CN113121639B

CN113121639B - Aureostatin analogue and conjugate thereof, preparation method and application thereof

Info

Publication number: CN113121639B
Application number: CN202011603851.7A
Authority: CN
Inventors: 许建烟; 章瑛; 贺峰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2019-12-30
Filing date: 2020-12-30
Publication date: 2024-05-14
Anticipated expiration: 2040-12-30
Also published as: CN113121639A

Abstract

The present disclosure relates to auristatin analogs and conjugates thereof, methods of making the same, and uses thereof. Specifically, the present disclosure provides an MMAE analog having a structure represented by formula (D), a conjugate, a preparation method thereof, a pharmaceutical composition containing the same, and use thereof in preparing a medicament for treating cancer through receptor modulation. Wherein each substituent in the general formula (D) is as defined in the specification.

Description

Aureostatin analogue and conjugate thereof, preparation method and application thereof

Technical Field

The present disclosure relates to a class of ligand-drug conjugates of drug Australian (MMAE) analogs. In particular, the present disclosure relates to an antibody-drug conjugate comprising a novel structural toxin, a method for its preparation, a pharmaceutical composition comprising the conjugate and the use of the conjugate or pharmaceutical composition.

Background

The antibody drug conjugate (antibody drug conjugate, ADC) connects monoclonal antibody or antibody fragment with cytotoxin with biological activity through stable chemical linker compound, makes full use of the specificity of antibody to normal cell and tumor cell surface antigen combination and cytotoxin high efficiency, and simultaneously avoids the defects of low curative effect and overlarge toxic and side effect of the former. This means that the antibody drug conjugate can bind tumor cells precisely and reduce the effect on normal cells compared with conventional chemotherapeutics (Mullard A,(2013)Nature Reviews Drug Discovery,12:329–332;DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814).

The first antibody drug conjugate, mylotarg (gemtuzumab ozogamicin (gemtuzumab ozogamicin), of the wheatstone pharmaceutical company, limited) was approved by the FDA in the united states for the treatment of acute myeloid leukemia in 2000 (Drugs of the Future(2000)25(7):686;US4970198;US 5079233;US 5585089;US 5606040;US 5693762;US 5739116;US 5767285;US 5773001).

Month 8 2011, adcetris (brentuximab vedotin, seattle genetic company) passed the U.S. FDA rapid review channel for the treatment of hodgkin's lymphoma and recurrent anaplastic large cell lymphoma (nat. Biotechnol (2003) 21 (7): 778-784; WO2004010957; WO2005001038; US7090843a; US7659241; WO 2008025020).Is a novel targeted ADC drug, and can lead the drug to directly act on target CD30 on lymphoma cells and then generate endocytosis so as to induce apoptosis of tumor cells.

Mylotarg and Adcetris are targeted therapies against hematological tumors, which are relatively simple in tissue structure compared to solid tumors. Month 2, 2013, kadcyla (ado-trastuzumab emtansine, T-DM 1) received U.S. FDA approval for the treatment of advanced or metastatic breast cancer subjects positive for HER2 and resistant to trastuzumab (Tratuzumab, trade name: herceptin) and paclitaxel (WO 2005037992; US 8088387). Kadcyla is the first ADC drug approved by the FDA in the united states to treat solid tumors.

There is still a need to further develop ADC drugs with better therapeutic effects.

Disclosure of Invention

The present disclosure provides a novel MMAE analog which is a compound represented by general formula (D):

or a tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

r ¹-R⁶ is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;

r ⁷ is selected from the group consisting of hydrogen, alkyl, alkoxy, and cycloalkyl;

Any two of R ⁸-R¹¹ form cycloalkyl, and the remaining two groups are either selected from hydrogen, alkyl, and cycloalkyl;

R ¹² is selected from the group consisting of a hydrogen atom and an alkyl group;

R ¹³-R¹⁵ is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, and halogen;

R ¹⁶ is selected from aryl or heteroaryl, which is optionally further substituted with a substituent selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl.

R ¹⁶ is selected from aryl or heteroaryl, which is optionally further substituted with one or more substituents selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl.

In some embodiments of the present disclosure, the compound of formula (D) as described previously is a compound of formula (D ₁):

Or a tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

R ⁹ and R ¹⁰ form cycloalkyl;

R ²-R⁸,R¹¹-R¹⁶ is as defined in formula (D).

In some embodiments of the present disclosure, the compound of formula (D) as set forth in any one of the preceding claims is:

Another aspect of the present disclosure relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate comprises a structure represented by formula (-D):

Or a tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, wherein:

R ²-R⁶ is selected from the group consisting of hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, and cycloalkyl;

R ¹⁶ is selected from aryl or heteroaryl, said aryl or heteroaryl optionally being further substituted with a substituent selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl;

wavy lines represent hydrogen atoms, or covalently attached to linker units or to antibodies that bind to antigens expressed by the target cells.

R ¹⁶ is selected from aryl or heteroaryl, said aryl or heteroaryl optionally being further substituted with one or more substituents selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl;

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof, as claimed in any one of the preceding claims, wherein the ligand-drug conjugate comprises a structure represented by formula (-D ₁):

R ⁹ and R ¹⁰ form cycloalkyl;

wavy line, R ²-R⁸,R¹¹-R¹⁶ is as defined in the general formula (-D).

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof, as set forth in any one of the preceding claims, wherein the ligand-drug conjugate comprises a structure of the formula:

Or a tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, wherein: wavy lines represent hydrogen atoms, or covalently attached to linker units or to antibodies that bind to antigens expressed by the target cells.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims is a ligand-drug conjugate or pharmaceutically acceptable salt thereof of the general formula (Pc-L-D):

Wherein:

R ²-R¹⁶ is as defined in formula (D);

n is 1 to 10, and may be an integer or a decimal number;

pc is a ligand; l is a linker unit.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims is a ligand-drug conjugate or pharmaceutically acceptable salt thereof of the general formula (Pc-L-D1):

Wherein:

R ²-R¹⁶ is as defined in formula (-D);

pc, L, n are as defined in the general formula (Pc-L-D).

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims is a ligand-drug conjugate or pharmaceutically acceptable salt thereof as shown below:

pc, L, n are as defined in the general formula (Pc-L-D).

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein n can be an integer or fraction between 1 and 10, and n can be an average of 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. n is 1 to 8, and may be an integer or a decimal number; preferably 2 to 8, and may be an integer or a decimal.

In some embodiments of the present disclosure, the ligand-drug conjugate of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the linker unit-L-is-Y-L ¹-L²-L³-L⁴,

Y is a stretching unit selected fromOr a chemical bond, X ₁ is selected from a hydrogen atom, an alkyl group, an alkoxy group, an aryl group, or a halogen, X ₂ is selected from an alkylene group, said alkylene group optionally being further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

l ¹ is a stretch unit selected from the group consisting of- (succinimid-3-yl-N) -W-C (O) -, -CH ₂-C(O)-NR¹⁷ -W-C (O) -, and-C (O) -W-C (O) -, wherein W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-cycloalkyl, and linear heteroalkyl of 1 to 8 atoms containing 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein each of said C _1-8 alkyl, cycloalkyl, and linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteroalkyl, alkoxy, and cycloalkyl;

L ² is selected from -NR¹⁸(CH₂CH₂O)_pCH₂CH₂C(O)-、-NR¹⁸(CH₂CH₂O)_pCH₂C(O)-、 -S(CH₂)_pC(O)- and a bond, wherein p is an integer from 1 to 20; preferably a chemical bond;

L ³ is a peptide residue consisting of 2 to 7 amino acids, preferably selected from valine, citrulline, methylvaline; wherein the amino acid is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

r ¹⁷ and R ¹⁸ are the same or different and are each independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

L ⁴ is an extension unit, preferably PAB.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof, wherein Y is

In some embodiments of the present disclosure, the ligand-drug conjugate of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L ¹ is selected from- (succinimidyl-3-yl-N) - (CH ₂)_s -C (O) -, wherein s is an integer from 2 to 8, preferably

In some embodiments of the present disclosure, the ligand-drug conjugate of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein L ³ is a dipeptide amino acid unit, preferably from valine-citrulline.

In some embodiments of the present disclosure, the ligand-drug conjugate of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the linker unit-L-is selected from the group consisting of:

Wherein the a end is connected with a ligand and the b end is connected with a drug.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof as described in any one of the preceding claims, selected from the following structural formulas:

Wherein:

n is 1 to 10, and may be an integer or a decimal number;

pc is a ligand.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein the Pc is an antibody or antigen-binding fragment thereof, wherein the antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, and a fully human antibody.

In some embodiments of the disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, and an anti-Mesothelin antibody, or antigen binding fragment thereof.

In some embodiments of the present disclosure, the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is selected from Trastuzumab、Pertuzumab、 Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、 Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96 and Glematumamab or antigen-binding fragment thereof.

Another aspect of the present disclosure relates to a method for preparing a compound represented by general formula (D) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

deprotection of the general formula (DA) to give a compound of the general formula (D),

Wherein: r ²-R¹⁶ is as defined in formula (D).

Another aspect of the present disclosure relates to a compound as follows:

Or a tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, may be used as an intermediate in the preparation of the ligand-drug conjugates of the present disclosure.

Another aspect of the present disclosure relates to a method of preparing compound 2, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

the compound 1 and the compound 2a undergo a condensation reaction to obtain a compound 2.

Another aspect of the present disclosure relates to a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in general formula (Pc-L-D), comprising the steps of:

After Pc is reduced, the Pc is coupled with a compound to obtain a compound shown in a general formula (ADC-1);

Wherein Pc, n is defined as in the general formula (Pc-L-D).

Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate as described in any one of the preceding formulas (Pc-L-D), or a pharmaceutically acceptable salt thereof, or a compound as described in any one of the preceding formulas (D), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect of the present disclosure, a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the step of linking a compound represented by general formula (D), compound 2, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, of the present disclosure to a ligand, preferably via a linker, preferably the ligand is a monoclonal antibody.

In another aspect of the present disclosure, further relates to a ligand-drug conjugate or compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use as a medicament.

In another aspect of the present disclosure, further relates to the use of a ligand-drug conjugate or compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating or preventing a tumor; preferably wherein the tumor is selected from the group consisting of cancers associated with HER2、HER3、B7H3、c-Met、HER4、 CD20、CD22、CD30、CD33、CD44、CD56、CD70、CD73、CD105、CEA、A33、 Cripto、EphA2、G250、MUCl、Lewis Y、VEGFR、GPNMB、Integrin、PSMA、 Tenascin-C、SLC44A4、Mesothelin and EGFR expression.

In another aspect of the present disclosure, further relates to the use of a ligand-drug conjugate or compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for the manufacture of a medicament for the treatment and/or prophylaxis of a cancer, preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, prostate cancer, and lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma or relapsed anaplastic large cell lymphoma).

In another aspect of the present disclosure, further relates to a method for treating and/or preventing a tumor, the method comprising administering to a subject in need thereof a therapeutically effective dose or a prophylactically effective dose of a ligand-drug conjugate or compound of any one of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same; preferably wherein the tumor is a cancer associated with HER2, HER3 or EGFR expression.

In another aspect of the present disclosure, further relates to a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective dose or a prophylactically effective dose of a ligand-drug conjugate or compound of any one of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same; the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma).

The active compound (e.g., a compound according to the present disclosure, or a pharmaceutically acceptable salt thereof, a ligand-drug conjugate, or a pharmaceutically acceptable salt thereof) may be formulated in a form suitable for administration by any suitable route, preferably in a unit dose manner, or in a manner such that the subject is able to administer itself in a single dose. The unit dosage form of the present disclosure may be in the form of a tablet, capsule, cachet, bottled liquid medicine, powder, granule, lozenge, suppository, reconstituted powder or liquid formulation.

The dosage of the active compound or composition to be administered in the disclosed methods of treatment will generally vary with the severity of the disease, the weight of the subject, and the efficacy of the active compound. However, as a general guideline, a suitable unit dose may be 0.1mg to 1000mg.

The pharmaceutical compositions of the present disclosure may contain, in addition to the active compound, one or more excipients selected from the following ingredients: fillers, diluents, binders, wetting agents, disintegrants or excipients, and the like. Depending on the method of administration, the compositions may contain from 0.1 to 99% by weight of the active compound.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral administration, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, such compositions may contain binders, fillers, lubricants, disintegrants or pharmaceutically acceptable wetting agents, etc., and such compositions may also contain one or more ingredients selected from the group consisting of: sweeteners, flavoring agents, coloring agents and preservatives to provide a pleasing and palatable pharmaceutical preparation.

The aqueous suspension contains the active substance and excipients suitable for the preparation of aqueous suspensions for mixing. The aqueous suspension may also contain one or more preservative examples, one or more coloring agents, one or more flavoring agents and one or more sweetening agents.

The oil suspensions may be formulated by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickener. The above-described sweeteners and flavoring agents may be added to provide a palatable preparation.

The pharmaceutical compositions may also be provided as dispersible powders and granules for the preparation of an aqueous suspension by the addition of one or more of water-miscible dispersing agents, wetting agents, suspending agents or preserving agents. Other excipients, for example sweetening, flavoring and coloring agents, may also be added. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the present disclosure may also be in the form of an oil-in-water emulsion.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable vehicles or solvents that may be used are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding it to a mixture of water and glycerol. The injection or microemulsion may be injected into the blood stream of the subject by local bolus injection. Or preferably the solution and microemulsion are administered in a manner that maintains a constant circulating concentration of the compound of the present disclosure. To maintain this constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump of the DELTEC CADD-PLUS. TM.5400 type.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspensions may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, nontoxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

The active compounds of the present disclosure may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As is well known to those skilled in the art, the amount of drug administered depends on a variety of factors, including, but not limited to, the following: the activity of the specific compound used, the age of the subject, the weight of the subject, the health condition of the subject, the behavior of the subject, the diet of the subject, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc.; in addition, the optimal mode of treatment, such as the mode of treatment, the daily amount of compound (I) of formula (I) or the type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

The present disclosure relates to conjugates for targeting different cancer cells, improving the ability of infectious organisms and/or for treating autoimmune diseases, the conjugates comprising a targeting (binding) moiety and a therapeutic moiety belonging to the drug. The antibody targeting moiety is linked to the therapeutic moiety of the compound via an intracellular cleavable linkage that increases the therapeutic efficacy.

For many years, one goal of scientists in the field of specific targeted drug therapies has been to use monoclonal antibodies (mabs) for the specific delivery of toxic agents to human cancers. Conjugates of tumor-associated MAbs with appropriate toxic agents have been developed, but are of half-way in the treatment of cancer, with little use in other diseases such as infectious and autoimmune diseases. Toxic agents are the most common chemotherapeutic agents. There is a further need to develop more effective antibody conjugates with intracellular cleavable linkers for the treatment of cancer, pathogens and other diseases.

The patent of antibody toxin conjugates currently disclosed is for example WO2016127790.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terminology will be used in accordance with the definitions set out below.

When trade names are used in this disclosure, it is intended to include formulations of the trade name product, non-patent drugs and active drug moieties of the trade name product.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

The term "ligand" is a macromolecular compound that is capable of recognizing and binding to an antigen or receptor associated with a target cell. The ligand functions to present the drug to the target cell population to which the ligand binds, including but not limited to, a protein hormone, lectin, growth factor, antibody or other molecule capable of binding to the cell. In embodiments of the present disclosure, the ligand is denoted Pc, and the ligand may form a bond with the linking unit through a heteroatom on the ligand, preferably an antibody or antigen binding fragment thereof, selected from chimeric, humanized, fully human or murine; monoclonal antibodies are preferred.

The term "drug" refers to a cytotoxic drug, indicated as D, which has chemical molecules within tumor cells that are strong to disrupt their normal growth. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they can also cause apoptosis in normal cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、 Bi²¹²、P³² and radioactive isotopes of Lu), chemotherapeutic agents, antibiotics and nucleolytic enzymes.

The term "linker unit", "linker" or linker fragment "refers to a chemical structural fragment or bond that is linked at one end to a ligand (e.g., an antibody or antigen binding fragment thereof) and at the other end to a drug, or can be linked to other linkers before being linked to a drug.

The joint may comprise one or more joint members. Exemplary linker members include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4- (2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). The linker may comprise a stretch unit, a spacer unit, an amino acid unit and an extension unit, and may be synthesized by methods known in the art, such as described in US 2005-023849 A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al CANCER RESEARCH 52:127-131 (1992); U.S. Pat. No.5,208,020) may be used.

The term "stretcher unit" refers to a fragment of a chemical structure that is covalently linked at one end to a ligand through a carbon atom and at the other end to a cytotoxic drug through a sulfur atom. In the present disclosure, the stretching unit is defined as general formula (Y). The stretcher unit is attached by reaction with amino groups in the antibody by means of reductive amination, preferably epsilon-amino groups on the N-terminal and/or lysine residues of the antibody. A further tension unit of the present disclosure includes structure MC.

The term "spacer" is a bifunctional fragment of a chemical structure that can be used to couple a linker unit to a cytotoxic drug to ultimately form a ligand-cytotoxic drug conjugate in such a way that the cytotoxic drug can be selectively attached to the linker unit.

The term "amino acid unit" refers to an amino acid that, if an extension unit is present, can attach the carbonyl group in the following structural formula Y _R to the extension unit, and if no extension unit is present, can attach Y _R directly to a cytotoxic drug, in embodiments of the present disclosure, the amino acid unit is represented as-K _k -:

-K _k -is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, -K-units each independently have the following structural formula K _a or K _b, K being an integer between 0 and 10:

Wherein:

R ₂₃ is-H or methyl;

R ₂₄ is H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl 、-CH₂OH、 -CH(OH)CH₃、-CH₂CH₂SCH₃、-CH₂CONH₂、-CH₂COOH、-CH₂CH₂CONH₂、 -CH₂CH₂COOH、-(CH₂)₃NHC(＝NH)NH₂、-(CH₂)₃NH₂、-(CH₂)₃NHCOCH₃、 -(CH₂)₃NHCHO、-(CH₂)₄NHC(＝NH)NH₂、-(CH₂)₄NH₂、-(CH₂)₄NHCOCH₃、 -(CH₂)₄NHCHO、-(CH₂)₃NHCONH₂、-(CH₂)₄NHCONH₂、-CH₂CH₂CH(OH)CH₂NH₂、2- pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

R ₂₅ is-aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-.

In one embodiment, -K _k -is a dipeptide, preferably-valine-citrulline-, -phenylalanine-lysine-or-N-methylvaline-citrulline-, further preferably-valine-citrulline-.

In another embodiment, -K _k -is a dipeptide, preferably

The term "amino acid" refers to an organic compound that contains both an amino group and a carboxyl group in the molecular structure, and both amino and carboxyl groups are directly attached to the-CH-structure. The general formula is H ₂ NCHRCOOH. The amino groups are classified into α, β, γ, δ, and ε … … -amino acids according to the positions of the carbon atoms in the carboxylic acid to which they are attached. In the biological world, amino acids constituting natural proteins have their specific structural characteristics, i.e., their amino groups are directly linked to α -carbon atoms, i.e., α -amino acids including Glycine (Glycine), alanine (Alanine), valine (Valine), leucine (Leucine), isoleucine (Isoleucine), phenylalanine (PHENYLALANINE), tryptophan (Tryptophan), tyrosine (Tyrosine), aspartic acid (ASPARTIC ACID), histidine (HISTIDINE), asparagine (ASPARAGINE), glutamic acid (Glutamic acid), lysine (Lysine), glutamine (Glutamine), methionine (Methionine), arginine (Arginine), serine (Serine), threonine (Threonine), cysteine (Cysteine), proline (Proline), and the like.

In one embodiment of the present disclosure, the amino acid is selected from the group consisting of

The term "extension unit" refers to a chemical structure that can couple an amino acid unit to a cytotoxic drug in the presence of the amino acid unit, or through coupling to a carbonyl group on Y _R in the absence of the amino acid unit. In embodiments of the present disclosure, the extension unit is represented by-Q _q -, Q is selected from 0,1,2.

In the present disclosure, the extension unit is PAB, the structure is as 4-iminobenzylcarbamoyl fragment, the structure is as shown in formula (VI), and is connected to D,

Abbreviations (abbreviations)

Joint assemblies include, but are not limited to:

mc=6-maleimidocaproyl, structure as follows:

Val-Cit or "vc" =valine-citrulline (an exemplary dipeptide in a protease cleavable linker),

Citrulline = 2-amino-5-ureidovaleric acid,

PAB = p-aminobenzyloxycarbonyl ("example of a self-sacrifice" linker assembly),

Me-Val-Cit = N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent cleavage by cathepsin B),

MC (PEG) 6-oh=maleimidocaproyl-polyethylene glycol (which may be attached to antibody cysteine),

SPP = N-succinimidyl 4- (2-pyridylthio) pentanoate,

Spdp=n-succinimidyl 3- (2-pyridyldithio) propionate,

Smcc=succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate,

It=iminothiolane.

The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug through a linking unit. In the present disclosure "ligand-drug conjugate" is preferably an antibody-drug conjugate (antibody drug conjugate, ADC), meaning that a monoclonal antibody or antibody fragment is linked to a biologically active toxin drug via a linking unit.

The term "drug loading" or "average drug load" refers to the average amount of cytotoxic drug loaded per ligand in the molecule of formula (I), which may also be expressed as a ratio of the amount of drug to the amount of antibody, and the drug loading may range from 0 to 12, preferably 1 to 10, cytotoxic drugs (D) per ligand (Pc). In embodiments of the present disclosure, the Drug loading is expressed as n, which may also be referred to as a DAR (Drug-agent Ratio) value, where n may be a non-zero integer or fraction of 0 to 12, preferably an integer or fraction between 1-10; more preferably an integer or fraction of 2 to 8; most preferably an integer or fraction of 3 to 8. An exemplary average value may be 1,2,3,4,5,6,7,8,9, 10. The number of drug molecules per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV/visible light spectrometry, CE-SDS, RP-HPLC, mass spectrometry, ELISA assays and HPLC. In embodiments of the present disclosure, the drug loading is expressed as y, which may be, for example, an average of 1,2,3,4,5,6,7,8,9, 10, ranging from 0 to 12, preferably 1 to 10, more preferably 1 to 8, or 2 to 7, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5.

The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

(1) The molar ratio of the connecting reagent to the monoclonal antibody is controlled,

(2) The reaction time and the temperature are controlled,

(3) Different reagents are selected.

The amino acid three-letter codes and one-letter codes in antibodies used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

The term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed by two heavy and two light chains joined by interchain disulfide bonds. Immunoglobulins can be classified into five classes, or isotypes called immunoglobulins, igM, igD, igG, igA and IgE, according to the amino acid composition and arrangement of the constant regions of the heavy chains of the immunoglobulins, the corresponding heavy chains being the mu, delta, gamma, alpha and epsilon chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The antibodies described in the present disclosure are preferably specific antibodies to cell surface antigens on target cells, non-limiting examples being the following antibodies: one or more of an anti-HER 2 (ErbB 2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-C-Met antibody, an anti-HER 3 (ErbB 3) antibody, an anti-HER 4 (ErbB 4) antibody, an anti-CD 20 antibody, an anti-CD 22 antibody, an anti-CD 30 antibody, an anti-CD 33 antibody, an anti-CD 44 antibody, an anti-CD 56 antibody, an anti-CD 70 antibody, an anti-CD 73 antibody, an anti-CD 105 antibody, an anti-CEA antibody, an anti-a 33 antibody, an anti-Cripto antibody, an anti-EphA 2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC 44A4 antibody, or an anti-Mesothelin antibody; trastuzumab (trade name Herceptin), pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), nituzumab (Nimotuzumab, trade names tenascin )、 Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、 Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96 and Glematumamab) are preferred.

The sequences of about 110 amino acids near the N-terminus of the full length antibody heavy and light chains vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

The term "murine antibody" is used in this disclosure to refer to antibodies prepared by murine methods according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with a specific antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce the immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the murine specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., into framework sequences of different types of human germline antibodies. The heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured for CDRs by phage display. Further references describing methods of using mouse antibodies for the humanization involved include, for example, queen et al, proc., natl. Acad. Sci. USA,88, 2869, 1991 and methods of Winter and co-workers [ Jones et al, nature, 321, 522 (1986), riechmann et al, nature,332, 323-327 (1988), verhoeyen et al, science,239, 1534 (1988) ].

The terms "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", are used to eliminate immunogenicity and toxic side effects by making both the variable and constant regions of the antibody human. Monoclonal antibody development has undergone four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (PHAGE DISPLAY), transgenic mouse antibody preparation technology (TRANSGENIC MOUSE), single B cell antibody preparation technology, and the like.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments contained in the "antigen-binding fragment" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') ₂ fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of the single arm of the antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature 341:544-546) consisting of VH domains; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, so that they can produce a single protein chain (known as a single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, e.g., bird et al (1988) Science242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

Typically, fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity in a fragment obtained by treating an IgG antibody molecule with protease papain (cleavage of amino acid residue 224 of the H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds.

Typically, F (ab') 2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and comprising two Fab regions linked at hinge positions, obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

In general, fab 'is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity obtained by cleavage of disulfide bonds in the hinge region of the above F (ab') 2.

In addition, the Fab ' may be produced by inserting DNA encoding a Fab ' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker. Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56, and Roovers et al (2001), cancer Immunol.

The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 ^-7 M, e.g., about less than 10 ^-8M、10^-9 M or 10 ^-10 M or less.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the expression vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the expression vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The expression vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. Antigen binding fragments can likewise be prepared by conventional methods. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained by alignment of IMGT human antibody variable region germline gene databases and MOE software from the ImMunoGeneTics (IMGT) website http:// IMGT. Cines. FR, or from the immunoglobulin journal, lefranc, g., the Immunoglobulin FactsBook, academic Press, 2001ISBN 012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that are susceptible to transformation include members of the enterobacteriaceae (enterobacteriaceae), such as strains of escherichia coli (ESCHERICHIA COLI) or Salmonella (Salmonella); the family of Bacillaceae (baciliaceae) such as bacillus subtilis (Bacillus subtilis); pneumococci (Pneumococcus); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the N-terminal site of the Fc region. Positive clones were grown up in bioreactor medium to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The term "peptide" refers to a compound fragment between an amino acid and a protein, which is formed by connecting 2 or more amino acid molecules to each other through peptide bonds, and is a structural and functional fragment of the protein.

The term "sugar" refers to a biological macromolecule composed of C, H, O elements, which can be classified into monosaccharides, disaccharides, polysaccharides, and the like.

The term "toxin" refers to any substance capable of producing a deleterious effect on the growth or proliferation of cells and may be a small molecule toxin and derivatives thereof from bacteria, fungi, plants or animals, including camptothecin derivatives such as isatecan, maytansinoids and derivatives thereof (CN 101573384) such as DM1, DM3, DM4, auristatin F (AF) and derivatives thereof such as MMAF, MMAE, 3024 (WO 2016/127790A1, compound 7), diphtheria toxin, exotoxin, ricin (ricin) a chain, abrin (abrin) a chain, modeccin, alpha-furin (sarcin), aleurite (Aleutites fordii) toxin, carnation (dianthin) toxin, pokeweed (Phytolaca americana) toxin (PAPI, PAPII and PAP-S), balsam (Momordica charantia) inhibitors, curcin (curcin), crotonin (crotin), saporin (sapaonaria officinalis) inhibitors, gelonin (gelonin), clindamycin (3825), and trichostatin (trichothecenes).

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 (e.g., 1,2, 3,4, 5,6,7,8, 9, 10, 11, and 12) carbon atoms, more preferably an alkyl group containing from 1 to 10 carbon atoms, and most preferably an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl 4, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, where alkyl is as defined above.

The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the same carbon atom or two different carbon atoms of the parent alkane, which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkylene group containing from 1 to 12 (e.g., 1,2, 3,4, 5,6,7,8, 9, 10, 11, and 12) carbon atoms, more preferably containing from 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH ₂ -), 1-ethylene (-CH (CH ₃) -), 1, 2-ethylene (-CH ₂CH₂) -, 1-propylene (-CH (CH ₂CH₃) -), 1, 2-propylene (-CH ₂CH(CH₃) -), 1, 3-propylene (-CH ₂CH₂CH₂ -), 1, 4-butylene (-CH ₂CH₂CH₂CH₂ -), 1, 5-butylene (-CH ₂CH₂CH₂CH₂CH₂ -), and the like. The alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably independently and optionally with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein the alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy. The alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 8 (e.g., 3, 4, 5, 6, 7, and 8) carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S (O) or S (O) _m (where m is an integer from 0 to 2), but excluding the ring portion of-O-, -O-S-or-S-, the remaining ring atoms being carbon. Preferably from 3 to 12 (e.g., 3,4,5,6,7,8,9,10,11, and 12) ring atoms, of which 1 to 4 (e.g., 1,2, 3, and 4) are heteroatoms; more preferably the cycloalkyl ring contains 3 to 10 ring atoms; more preferably 3 to 8 ring atoms (e.g., 3,4,5,6,7, and 8), of which 1-3 (e.g., 1,2, and 3) are heteroatoms; more preferably 3 to 6 ring atoms, of which 1-3 are heteroatoms; most preferably 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl groups include spiro, fused and bridged heterocyclic groups.

The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group having one atom (referred to as a spiro atom) shared between 5-to 20-membered monocyclic rings, wherein one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen or S (O) _m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Which may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The spiroheterocyclyl groups are classified into a single spiroheterocyclyl group, a double spiroheterocyclyl group or a multiple spiroheterocyclyl group according to the number of common spiro atoms between rings, and preferably a single spiroheterocyclyl group and a double spiroheterocyclyl group. More preferably a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered single spiro heterocyclic group. Non-limiting examples of spiroheterocyclyl groups include:

The term "fused heterocyclyl" refers to a 5 to 20 membered, polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system, one or more of which may contain one or more double bonds, but none of which has a fully conjugated pi electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S (O) _m (where m is an integer from 0to 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. The number of constituent rings may be classified as a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclyl groups include:

the term "bridged heterocyclyl" refers to a5 to 14 membered, polycyclic heterocyclic group in which any two rings share two atoms which are not directly connected, which may contain one or more double bonds, but none of the rings has a fully conjugated pi electron system in which one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen or S (O) _m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14 membered, more preferably 7 to 10 membered. Heterocyclic groups which may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged according to the number of constituent rings are preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups include:

the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is heterocyclyl, non-limiting examples of which include:

Etc. /(I)

The heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (fused polycyclic being a ring sharing adjacent pairs of carbon atoms) group having a conjugated pi electron system, preferably 6 to 10 membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 (e.g., 1,2,3, and 4) heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10 membered (e.g., 5, 6, 7, 8, 9, or 10 membered), more preferably 5 or 6 membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

The term "amino protecting group" is intended to mean an amino group that is protected by an easily removable group in order to keep the amino group unchanged when the reaction is carried out at other positions of the molecule. Non-limiting examples include 9-fluorenylmethoxycarbonyl, t-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1 to 3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

The term "cycloalkylalkyl" refers to an alkyl group having hydrogen substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

The term "haloalkyl" refers to a hydrogen on an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group in which the hydrogen on the alkyl group is replaced with one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to-NH ₂.

The term "nitro" refers to-NO ₂.

The term "mercapto" refers to-SH.

The term "amide" refers to-C (O) N (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

The present disclosure also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art are able to refer to the relevant literature for the synthesis of deuterated forms of the compounds of formula (I). Commercially available deuterated starting materials may be used in preparing the deuterated form of the compound of formula (I) or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may be, but is not necessarily, present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

The term "pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, which is safe and effective when used in a mammal, and which has the desired biological activity, an antibody-antibody drug conjugate compound of the present disclosure containing at least one amino group and thus being capable of forming a salt with an acid, non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

The preparation of the conventional pharmaceutical composition is shown in Chinese pharmacopoeia.

The term "carrier" as used in this disclosure refers to a system that alters the manner and distribution of a drug into the body, controls the release rate of the drug, and delivers the drug to a targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects and improve bioavailability. For example, a polymer surfactant which can be used as a carrier can be self-assembled due to the unique amphiphilic structure of the polymer surfactant to form various forms of aggregates, and preferable examples are micelles, microemulsions, gels, liquid crystals, vesicles and the like. These aggregates have the ability to entrap drug molecules while having good permeability to membranes and can be used as good drug carriers.

The term "excipient" is an addition to the primary drug in a pharmaceutical formulation, and may also be referred to as an adjuvant. Such as binders, fillers, disintegrants, lubricants in the tablet; a base portion in a semisolid formulation ointment, cream; preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid formulations may be referred to as excipients.

The term "diluent" is also known as filler and its primary purpose is to increase the weight and volume of the tablet. The diluent is added to ensure a certain volume, reduce the dosage deviation of main components, improve the compression molding property of the medicine, and the like. When the drug in the tablet contains an oily component, an absorbent is added to absorb the oily substance so as to maintain the "dry" state, thereby facilitating the preparation of the tablet. Such as starch, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

The present disclosure relates to a class of cleavable linker arms of specific structure and actives of specific structure, and Antibody Drug Conjugates (ADCs) consisting of the linker arms, the actives (toxins) and antibodies. Such ADCs are complexes formed by attaching a toxic substance (toxin) to an antibody via a spacer. The antibody coupled drug (ADC) is degraded in vivo to release active molecules (toxins), thereby playing an anti-tumor role.

Synthetic methods of the present disclosure

In order to accomplish the synthesis purpose of the present disclosure, the present disclosure adopts the following synthesis technical scheme:

scheme one:

A method of the present disclosure for the preparation of a compound of formula (D), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

The general formula (DA) is subjected to deprotection reaction under alkaline conditions to obtain a compound shown as the general formula (D),

Wherein: r ²-R¹⁶ is as defined in formula (D).

Reagents that provide basic conditions include organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide; diethylamine is preferred.

Scheme II:

A process for preparing compound 2 of the present disclosure, or a pharmaceutically acceptable salt thereof, which comprises:

and (3) adding a condensing agent into the compound 1 and the compound 2a under alkaline conditions to perform condensation reaction to obtain the compound 2.

Reagents for providing basic conditions include organic bases including, but not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N-diisopropylethylamine, N-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide, and inorganic bases including, but not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably N, N-diisopropylethylamine.

The condensing agent is selected from 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methyl morpholine chloride, 1-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N '-dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate, 2- (7-azobenzotriazol-N, N ', N' -tetramethylurea hexafluorophosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate or benzotriazol-1-yl-oxy-tripyrrolidinylphosphos-ate, preferably 4- (4, 6-dimethoxy-1, 3, 5-triazin-4-methylbenzotriazole) -1-hydroxy-benzotriazol-3-e hydrochloride, and preferably 1-hydroxy-3-ethylmorpholine chloride.

Scheme III:

A method of ligand-drug conjugate or pharmaceutically acceptable salt thereof, represented by the general formula (Pc-L-D) of the present disclosure, comprising the steps of:

/>

after Pc is reduced, coupling reaction is carried out with a compound 2 to obtain a compound shown in a general formula (ADC-1); the reducing agent is preferably TCEP, in particular, preferably reduces disulfide bonds on the antibody;

Wherein Pc, n is defined as in the general formula (Pc-L-D).

Detailed Description

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure.

Experimental methods for which specific conditions are not noted in the examples of the present disclosure are generally performed according to conventional conditions or according to conditions suggested by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

1. Synthesis of Compounds

Examples

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). The NMR shift (. Delta.) is given in units of 10 ^-6 (ppm). NMR was performed using a Bruker AVANCE-400 nuclear magnetic resonance apparatus using deuterated dimethyl sulfoxide (DMSO-d ₆), deuterated chloroform (CDCl ₃), deuterated methanol (CD ₃ OD) as the solvent and Tetramethylsilane (TMS) as the internal standard.

MS was determined using an Agilent 1200/1290DAD-6110/6120 Quadrapol MS liquid chromatography-mass spectrometry (manufacturer: agilent, MS model: 6110/6120 Quadrapol MS).

Waters ACQuity UPLC-QD/SQD (manufacturers: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector) THERMO Ultimate 3000-Q Exactive (manufacturers: THERMO, MS model: THERMO Q Exactive)

High Performance Liquid Chromatography (HPLC) analysis used AGILENT HPLC DAD, AGILENT HPLC VWD, and WATERS HPLC E2695-2489 high pressure liquid chromatographs.

Chiral HPLC analysis was determined using an Agilent 1260DAD high performance liquid chromatograph.

The high performance liquid phase was prepared by using Waters 2545-2767, waters 2767-SQ Detecor2, shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.

The CombiFlash rapid preparation instrument used CombiFlash Rf200 (TELEDYNE ISCO).

The thin layer chromatography silica gel plate uses a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The silica gel column chromatography generally uses 200-300 mesh silica gel of yellow sea of the tobacco stand as a carrier.

The average inhibition rate of kinase and IC ₅₀ were measured by NovoStar microplate reader (BMG, germany).

Known starting materials of the present disclosure may be synthesized using or following methods known in the art, or may be purchased from ABCR GmbH & co.kg, acros Organics, ALDRICH CHEMICAL Company, shao Yuan chemical technology (Accela ChemBio Inc), dary chemicals, and the like.

The examples are not particularly described, and the reaction can be carried out under an argon atmosphere or a nitrogen atmosphere.

An argon or nitrogen atmosphere means that the reactor flask is connected to a balloon of argon or nitrogen of about 1L volume.

The hydrogen atmosphere is defined as the reaction flask being connected to a balloon of hydrogen gas of about 1L volume.

The pressure hydrogenation reaction uses Parr 3916EKX type hydrogenometer and clear blue QL-500 type hydrogen generator or HC2-SS type hydrogenometer.

The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times.

The microwave reaction used was a CEM Discover-S908860 type microwave reactor.

The examples are not specifically described, and the solution refers to an aqueous solution.

The reaction temperature is room temperature and is 20-30 deg.c without specific explanation in the examples.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the system of eluent for column chromatography employed for purifying the compound and the developing reagent system of thin layer chromatography included: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: the volume ratio of the petroleum ether to the ethyl acetate is adjusted according to the polarity of the compound, and small amount of alkaline or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

Example 1

(S) -N- ((3R, 4S, 5S) -1- ((1S, 3S, 5S) -3- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) -2-azabicyclo [3.1.0] hex-2-yl) -3-methoxy-5-methyl-1-oxohept-4-yl) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methylamino) butyrylamino) butyramide 1

First step

(9H-fluoren-9-yl) methyl (1S, 3S, 5S) -3- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) -2-azabicyclo [3.1.0] hexane-2-carboxylate 1c

(2R, 3R) -3- ((1S, 3S, 5S) -2- (((9H-fluoren-9-yl) methoxy) carbonyl) -2-azabicyclo [3.1.0] hex-3-yl) -3-methoxy-2-methylpropanoic acid 1a (1.05 g,2.49mmol, prepared by the method disclosed in step 7 on page 20 of the specification in patent application "U.S. Pat. No. 5,223") and (1S, 2R) -2-amino-1-phenylpropan-1-ol 1b (0.42 g,2.78mmol, prepared by the known method "Journal of Organic Chemistry,2012, vol.77, # 12, p.5454-5460") were added to the reaction flask, 10mL of dichloromethane and 2mL of N, N-dimethylformamide were added three times under stirring, 2- (7-benzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.g, 3.00 mmol) and N-isopropyl amine were added under stirring, and sodium chloride (1.35 g,2.78mmol, prepared by the method "Journal of Organic Chemistry.78 mmol), the filtrate was concentrated by the method of saturated aqueous solution of 1 mL (30 mL of sodium chloride, 35 mL, saturated aqueous solution was added to the solution was dried under a saturated solution of the aqueous phase (38 mL, saturated aqueous solution of the aqueous solution was concentrated under reduced pressure, and saturated aqueous solution was saturated with saturated aqueous solution, and saturated aqueous solution.

MS m/z(ESI):555.2[M+1]

Second step

(2R, 3R) -3- ((1S, 3S, 5S) -2-azabicyclo [3.1.0] hex-3-yl) -N- ((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) -3-methoxy-2-methylpropanamide 1d

1C (1.38 g,2.49 mmol) was dissolved in 10mL of dichloromethane, 20mL of diethylamine was added, replaced with argon three times, and the reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography with a developer system A to give the title product 1d (805 mg, yield: 97.3%). MS m/z (ESI): 333.2[ M+1]

Third step

(9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- (((3R, 4S, 5S) -1- ((1S, 3S, 5S) -3- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) -2-azabicyclo [3.1.0] hex-2-yl) -3-methoxy-5-methyl-1-oxohept-4-yl) (methyl) amino) -3-methyl-1-oxobutan-2-yl) carbamate 1f

(5S, 8S,11S, 12R) -11- ((S) -sec-butyl) -1- (9H-fluoren-9-yl) -5, 8-diisopropyl-12-methoxy-4, 10-dimethyl-3, 6, 9-trioxo-2-oxa-4, 7, 10-triaza-tetradeca-14-oic acid 1e (1.54 g,2.41mmol, supplier: happy element) was added to the reaction flask, 30mL of acetonitrile was added, argon was replaced three times, the ice water bath was cooled to 0-5 ℃, 2- (7-benzotriazol-oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.10 g,2.89 mmol) and N, N-diisopropylethylamine (0.94 g,7.27 mmol) were added, and the ice bath was stirred for 10 minutes. 10mL of an acetonitrile suspension of 1d (805 mg,2.42 mmol) was added and the reaction stirred in an ice bath for 40 minutes. 60mL of water was added, extracted with ethyl acetate (20 mL. Times.4), the organic phase was washed with saturated sodium chloride solution (60 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developer system A to give crude product 1f (2.9 g).

MS m/z(ESI):952.3[M+1]

Fourth step

The crude 1f (510 mg,0.53 mmol) was dissolved in 2mL of dichloromethane, 4mL of diethylamine was added, replaced three times with argon, and the reaction was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the resulting residue was purified by silica gel column chromatography with a developer system A to give the title product 1 (266 mg, yield: 68.0%).

MS m/z(ESI):730.4[M+1]

¹H NMR(400MHz,CD₃OD):δ7.36-7.40(m,2H),7.31(t,2H),7.24(d,1H),4.69(d, 1H),4.56(d,1H),4.17-4.28(m,2H),4.06-4.14(m,1H),3.91(d,1H),3.78(t,1H),3.27-3.44(m,7H),3.15(s,3H),2.84-2.93(m,1H),2.60-2.67(m,2H),2.30-2.37(m, 3H),2.02-2.10(m,2H),1.79-1.95(m,4H),1.38-1.53(m,2H),1.25-1.36(m,2H),1.21(d,1H),1.13-1.17(m,2H),1.07-1.11(m,2H),0.93-1.05(m,15H),0.83-0.89(m,4H), 0.70-0.79(m,1H).

Example 2

4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanoylamino) -5-ureidopentanoylamino) benzyl

((S) -1- (((3R, 4S, 5S) -1- ((1S, 3S, 5S) -3- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) -2-azabicyclo [3.1.0] hex-2-yl) -3-methoxy-5-methyl-1-oxohept-4-yl) (methyl) amino) -3-methyl-1-oxobutan-2-yl) (methyl) carbamate 2

1 (30 Mg,0.041 mmol) was added to 1mL of N, N-dimethylformamide, 4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide) -3-methylbutanoylamino) -5-ureidopentanoyl-ammo-m) benzyl (4-nitrophenyl) carbonate 2a (45 mg,0.061mmol, supplier: ark), 0.25mL of pyridine was further added, argon was replaced three times, 1-hydroxybenzotriazole (12 mg,0.089 mmol) and N, N-diisopropylethylamine (16 mg,0.123 mmol) were further added, and after stirring at room temperature for 4 hours, 2a (45 mg,0.061 mmol) was added and stirring was continued for 16 hours. The reaction solution was purified by high performance liquid chromatography (separation conditions: column XB ridge Prep C18 OBD 5um 19X 250mm; mobile phase: A-water (10 mmol NH ₄ OAc): B-acetonitrile, gradient elution, flow rate: 18 mL/min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product 2 (18 mg, yield: 33.0%).

MS m/z(ESI):1329.3[M+1]

¹H NMR(400MHz,CD₃OD):δ7.58(d,2H),7.29-7.42(m,6H),7.20-7.26(m,1H), 6.79(s,2H),5.04-5.20(m,4H),4.47-4.61(m,3H),4.13-4.28(m,3H),4.06-4.12(m,1H),3.91(d,1H),3.75-3.82(m,1H),3.48(t,3H),3.27-3.41(m,7H),3.16-3.25(m,2H), 3.18(s,3H),2.91-2.97(m,2H),2.60-2.65(m,2H),2.27(t,2H),2.20(t,1H),2.01-2.10(m,3H),1.69-1.94(m,6H),1.63-1.68(m,6H),1.46-1.51(m,1H),1.27-1.37(m,5H), 1.12-1.21(m,3H),1.09(d,2H),0.93-1.04(m,11H),0.80-0.92(m,11H),0.70-0.77(m, 2H).

2. Synthesis of antibodies

Antibodies (including but not limited to Pertuzumab, nituzumab Nimotuzumab, trastuzumab, anti-B7H 3 antibodies, anti-CD 79B antibodies) are prepared according to conventional methods of antibodies, e.g., following vector construction, eukaryotic cells such as HEK293 cells (Life Technologies cat.no. 11625019) are transfected and purified for expression. Exemplary antibody sequences are as follows:

(1) The following is the sequence of Trastuzumab (Trastuzumab) for Trastuzumab (Trastuzumab for Trastuzumab in WO2016/127790 A1)

Light chain

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.1

Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.2

(2) The following is the sequence of Pertuzumab (Pertuzumab) for Pertuzumab (Pertuzumab in WO 2016/127790)

Light chain:

DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRY TGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO.3

heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVN PNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

SEQ ID NO.4

(3) The sequence of the anti-B7H 3 antibody 1F9DS (antibody H1702-DS in WO2019/024911A 1) is as follows: light chain

DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTN TRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKA GVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE C

SEQ ID NO.5

Heavy chain

QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISY DGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.6

(4) The following is the sequence of CD 79B antibody hAb015 (antibody hAb015 in WO2020/156439A 1) light chain:

DFVMTQTPLSLPVTPGEPASISCRSSQSIVHSDGNTYFEWYLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO.7

heavy chain:

EVQLVQSGAEVKKPGSSVKVSCKASGSSFSSYGINWVKQAPGQGLEWIGEIFPR SGNTYYNEKFEGRATLTADKSTSTAYMELRSLRSEDTAVYYCAKGDLGDFDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

SEQ ID NO:8

3. Preparation of antibody ADC conjugates

ADC stock drug load analysis

1. RP-HPLC analysis of DAR

Reagents and instrumentation:

trifluoroacetic acid (TFA), sigma production, 100 mL/vial; acetonitrile, LC grade, 4L/bottle, fisher;

DTT (0.25M), 1 g/bottle, sigma production. High performance liquid chromatograph: agilent 1200.

Preparing a solution:

0.25M DTT solution: the solution of DTT 5.78mg was dissolved in 150. Mu.L of purified water to prepare a 0.25M solution of DTT, which was stored at-20 ℃. Mobile phase a (0.1% tfa in water): 1000mL of purified water was measured in a measuring cylinder, 1mL of TFA was added, and the mixture was used after thorough mixing, and stored at 2-8℃for 14 days. Mobile phase B (0.1% tfa in acetonitrile): 1000mL of acetonitrile is measured by a measuring cylinder, 1mLTFA is added, and the mixture is fully and uniformly mixed for use, and is stored for 14 days at the temperature of 2-8 ℃.

Bare antibody and sample (concentration 1mg/mL, about 200. Mu.L) for test, 4. Mu.L DDT was added for reduction, and water bath at 37℃for 1 hour.

Chromatographic conditions:

Chromatographic column: AGILENT PLRP-S1000 a 8 μm 4.6 x 250mm; column temperature: 80 ℃; DAD detector: detection wavelength 280nm; sample cell temperature: 4 ℃; flow rate: 1mL/min; the sample injection amount is as follows: 40 μl; chromatographic gradient: b% (initial 20% -5min36% -32min 60%)

Data analysis:

And (3) distinguishing the positions of the light chains and the heavy chains through the comparison of the spectrograms of the sample and the naked antibody, and then integrating the spectrograms of the detection sample to calculate the DAR value. The calculation formula is as follows: LC:0 (number of linked drugs), LC+1:2 (number of linked drugs), HC:0 (number of linked drugs), HC+1:2 (number of linked drugs), HC+2:4 (number of linked drugs), HC+3:6 (number of linked drugs). LC peak area sum=lc peak area+lc+1 peak area; total HC peak area = HC peak area + HC +1 peak area + HC +2 peak area + HC +3 peak area; LC DAR = Σ (number of linked drugs x peak area percent)/LC peak area sum; HC DAR = Σ (number of linked drugs x peak area percent)/HC peak area sum; dar=lc dar+hc DAR

2. CE-SDS analysis of drug loading DAR

Reagents and instrumentation:

SDS-MW ANALYSIS KIT Beckman, cat. No. 390953, the kit comprises SDS-MW gel separation buffer, SDS-MW sample buffer (sample dilution), acidic washing solution (0.1 mol/L hydrochloric acid solution), alkaline washing solution (0.1 mol sodium hydroxide solution), and internal standard substance (10 kDa internal standard). SDS kit manufactured by Beijing Bosi Biochemical technology institute, cat# BSYK018, which contains CE-SDS Gel Buffer, CE-SDS SampleBuffer (sample diluent) may also be used.

The alkylating solution (0.25 mo of iodoacetamide solution) is prepared by weighing about 0.046g of iodoacetamide, adding lmL ultrapure water, dissolving, mixing, and storing at 2-8deg.C in dark place for 7 days.

Capillary electrophoresis apparatus: SCIEX Co., ltd., PA800plus

Capillary tube uncoated fused silica capillary tube (inner diameter 50 μm), cut to a total length of 30.2cm, and high resolution method effective separation length of 20cm

Sample solution preparation: the test sample was diluted to 1mg/mL with SDS sample buffer. And (5) sampling. 95. Mu.L of the sample solution (1 mg/mL) was added to 5. Mu.L of a 0.8mol/L iodoacetamide aqueous solution, and the mixture was vortexed and homogenized. A blank control (95. Mu.L) was added with 5. Mu.L of a 0.8mol/L iodoacetamide aqueous solution, and the mixture was vortexed and homogenized, and 75. Mu.L of each solution was taken out of the sample tube into a sample bottle, followed by immediate analysis.

Assay:

(1) Pretreatment of the capillary: the 0.1mol/L sodium hydroxide solution was rinsed at 60psi for 3 minutes, then 0.1mol/L hydrochloric acid solution was rinsed at 60psi for 2 minutes, and finally pure water was rinsed at 70psi for 1 minute. Each run should be preceded.

(2) Prefilling of the capillary: SDS gel separation buffer was washed at 50psi pressure for 15 minutes. Each run should be preceded.

Sample injection: 10kV reversed-phase polarity electric sample injection and reduction sample injection for 20 seconds

Separating: run at 15kV for 40 min with reversed polarity.

Sample cell temperature: 18-25 ℃.

Capillary temperature: 18-25 ℃.

Analysis of results:

Data analysis: the sulfhydryl groups released based on the disulfide bonds opened in the antibody are coupled with corresponding Drugs, and the data are analyzed by Beckman software, so that the corrected peak areas of heavy chains, non-glycosylated heavy chains, light chains and the like respectively occupy all the corrected peak areas. According to the formula DAR= [4 x heavy chain (H) peak area+2 x half antibody (H-L) peak area+4 x double chain (H-H) peak area+2 x heavy light chain (H-H-L) peak area ]/[ heavy chain (H) peak area/2+half antibody (H-L) peak area/2+double chain (H-H) peak area+heavy light chain (H-H-L) peak area+full antibody peak area ], finally calculating the weighted average value of the ADC medicine

Example 3

The synthesis of ADC-1 can be carried out with reference to known literature, such as example 12 at page 173 of the specification in CN1938046B, or example 2 at page 146 of the specification in WO 2014057687. The synthetic methods disclosed therein are incorporated into the present disclosure by reference, wherein the toxin compound moiety used is compound 2 of the present disclosure.

Example 4

ADC-2 can be synthesized with reference to example 18 on page 65 of the specification in WO 2016127790. The synthetic methods disclosed therein are incorporated into the present disclosure by reference, wherein the toxin compound moiety used is compound 2 of the present disclosure.

Example 5ADC-3

To an aqueous solution of antibody Trastuzumab in PBS (0.05M in PBS at pH=6.5; 10.0mg/mL,1.14mL,77.03 nmol) was added an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10 mM, 20. Mu.L, 200 nmol) prepared, and the mixture was placed in a water bath shaker and allowed to react with shaking at 37℃for 3 hours, thereby stopping the reaction. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 2 (1.02 mg,767.7 nmol) was dissolved in 50. Mu.L of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25℃for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified by Sephadex G25 gel column (eluent: 0.05M PBS buffer aqueous solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (0.90 mg/mL, 12.7 mL) of an exemplary product ADC-3 of conjugate FADC-3, which was stored at 4 ℃.

Average drug loading calculated by RP-HPLC: n=4.47.

Example 6ADC-4

To an aqueous solution of antibody Pertuzumab in PBS (0.05M aqueous solution of PBS buffer at pH=6.5; 10.0mg/mL,1.14mL,77.03 nmol) was added an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) prepared (10 mM, 20. Mu.L, 200 nmol) at 37℃and the mixture was placed in a water bath shaker and reacted for 3 hours with shaking at 37℃to stop the reaction. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 2 (1.02 mg,767.7 nmol) was dissolved in 50. Mu.L of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted at 25℃for 3 hours with shaking, and the reaction was stopped. The reaction was desalted and purified by Sephadex G25 gel column (eluent: 0.05M PBS buffer aqueous solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.00 mg/mL,11.9 mL) of an exemplary product ADC-4 of conjugate FADC-4, which was stored at 4 ℃.

Average drug loading calculated by RP-HPLC: n=4.57.

Example 7ADC-5

To an aqueous solution of antibody 1F9DS in PBS (0.05M in PBS buffer at ph=6.5; 10.0mg/mL,1.26mL,85.14 nmol) was added an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) prepared (10 mm,22.1 μl,221 nmol) at 37 ℃, placed in a water bath shaker, and reacted for 3 hours with shaking at 37 ℃. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 2 (1.13 mg,850.49 nmol) was dissolved in 50. Mu.L of DMSO, added to the reaction solution, and placed in a water bath shaker to perform a shaking reaction at 25℃for 3 hours, and the reaction was stopped. The reaction was desalted and purified by Sephadex G25 gel column (eluent: 0.05M PBS buffer aqueous solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (0.99 mg/mL, 13.3 mL) of an exemplary product ADC-5 of conjugate FADC-5, which was stored at 4 ℃.

Average drug loading calculated by RP-HPLC: n=3.76.

Example 8ADC-6

To an aqueous solution of antibody hAb015 in PBS (0.05M aqueous solution of PBS buffer at ph=6.5; 10.0mg/mL,5.00mL,337.84 nmol) was added an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) prepared (10 mm,85.0 μl,850 nmol) at 37 ℃, and the mixture was placed in a water bath shaker and allowed to react with shaking at 37 ℃ for 3 hours, to stop the reaction. The reaction solution was cooled to 25 ℃ with a water bath.

The compound MC-vc-PAB-MMAE (4.45 mg,3379.79nmol, Hanxiang biotechnology, batch NO: 20190322) was dissolved in 250. Mu.L of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted for 3 hours with shaking at 25℃to stop the reaction. The reaction was desalted and purified by Sephadex G25 gel column (eluent: 0.05M PBS buffer aqueous solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (2.79 mg/mL,17.4 mL) of an exemplary product ADC-6 of conjugate FADC-6, which was stored at 4 ℃.

Average drug loading calculated for CE-SDS: n=3.09.

Example 9ADC-7

To an aqueous solution of antibody hAb015 in PBS (0.05M aqueous PBS buffer at ph=6.5; 10.0mg/mL,1.80mL,121.62 nmol) was added an aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) prepared (10 mm,30.4 μl,304 nmol) at 37 ℃, placed in a water bath shaker, and reacted for 3 hours with shaking at 37 ℃. The reaction solution was cooled to 25 ℃ with a water bath.

Compound 2 (1.62 mg,1219.29 nmol) was dissolved in 90. Mu.L of DMSO, added to the reaction solution, and placed in a water bath shaker to perform a shaking reaction at 25℃for 3 hours, and the reaction was stopped. The reaction was desalted and purified by Sephadex G25 gel column (eluent: 0.05M PBS buffer aqueous solution at pH 6.5, containing 0.001M EDTA) to give PBS buffer (1.37 mg/mL, 12.0 mL) of an exemplary product ADC-7 of conjugate FADC-7, which was stored at 4 ℃.

Average drug loading calculated for CE-SDS: n=3.21.

Biological evaluation

Test example 1: in vitro proliferation inhibition test of compounds of general formula (D) on tumor cells HepG2 and SK-BR-3

1. Test purpose:

The samples of the present disclosure were tested for inhibition of HepG2 and SK-BR-3 cell proliferation.

2. Test materials and instruments:

EMEM: purchased from LONZA, cat: BE12-611F;

McCoy' S5a: purchased from Gibco under accession number 16600108;

FBS: purchased from Gibco, cat No.: 10099-141;

CELLTILTER-Glo: purchased from Promega, cat: 7572;

PBS: purchased from Shanghai source culture Biotech Co., ltd., product number: b320;

HepG2 human liver cancer cell line: purchasing from cell banks of the department of Chinese sciences;

SK-BR-3 human breast cancer cell line: purchased from ATCC under the designation HTB-30;

75cm TC-Treated Culture Flask, available from Corning Incorporated under the trade designation: 430641;

instrument PHERASTAR FS: purchased from BMG labtech.

3. The test method comprises the following steps:

1. Cell plating

HepG2 and SK-BR-3 cells were cultured in EMEM and McCoy' S5a medium containing 10% FBS, respectively. In the experiment, hepG2 cell density was adjusted to 4.4X10 ⁴ cells/mL, SK-BR-3 cell density was adjusted to 3.7X10 ⁴ cells/mL, 135. Mu.L of cell suspension was added to each well of columns 2 to 11 of 96 well plates, and column 12 was blank. The cells were incubated in a 37℃incubator with 5% CO ₂ for 24 hours.

2. Compound medicine preparation

1) Preparation of compound drug stock solution: the drug was dissolved in DMSO to give a stock solution concentration of 20mM.

2) Dispensing plate 1: initial column 1 dilutes stock 40-fold, and columns 2 through 11 serially dilute 3-fold in a gradient. Column 12 is DMSO.

3) Dispensing plate 2: in columns 2 to 11, 95. Mu.L of the corresponding culture medium was added, and 5. Mu.L was aspirated from columns 3 to 12 of the dispensing plate 1 to columns 2 to 11 of the dispensing plate 2. Mixing well.

3. Treatment of cells

Mu.L of the added cells were aspirated from the dispensing plate 2. The incubation was continued for 3 days in a 37℃incubator with 5% CO ₂.

After 4.72 hours, 75 mu L of CTG is added into each hole, the reaction is carried out for 10 minutes at room temperature, the luminescence value is read by PHERASTAR FS, and the IC ₅₀ can be obtained after GRAPHPAD PRISM software treatment.

TABLE 1 IC of proliferation inhibition of HepG2 and SK-BR-3 cells by the compounds of the present disclosure ₅₀

Numbering of compounds	IC₅₀(HepG2)/nM	IC₅₀(SK-BR-3)/nM
			1	0.81	0.17

Conclusion: the compounds of the present disclosure have significant proliferation inhibiting activity on both HepG and SK-BR-3 cells.

Test example 2: ligand-drug conjugate of the present disclosure for in vitro proliferation inhibition test of tumor cells

1. Purpose of testing

The purpose of this experiment was to examine the inhibitory activity of the ligand-drug conjugates of the present disclosure on the proliferation of MCF-7 tumor cells (human breast cancer cells, ATCC, cat# HTB-22) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, cat# HTB-30) in vitro. Treating cells in vitro with compounds of different concentrations, culturing for 6 days, and adopting CTG%Luminescent Cell Viability Assay, promega, cat: g7573 The proliferation of cells is examined by the reagent and the in vitro activity of the compound is evaluated based on the IC50 value.

2. Experimental method

The following is an example of an in vitro proliferation inhibition test method for SK-BR-3 cells or MCF-7 cells, and is used to illustrate the method of the ligand-drug conjugate of the present disclosure for in vitro proliferation inhibition activity testing of tumor cells. The method is equally applicable to, but not limited to, in vitro proliferation inhibition activity assays for other tumor cells.

1. Cell culture: MCF-7 or SK-BR-3 cells were cultured in EMEM medium (GE, cat# SH 30024.01) with 10% FBS and McCoy's 5A medium (Gibco, cat# 16600-108) with 10% FBS.

2. Cell preparation. MCF-7 or SK-BR-3 cells in the logarithmic growth phase were washed 1 time with PBS (phosphate buffer, shanghai source culture Biotechnology Co., ltd.), then digested 2-3mL of trypsin (0.25% Trypsin-EDTA (1X), gibico, life Technologies Co.) for 2-3 minutes, after complete cell digestion, 10-15mL of cell culture broth was added, the digested cells were eluted, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and then 10-20mL of cell culture broth was added to resuspend the cells to prepare a single cell suspension.

3. Cell plating. The single cell suspension of MCF-7 or SK-BR-3 is uniformly mixed, the cell culture solution is used for respectively adjusting the density of living cells to 2.75X10 ³ cells/mL and 8.25X10 ³ cells/mL, the cell suspension with the adjusted density is uniformly mixed, and 180 mu L/hole is added into a 96-well cell culture plate. 200. Mu.L of medium was added to the wells outside Zhou Kongzhi. The plates were incubated in an incubator for 24 hours (37 ℃,5% co ₂).

4. Ligand-drug conjugate samples were formulated. The initial concentration was adjusted to 5uM with PBS, then 4-fold dilutions were made with PBS, 9 concentration spots, each with 2 multiplex wells.

5. And (5) sample adding operation. To the plate of step 3, 20. Mu.L of the samples to be tested prepared in step 4 were added, each sample was double-well incubated in an incubator for 6 days (37 ℃,5% CO ₂).

6. And (5) color development operation. The 96-well cell culture plate was removed, 90. Mu.L of CTG solution was added to each well, and incubated at room temperature for 10 minutes.

7. And (5) reading the board. The 96-well cell culture plate was removed, placed in an enzyme-labeled instrument (BMG labtech, PHERASTAR FS), and chemiluminescence was measured using the enzyme-labeled instrument.

3. Data analysis

Data were analyzed by Microsoft Excel, GRAPHPAD PRISM. The results of the examples are given in the following table.

TABLE 2 IC ₅₀ values for in vitro proliferation inhibition of SK-BR-3 cells by ligand-drug conjugates of the present disclosure

Sample of	SK-BR-3 cell IC ₅₀ (nM)	Imax％
			ADC-3	0.069	94.73
ADC-4	0.065	94.6
			Trastuzumab	>500	47
Pertuzumab	>500	8.11

TABLE 3 IC ₅₀ values for inhibition of MCF-7 cell proliferation in vitro by ligand-drug conjugates of the present disclosure

Sample of	MCF-7 cell IC ₅₀ (nM)	Imax％
			ADC-5	2.71	88.28
1F9DS	>500	0

Conclusion: the ligand-drug conjugates of the present disclosure have significant proliferation inhibiting activity on SK-BR-3 cells and MCF-7 cells.

Test example 3 efficacy of the active Compounds of the present disclosure against human diffuse large B cell lymphoma WSU-DLCL2 nude mice subcutaneously transplanted tumor

1. Experimental animals and feeding conditions

Nude mice, BALB/c-nu, females, were purchased from Beijing Fukang Biotech Co. Production license number: SCXK (Beijing) 2019-0008, animal eligibility number No.1103222011004014. Feeding environment: SPF stage.

2. Experimental design and experimental method

Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from AMERICAN TYPE Culture Collection. The culture conditions were RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum and a mixture of green and streptomycin (Gibco, 15140-122) and incubated in an incubator with 5% CO ₂ air at 37 ℃. Passaging for 2-3 times a week, collecting cells when the cells are in exponential growth phase, counting, and inoculating.

Each nude mouse was inoculated subcutaneously with 2.1X10 ⁷ WSU-DLCL2 cells and animals were grouped according to tumor volume (D0) after tumor growth to 100-150mm ³. 6/group, 4 total groups. Mice were given by Intravenous (IV) administration at a volume of 10mL/kg. Tumor volumes were measured 2 times per week, mice weights were weighed, and data were recorded.

3. Experimental results

ADC-6 (3 mg/kg, IV, D0) had a tumor inhibition of 65% for WSU-DLCL2 with 1/6 tumor fraction regression; the tumor inhibition rates of ADC-7 (3, 10mg/kg, IV, D0) on WSU-DLCL2 are 88% and 126%, respectively, and the 10mg/kg dose group has 6/6 tumor partial regression; the tumor-bearing mice can better tolerate the drugs, and no obvious symptoms such as weight loss occur. See in particular table 4 below.

Table 4. Results of ADC tumor inhibition experiments

The tumor inhibition rate calculation method comprises the following steps:

Tumor Volume (TV) calculation formula: tv=1/2×l _{Long length}×L_{Short length} ²

Relative tumor proliferation rate T/C (%) = (T-T ₀)/(C-C₀) ×100

Tumor inhibition rate TGI (%) =1-T/C (%)

Wherein T, C is the tumor volume of the treatment group and the control group at the end of the experiment; t ₀、C₀ is the tumor volume at the beginning of the experiment.

5. Conclusion of experiment:

the ADC-7 conjugate disclosed by the disclosure has obvious curative effects on human diffuse large B cell lymphoma WSU-DLCL2 nude mice subcutaneous transplantation tumor by single intravenous injection of 3mg/kg or 10mg/kg, and causes partial regression of the tumor.

Sequence listing

<110> Jiangsu Hengrui medicine Co., ltd

Shanghai Hengrui medicine Co., ltd

<120> Austrastatin analogue and conjugate thereof, preparation method and application thereof

<150> 201911390425.7

<151> 2020-12-30

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Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

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305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 5

<211> 215

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<221> CHAIN

<223> H1702-DS light chain

<400> 5

Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg

85 90 95

Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys

210 215

<210> 6

<211> 449

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<221> CHAIN

<223> H1702-DS heavy chain

<400> 6

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 7

<211> 219

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<221> CHAIN

<223> HAb015 light chain

<400> 7

Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 8

<211> 447

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<220>

<221> CHAIN

<223> HAb015 heavy chain

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Ser Phe Ser Ser Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Glu Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Asp Leu Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

Claims

1. A compound of the general formula (D):

Or a pharmaceutically acceptable salt thereof, wherein:

R ¹-R⁶ is each independently selected from a hydrogen atom or a C _1-6 alkyl group;

R ⁷ is selected from a hydrogen atom or a C _1-6 alkyl group;

Any two groups among R ⁸-R¹¹, together with the atoms to which they are bonded, form a C _3-6 cycloalkyl group, the remaining two groups being hydrogen atoms;

r ¹² is selected from a hydrogen atom or a C _1-6 alkyl group;

each R ¹³-R¹⁵ is independently selected from a hydrogen atom, a hydroxyl group, or a C _1-6 alkyl group;

R ¹⁶ is phenyl.

2. The compound represented by the general formula (D) according to claim 1, which is a compound represented by the general formula (D ₁):

Or a pharmaceutically acceptable salt thereof,

R ⁹ and R ¹⁰ together with the atom to which they are bound form C _3-6 cycloalkyl;

R ²-R⁸,R¹¹-R¹⁶ is as defined in claim 1.

3. The compound represented by the general formula (D) according to claim 1, which is:

4. a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, which is a ligand-drug conjugate represented by the general formula (Pc-L-D):

Wherein:

R ²-R⁶ is each independently selected from a hydrogen atom or a C _1-6 alkyl group;

R ⁷ is selected from a hydrogen atom or a C _1-6 alkyl group;

r ¹² is selected from a hydrogen atom or a C _1-6 alkyl group;

R ¹⁶ is phenyl;

n is an integer or fraction of 1 to 10;

Pc is an antibody; l is a linker unit.

5. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, which is a ligand-drug conjugate or pharmaceutically acceptable salt thereof represented by the general formula (Pc-L-D1):

Wherein:

r ²-R¹⁶, pc, L, n are as defined in claim 4.

6. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, which is a ligand-drug conjugate or pharmaceutically acceptable salt thereof as shown below:

pc, L, n are as defined in claim 4.

7. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 4, wherein n is an integer or fraction of 1 to 8.

8. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 4, wherein n is an integer or fraction from 2 to 8.

9. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4 wherein the linker unit-L-is-Y-L ¹-L²-L³-L⁴,

Y is selected fromOr a chemical bond, X ₁ is selected from a hydrogen atom or a C _1-6 alkyl group, and X ₂ is selected from a C _1-6 alkylene group;

L ¹ is selected from the group consisting of- (succinimidyl-3-yl-N) -W-C (O) -, -CH ₂-C(O)-NR¹⁷ -W-C (O) -and-C (O) -W-C (O) -, wherein W is selected from the group consisting of C _1-8 alkyl and C _1-8 alkyl-C _3-6 cycloalkyl;

L ² is a bond;

L ³ is a peptide residue consisting of 2 to 7 amino acids selected from valine, citrulline, methylvaline;

L ⁴ is PAB.

10. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein Y is

11. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein L ¹ is selected from- (succinimid-3-yl-N) - (CH ₂)_s -C (O) -, wherein s is an integer from 2 to 8.

12. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 9, wherein L ³ is a dipeptide amino acid unit.

13. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 9, wherein L ³ is valine-citrulline.

14. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein linker unit-L-is selected from:

15. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, selected from the following structural formulas:

Wherein:

n is an integer or fraction of 1 to 10;

pc is an antibody.

16. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 15, wherein the antibody is selected from the group consisting of an anti-HER 2 (ErbB 2) antibody and an anti-B7-H3 antibody.

17. A ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 16, wherein the antibody is selected from Trastuzumab and Pertuzumab.

18. A process for preparing a compound of formula (D) or a pharmaceutically acceptable salt thereof, comprising the steps of:

The compound of the general formula (DA) is subjected to deprotection reaction under alkaline conditions to obtain a compound of the general formula (D),

Wherein: r ²-R¹⁶ is as defined in claim 1.

19. A method of preparing a ligand-drug conjugate of the general formula ADC-1, or a pharmaceutically acceptable salt thereof, comprising the steps of:

After Pc is reduced, coupling reaction is carried out with a compound 2 to obtain a compound shown in a general formula (ADC-1);

wherein Pc, n is as defined in claim 4.

20. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to formula (D) of any one of claims 1 to 3, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

21. A pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate according to any one of claims 4 to 17, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

22. Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 4 to 17, or a pharmaceutical composition according to claim 21, in the manufacture of a medicament for the treatment and/or prophylaxis of cancer selected from breast cancer, ovarian cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, bone cancer, skin cancer, pancreatic cancer and lymphoma.

23. The use according to claim 22, wherein the skin cancer is melanoma and the colorectal cancer is colon cancer or rectal cancer.