd R_bEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl) and-C_1- ₆Alkylene- (5-12 membered heteroaryl); n is₁Is 1, 2 or 3;

preferably, R₁Each occurrence is independently selected from H (hydrogen), F, nitro, -NR_aR_b、C_1-4Alkyl, -CF₃、-OCH₃and-OCF₃；R_aAnd R_bEach independently selected from H (hydrogen), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl and 5-12 membered heteroaryl; n is₁Is 1 or 2; and is

More preferably, R₁Independently at each occurrence, selected from H (hydrogen), nitro, -NH₂、-N(CH₃)₂and-OCH₃；n₁Is 1 or 2.
The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein R is₂Selected from H (hydrogen), D (deuterium), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl radical, C_3-6Cycloalkyl, 6-10 membered aryl, 5-12 membered heteroaryl, -C_1-6alkylene-C_3-6Cycloalkyl, -C_1-6Alkylene- (6-to 10-membered aryl), -C_1-6Alkylene- (5-12 membered heteroaryl), C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl substitution; n is₂Is 0, 1, 2, 3 or 4;

preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), D (deuterium), C_1- ₆Alkyl radical, C_3-6Cycloalkyl radical, C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, 5-12 membered heteroaryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂6-to 10-membered aryl-SO₂And 5-12 membered heteroaryl-SO₂-; said 6-10 membered aryl and 5-12 membered heteroaryl are optionally substituted with H (hydrogen), D (deuterium), halogen or C_1-6Alkyl substitution; n is₂Is 0, 1, 2 or 3;

more preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), C_1-6Alkyl radical, C_1-6alkyl-C (═ O) -, halo-C_1-6alkyl-C (═ O) -, 6-10 membered aryl-C (═ O) -, C_1-6alkyl-SO₂-, halo C_1-6alkyl-SO₂And 6-to 10-membered aryl-SO₂-; said 6-to 10-membered aryl group being optionally substituted by H (hydrogen) or C_1-6Alkyl substitution; n is₂Is 0, 1, 2 or 3;

more preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), C_1-6Alkyl radical, CH₃C(＝O)-、CF₃C (═ O) -, benzoyl, CH₃SO₂-、CF₃SO₂-and p-toluenesulfonyl; n is₂Is 1 or 2; and is

Most preferably, R₂Selected from H (hydrogen), C_1-6Alkyl and-NR_cR_d；R_cAnd R_dEach independently selected from H (hydrogen), methyl, isopropyl, CH₃C (═ O) -, benzoyl, CH₃SO₂-and p-toluenesulfonyl; n is₂Is 2.
The compound of any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein R is, at each occurrence, independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl, haloGeneration C_1-6Alkyl (e.g. -CF)₃)、C_1-6Alkoxy and halo C_1-6Alkoxy (e.g., -OCF)₃) (ii) a n is 1, 2 or 3;

preferably, R is independently selected for each occurrence from H (hydrogen), C_1-6Alkyl and halo C_1-6Alkyl (e.g. -CF)₃) (ii) a n is 1 or 2; and is

More preferably, R is independently selected for each occurrence from H (hydrogen), methyl and-CF₃(ii) a n is 1 or 2.
The compound of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein AA is selected from the group consisting of

One of the two positions of the above groups, marked by 1 or 2, and-L₁-the group is attached and linked via another position to
Attached, preferably, the above group is via the position marked with 1 and-L₁-the group is linked and is linked via the 2-labelled position
Connecting;

more preferably, AA is selected from

The above groups being linked to the-L1-group via one of the two positions marked 1 or 2 and via the other position
Attached, preferably, the above group is via the position marked with 1 and-L₁-the group is linked and is linked via the 2-labelled position
Connecting; and is

Most preferably, AA is selected from

The above groups being linked to the-L1-group via one of the two positions marked 1 or 2 and via the other position
Attached, preferably, the above group is attached to the-L1-group via the 1-labeled position and via the 2-labeled position
And (4) connecting.
The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein, L₁Is selected from
(e.g. using
)、

The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl, cyano-substituted C_1-6Alkyl radical, C_1-6Alkoxy and C_3-6A cycloalkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

preferably, L₁Is selected from
(e.g. using
)、
The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl and cyano-substituted C_1-6An alkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

more preferably, L₁Is selected from
(e.g. using
)、
The above groups are linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂Connecting; r₃、R₄、R₅And R₆Each independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid group, cyano, C_1-6Alkyl, halo C_1-6Alkyl and cyano-substituted C_1-6An alkyl group; y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and is

Most preferably, L₁Is selected from
It is linked to AA via one of the two positions marked 1 or 2 and to L via the other position₂Linking, preferably, the above group is linked to AA via the 1-labeled position and to L via the 2-labeled position₂And (4) connecting.
A compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereofA salt, solvate, hydrate, isomer of (I), or any crystal form or racemate thereof, wherein L is₂Is absent or is
Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁Selected from O and-NH-, Z₂Absent or selected from O and-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

preferably, L₂Is absent or is
Which is marked by one of the two positions 1 or 2 and L₁Is connected to L via another position₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃Connection of, Z₁is-NH-, Z₂Absent or-NH-, m1 and m2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and is

More preferably, L₂Is absent or selected from

Which is marked by one of the two positions 1 or 2 and L₁Is connected to and passes through another location and L₃Is connected, preferably by 1-marked position with L₁Is connected and passes the position marked by 2 and L₃And (4) connecting.
Claim 1-7 or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, wherein L₃Is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups: c_3-8Cycloalkylene, phenylene, naphthylene, 5-12 membered heteroarylene, 3-8 membered heterocyclene, 6-12 membered bridged heterocyclene, 6-12 membered spiroheterocyclene, and 6-12 membered fused heterocyclene, when the above groups contain a nitrogen atom, the nitrogen atom is optionally quaternized; r₇Each occurrence is independently selected from H (hydrogen), D (deuterium), halogen, carboxyl, sulfonic acid, C_1-4Alkyl, -CF₃、C_1-4Alkoxy radical, C_2-6Alkenyl and C_2-6An alkynyl group;

preferably, L₃Is absent or selected from-NR₇-and optionally substituted with one or more R₇Substituted of the following groups:

counterion, R₇Each independently at each occurrence is selected from H (hydrogen), D (deuterium), methyl and-CF₃One of the two positions of the above radicals marked by 1 or 2 is linked to L₂Is connected to L via another position₄Linking, preferably, the above group via the 1-labelled position with L₂Is connected and passes the position marked by 2 and L₄Connecting;

more preferably, L₃Absent or selected from:

e is a counterion, R₇Each independently at each occurrence is selected from H (hydrogen), D (deuterium), methyl and-CF₃One of the two positions of the above radicals marked by 1 or 2 is linked to L₂Is connected to L via another position₄Linking, preferably, the above group via the 1-labelled position with L₂Is connected and passes the position marked by 2 and L₄Connecting; and is

Most preferably, L₃Is absent or selected from
E is a counterion, preferably a halide anion, more preferably chloride, bromide or iodide, in one of the two positions of which the above radical is marked by 1 or 2, with L₂Is connected to L via another position₄Linking, preferably, the above group via the 1-labelled position with L₂Is connected and passes the position marked by 2 and L₄And (4) connecting.
The compound of any one of claims 1-8, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein, L₄Is absent or selected from C_1-6Alkylene, -NR₉-C_1-6Alkylene-, -C_1-6alkylene-NR₉-and-NR₉-；R₉Independently selected from H (hydrogen), D (deuterium), C_1-6Alkyl and C_3-6A cycloalkyl group; said C is_1-6Alkyl or C_1- ₆Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium), or halogen; and is

Preferably, L₄Is absent or selected from-CH₂CH₂-、-NHCH₂-、-CH₂NH-and-NH-.
The compound of any one of claims 1-9, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein L₅Is selected from

And is

Preferably, L₅Is selected from
The compound of any one of claims 1-10, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein the compound is selected from the group consisting of:

and

preferably, the compound is selected from:
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 after gamma sulfydryl is removed from the target or a group obtained after gamma sulfydryl is removed after a disulfide bond in the target is reduced;

L₅' is selected from

Which is marked by one of the two positions 1 or 2 and L₄Attached to the thiol group of the target via another position; preferably, it is marked by 1 position and L₄Connecting and connecting with the sulfhydryl group of the target through the position marked by 2; r is 0, 1, 2, 3, 4 or 5; γ is an integer from 1 to 10, e.g., 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 is

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

L₅' is selected from

Which is marked by one of the two positions 1 or 2 and L₄Attached to the thiol group of the target via another position; preferably, it is marked by 1 position and L₄Connecting and connecting with the sulfhydryl group of the target through the position marked by 2; and is

Preferably, L₅' is selected from
Which is marked by one of the two positions 1 or 2 and L₄Attached to the thiol group of the target via another position; preferably, it is marked by 1 position and L₄Attached to the thiol group of the target via the 2-labeled position.
The conjugate of claim 12 or 13, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, wherein said target is selected from the group consisting of a small molecule ligand, a protein, a polypeptide, and a non-protein agent (such as a sugar, RNA, or DNA);

preferably, the target is a substance that targets: epidermal growth factor, Trop-2, CD37, Her2, CD70, EGFRvIII, mesothelin, folate receptor 1, CEA CAM5, mucins (such as 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 cotransporter 2B, GPNMB, trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66 33, CD33, PD-L33, DR 33, E33, FcFcRH 72P, MPF, Napi3 33, Sema 5b, PSCAhlgBR, MSG783, STEP 33, TrpM 33, CD33, CRIPT 33, CRIPTC 33, CD 6572, CD33, CD, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, sealprotein 18.2, BMPR1B, Tyro7, C-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, GREIL 10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEL, STAT 36 1, TGTGR 1, CTSS, PGF, VEGFA, VEGFC 1, TGFACD 1QA, TGFAC QA, TGFAN QA, TGFAAK QA, TGFAN QA, TGFALSF QA, TGADTFAS QA, TFAS QA, TFD QA, TFPI QA, TFAK QA, TFS QA, TFAK QA, TFS QA;

preferably, the target is selected from small molecule ligands such as folic acid derivatives, glutamic urea derivatives, somatostatin derivatives, arylsulfonamide derivatives (e.g. carbonic anhydrase IX inhibitors), polyenes linking two aliphatic indoles, cyanine dyes and IR-783 or derivatives thereof; an antibody, e.g., a monoclonal antibody or 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 (e.g., scFv), nonhuman antibody, humanized antibody, chimeric antibody, fully human antibody, preanti, bispecific antibody, or multispecific antibody; RGD peptides that recognize cell surface integrin receptors; growth factors that recognize cell surface growth factor receptors such as EGF, PDGF or VEGF; peptides capable of recognizing functional cell surface plasminogen activator, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptor; CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACTD 2 ligand, DR5 ligand and Trop-2 ligand; and is

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

wherein a is a group obtained by removing γ thiol groups from Sacituzumab, trastuzumab or pertuzumab, or a group obtained by removing γ thiol groups from Sacituzumab, trastuzumab or pertuzumab after reduction of disulfide bonds and removal of 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:

wherein a1 is a group obtained by removing γ thiol groups from Sacituzumab or a group obtained by removing γ thiol groups from Sacituzumab after reduction of the disulfide bond, 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) conjugates of any one of claims 12-15, or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof, and optionally one or more pharmaceutically acceptable carriers, and optionally further comprising one or more other anticancer drugs, such as a chemotherapeutic agent and/or an antibody, wherein the pharmaceutical composition has an average DAR value of 1 to 10 integer or fractional, e.g., 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, 6.6, 6.7, 6.7.7, 6.7, 6.7.7, 6, 6.7.7, 6.7, 7.7, 7, 7.8, 7.6, 6, 6.7, 7.7, 7, 7.8, 7, 7.8, 6, 6.0, 6, 8.5, 9.0, 9.5 or 10.0, preferably an integer or decimal number from 4 to 8, for example 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.
Use of a conjugate according to any one of claims 12 to 15, or a pharmaceutically acceptable salt, solvate, hydrate, isomer thereof, or any crystalline form or racemate thereof, for the manufacture of a medicament for the prevention or treatment of a cancer disease;

preferably, the cancer disease is selected from breast cancer (e.g., triple negative breast cancer), gastric cancer, esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, salivary gland cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell cancer, bladder cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, renal cancer, colon cancer, solid tumors, non-hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, and thyroid cancer;

more preferably, the cancer disease is selected from breast cancer (e.g., triple negative breast cancer) and gastric cancer;

further preferably, the cancer disease is selected from gastric cancer.
A process for preparing a compound of formula (Γ) according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof:

wherein:

R_d' is R_dOr PG₁，

PG₁And PG₂Each independently an amino protecting group, preferably Boc or MMT,

the remaining groups being as defined in any one of claims 1 to 11,

the method comprises the following steps:

the method comprises the following steps: reacting the compounds (Ia) -d with the compounds (Ia) -e in the presence of a metal catalyst to give the compounds of the formula (Ia), and

step two: reacting a compound of formula (Ia) under conditions to remove the amino protecting group to give a compound of formula (I') or a pharmaceutically acceptable salt thereof;

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

preferably, said step one of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

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

preferably, said step one of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof;

preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably conditions in the presence of trifluoroacetic acid;

preferably, said second step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, said step two of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
A process for preparing a compound of formula (Γ) according to claim 11 or a pharmaceutically acceptable salt, solvate, hydrate, isomer, or any crystalline form or racemate thereof:

wherein:

R_dis "R_dOr a hydrogen atom,

R_d' is R_dOr PG₁，

PG₁And PG₂Each independently an amino protecting group, preferably Boc or MMT,

the remaining groups being as defined in any one of claims 1 to 11,

the method comprises the following steps:

the method comprises the following steps: reacting the compound (Ia) -a in the presence of a base to obtain the compound (Ia) -b, with the proviso that when R is_d"and R_d' are both R_dIn the process, the reaction in the step one is not needed;

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

step three: reacting the compounds (Ia) -d with the compounds (Ia) -e in the presence of a metal catalyst to give the compounds of the formula (Ia), and

step four: reacting a compound of formula (Ia) under conditions to remove the amino protecting group to give a compound of formula (I') or a pharmaceutically acceptable salt thereof;

preferably, the base of step one of the process for the preparation of the compound of formula (I') is selected from organic and inorganic bases, such as triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, preferably triethylamine, diisopropylethylamine;

preferably, said step one of the process for the preparation of the compound of formula (I') is carried out at a temperature of from 0 ℃ to 40 ℃, preferably from 10 ℃ to 30 ℃;

preferably, said step one of the process for the preparation of the compound of formula (I') is carried out in an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidinone, saturated hydrocarbons (e.g. cyclohexane or hexane), halogenated hydrocarbons (e.g. dichloromethane, chloroform or1, 2-dichloroethane), nitriles (e.g. acetonitrile) and any combination thereof, preferably dichloromethane;

preferably, the condensation reagent of step two of the process for the preparation of the compound of formula (I') is selected from carbonyldiimidazole, triphosgene, preferably triphosgene;

preferably, the base of step two of the process for the preparation of the compound of formula (I') is selected from organic and inorganic bases, such as triethylamine, diisopropylethylamine, p-dimethylaminopyridine, pyridine, 2, 4, 6-trimethylpyridine and the like, preferably p-dimethylaminopyridine;

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

preferably, in the process for preparing the compound of formula (I'), in step two, the molar ratio of the compounds (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 two of the process for the preparation of the compound of formula (Γ) is carried out in an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidinone, saturated hydrocarbons (e.g. cyclohexane or hexane), halogenated hydrocarbons (e.g. dichloromethane, chloroform or1, 2-dichloroethane), nitriles (e.g. acetonitrile) and any combination thereof, preferably dichloromethane;

preferably, the metal catalyst in step three of the process for preparing the compound of formula (I') is a monovalent copper salt, such as cuprous halide (e.g., cuprous chloride, cuprous bromide, or cuprous iodide); or a divalent copper salt/reducing agent, such as copper sulfate/sodium ascorbate;

preferably, the process for the preparation of the compound of formula (I') said step three is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

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

preferably, the process for the preparation of the compound of formula (I') said step three is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof;

preferably, the conditions for removing the amino protecting group in step four of the process for preparing the compound of formula (I') are acidic conditions, more preferably in the presence of trifluoroacetic acid;

preferably, said step four of the process for the preparation of the compound of formula (Γ) is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, said step four of the process for the preparation of the compound of formula (Γ) is carried out in water and/or an organic solvent; the organic solvent is selected from the group consisting of N, N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
A process for preparing 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:

wherein:

R_d' is R_dOr PG₁，

R₁₀Is absent or is C_1-6Alkylene radical of the formula C_1-6Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium) or halogen,

PG₁and PG₂Each independently an amino protecting group, preferably Boc or MMT,

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

the remaining groups being as defined in any one of claims 1 to 11,

the method comprises the following steps:

the method comprises the following steps: reacting compound (Ib) -a with compound (Ib) -b in the presence of a base, optionally in the presence of a condensing agent, to give a compound of formula (Ib), and

step two: reacting a compound of formula (Ib) under conditions to remove the amino protecting group to give a compound of 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 triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from NaH, NaOH and Na₂CO₃And K₂CO₃；

Preferably, the base is DIPEA;

preferably, the condensation reagent is selected from the group consisting of phosgene, phosgene solids, phosgene, and mixtures thereof,

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

preferably, said first step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, in said step one of the process for the preparation of the compound of formula (I'), the molar ratio of compound (Ib) -a to base is from 1: 0.5 to 4, preferably from 1: 0.8 to 3, more preferably from 1: 1 to 2;

preferably, said step one of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof;

preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably conditions in the presence of trifluoroacetic acid;

preferably, said second step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, said second step of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
A process for preparing 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:

wherein:

R_d' optionally R_dOr PG₁，

R₁₀Is absent or is C_1-6Alkylene radical of the formula C_1-6Alkylene is optionally substituted with one or more H (hydrogen), D (deuterium) or halogen,

PG₁and PG₂Each independently an amino protecting group, preferably Boc or MMT,

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

the remaining groups being as defined in any one of claims 1 to 11,

the method comprises the following steps:

the method comprises the following steps: reacting the compounds (Ia) to (d) in the presence of a reducing agent to obtain compounds (Ib) to a;

step two: reacting compound (Ib) -a with compound (Ib) -b in the presence of a base, optionally in the presence of a condensing agent, to give a compound of formula (Ib), and

step three: reacting a compound of formula (Ib) under conditions to remove the amino protecting group to give a compound of formula (I ") or a pharmaceutically acceptable salt thereof;

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

preferably, the base is an organic base or an inorganic base; the organic base is selected from triethylamine, DIPEA, pyridine, NMM and DMAP; the inorganic base is selected from NaH, NaOH and Na₂CO₃And K₂CO₃；

Preferably, the base is DIPEA;

preferably, the condensation reagent is selected from the group consisting of phosgene, phosgene solids, phosgene, and mixtures thereof,

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

preferably, said step one of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 50 ℃, preferably 10 ℃ to 30 ℃;

preferably, said first step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, said step one of the process for the preparation of the compound of formula (I ") is carried out in an organic solvent; the organic solvent is selected from ethers (such as tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), alcohols (such as methanol, ethanol, isopropanol or tert-butanol), ethyl acetate and any combination thereof, preferably tetrahydrofuran;

preferably, in said step one of the process for the preparation of the compound of formula (I'), the molar ratio of compound (Ib) -a to base is from 1: 0.5 to 4, preferably from 1: 0.8 to 3, more preferably from 1: 1 to 2;

preferably, said step one of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof;

preferably, the conditions for removing the amino protecting group are acidic conditions, more preferably conditions in the presence of trifluoroacetic acid;

preferably, said second step of the process for the preparation of the compound of formula (I') is carried out at a temperature of-10 ℃ to 60 ℃, preferably 0 ℃ to 35 ℃, more preferably 0 ℃ to 25 ℃;

preferably, said second step of the process for the preparation of the compound of 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, dimethylsulfoxide, N-methylpyrrolidone, saturated hydrocarbons (e.g., cyclohexane or hexane), halogenated hydrocarbons (e.g., dichloromethane, chloroform or1, 2-dichloroethane), ethers (e.g., tetrahydrofuran, diethyl ether, dioxane or1, 2-dimethoxyethane), nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol, isopropanol or tert-butanol), and any combination thereof.
A process for preparing a conjugate of formula (II) according to any one of claims 12 to 15 or a pharmaceutically acceptable salt, solvate, hydrate, isomer or any crystalline form or racemate thereof,

the method comprising coupling a compound of formula (I) to a target,

wherein each group is as defined in any one of claims 1 to 15,

preferably, the method comprises mixing a compound of formula (I) with a targeting agent; optionally reducing the disulfide bond in the targets prior to mixing;

preferably, the disulfide bond reduction is performed 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 process is carried out at a temperature of from 0 ℃ to 50 ℃, preferably from 15 ℃ to 25 ℃;

preferably, the process is carried out in water and/or an organic solvent; the organic solvent is selected from N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide and N-methylpyrrolidone; and is

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