Topoisomerase inhibitor as well as conjugate and application thereof

Abstract

The invention relates to a topoisomerase inhibitor as well as a conjugate and application thereof. Specifically, the invention discloses a camptothecin derivative with a brand new structure, a linker conjugate and an antibody-drug conjugate. Experimental results show that by using the camptothecin derivative with high lipophilicity, the anti-tumor effect of the ADC drug can be remarkably improved, so that linker conjugates and cytotoxic small molecules are expected to be applied to the field of biological medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a topoisomerase inhibitor and a conjugate thereof and application thereof. BACKGROUND

[0002] The basic components of an antibody-drug conjugate (ADC) include an antibody or antibody-like ligand, a linker, and a cytotoxic small molecule. The antibody-drug conjugate utilizes the specific recognition of an antibody to an antigen to transport a small molecule drug into or near a target cell and effectively release a toxic molecule, achieving a therapeutic purpose. In 2000, Mylotrag (Gemtuzumab Ozogamicin) of Pfizer was first marketed, and the ADC field entered the public eye, triggering a market boom. Currently, there are 16 ADC drugs successfully marketed worldwide.

[0003] The cytotoxic small molecule in the antibody-drug conjugate can be a camptothecin derivative, which has an antitumor effect by inhibiting topoisomerase I. Recently, Immunomedics developed a new ADC drug IMMU-132 (Sacituzumab Govitecan) using camptothecin derivative SN-38 (the active metabolite of irinotecan) as a toxin molecule, which showed good antitumor effect. IMMU-132 uses a pH-sensitive linker to release toxins near tumor cells, killing tumor cells, but its stability is poor, and the released SN-38 is prone to phenolic hydroxyl glycosylation, thereby producing unnecessary toxicity. Another camptothecin derivative Dxd (amide derivative formed by irinotecan and hydroxyacetic acid) was developed by Daiichi Sankyo / AstraZeneca as a toxin molecule for ADC drug DS 8201a (Enhertu), which also showed good antitumor effect. DS-8201a uses a tetrapeptide linker-Gly-Gly-Phe-Gly-, which has good stability, but still has problems such as poor activity, low safety, and tumor drug resistance.

[0004] Therefore, there is an urgent need in the art to develop a camptothecin derivative with more optimal antitumor activity, which can improve its safety and effectiveness in the application of ADC drugs, thereby obtaining an antitumor drug with excellent efficacy. SUMMARY

[0005] The purpose of the present application is to provide an antibody-drug conjugate containing a camptothecin compound as a drug molecule, and an intermediate that can efficiently conjugate a camptothecin derivative with an antibody, thereby improving the efficacy of the antibody-drug conjugate.

[0006] In a first aspect, the present application provides a compound of formula (I), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the compound has the general formula:

[0007]

[0008] Y is selected from the group consisting of:

[0009] In another preferred embodiment, Y is selected from the group consisting of:

[0010] In another preferred embodiment, the compound is selected from the group consisting of:

[0011]

[0012] In a second aspect, the present application provides a linker conjugate of formula (II), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the compound has the general formula:

[0013]

[0014] Y is as defined in the first aspect of the present application;

[0015] L1 is a polypeptide residue selected from the group consisting of -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, preferably -Gly-Gly-Phe-Gly-;

[0016] L2 is a linker unit for linking to an antibody, and is

[0017] In another preferred embodiment, the compound is selected from the group consisting of:

[0018]

[0019]

[0020] In a third aspect, the present application provides an antibody-drug conjugate (ADC) of formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein Ab is an antibody or an antibody fragment; n is 2-8; and the ADC drug is selected from the group consisting of:

[0021]

[0022]

[0023]

[0024] In another preferred embodiment, n is 3.3-4.4, preferably 3.3-4.0, preferably 3.3-3.8, and more preferably 3.3-3.6.

[0025] Y, L1, and L2 are as defined in the second aspect of the present application.

[0026] In another preferred embodiment, the ADC drug is selected from the group consisting of:

[0027]

[0028] In a fourth aspect, the present application provides a linker conjugate of formula (II) for use in the preparation of an antibody-drug conjugate (ADC).

[0029] In another preferred embodiment, the antibody is an antibody capable of binding to a tumor-associated antigen.

[0030] In another preferred embodiment, the antibody-drug conjugate is formed by conjugating an antibody with a compound of formula (II) as defined in the second aspect of the present application.

[0031] In another preferred embodiment, the present application further provides a method for preparing an antibody-drug conjugate, comprising the following steps:

[0032] (1) providing a reaction system, and connecting a drug small molecule and a polypeptide residue at a specific site, and connecting a specific linker unit at the N-terminus of the polypeptide residue, thereby preparing a linker conjugate;

[0033] (2) providing another reaction system, wherein the reaction system comprises an antibody and a linker conjugate, and the linker conjugate comprises a drug small molecule, a polypeptide residue, and a linker unit;

[0034] (3) in the reaction system, conjugating the antibody and the linker conjugate, thereby preparing an antibody-drug conjugate as defined in the third aspect of the present application.

[0035] ​​In a fifth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0036] (i) an antibody-drug conjugate according to the third aspect of the present application, and

[0037] (ii) a pharmaceutically acceptable carrier.

[0038] In a sixth aspect of the present application, there is provided the use of an antibody-drug conjugate according to the third aspect of the present application or a pharmaceutical composition according to the fifth aspect of the present application for the manufacture of a medicament for the treatment of a tumor.

[0039] In another preferred embodiment, the tumor is selected from the group consisting of colorectal cancer, pancreatic cancer, cervical cancer, brain cancer, lung cancer, head and neck cancer, cholangiocarcinoma, gastric cancer.

[0040] It should be understood that, within the scope of the present application, all the technical features described above and in the following (e.g. in the examples) of the present application can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Chromatogram of P1 compound (DAD 18, Sig = 254 nm) is shown.

[0042] Figure 2 Chromatogram of P2 compound (DAD 18, Sig = 254 nm) is shown.

[0043] Figure 3 Chromatogram of P3 compound (DAD 18, Sig = 254 nm) is shown.

[0044] Figure 4 Chromatogram of P4 compound (DAD 18, Sig = 254 nm) is shown.

[0045] Figure 5 Chromatogram of A1 (DAD 18, Sig = 254 nm) is shown.

[0046] Figure 6 Chromatogram of A2 (DAD 18, Sig = 254 nm) is shown.

[0047] Figure 7 Chromatogram of A3 (DAD 18, Sig = 254 nm) is shown.

[0048] Figure 8 Chromatogram of A4 (DAD 18, Sig = 254 nm) is shown.

[0049] Figure 9 The results show that compounds P1, P2, P3, P4 effectively kill HCT-116 (Figure A) and HT-29 (Figure B) cells.

[0050] Figure 10 The results show that ADC-1, ADC-2, ADC-3, ADC-4 effectively kill HCT116 (Figure A), HT29 (Figure B), RKO (Figure C), SW480 (Figure D) cells.

[0051] Figure 11 The results show that ADC-1, ADC-2, ADC-3, ADC-4 effectively kill BxPC-3 cells.

[0052] Figure 12 The results show that ADC-1, ADC-2, ADC-3, ADC-4 effectively kill SiHa cells.

[0053] Figure 13 The results show that ADC-1, ADC-2, ADC-3, ADC-4 effectively kill U-87MG (Figure A), U-118MG (Figure B) cells.

[0054] Figure 14 The results show that ADC-1, ADC-2, ADC-3, ADC-4 effectively kill HCC827 (Figure A), EBC-1 (Figure B) cells at certain concentrations.

[0055] Figure 15 The results show that ADC-3 has significant in vivo tumor inhibition activity on colorectal cancer cell HT29 cells. DETAILED DESCRIPTION

[0056] The present inventors have made extensive and in-depth research, and unexpectedly found a class of topoisomerase inhibitors shown as formula I, a linker conjugate for antibody-drug conjugate shown as formula II, and an antibody-drug conjugate shown as formula III. Experimental results show that the antibody-drug conjugate described in the present application uses a highly lipophilic camptothecin derivative (i.e. a compound of formula I) as a drug small molecule, which has good membrane penetration, thereby greatly improving the efficacy of the ADC drug. On this basis, the present inventors have completed the present application.

[0057] TERMS

[0058] As used herein, the terms "antibody-drug conjugate (ADC) of the present application", "antibody-drug conjugate of the present application", "conjugate of the present application" or "ADC of the present application" are used interchangeably to refer to an antibody-drug conjugate having the formula (III).

[0059] As used herein, the terms "small molecule drug", "drug small molecule" and "camptothecin derivative" are used interchangeably and refer to a compound having the formula (I).

[0060] As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the compounds of the present application which are suitable for use as a medicament with the acid or base. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of the present application with an acid. Suitable acids for salt formation include, but are not limited to, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and the like inorganic acids; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like organic acids; and proline, phenylalanine, aspartic acid, glutamic acid and the like amino acids.

[0061] Another preferred class of salts is the salts of the compounds of the present application with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkylammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, t-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts of morpholine, piperazine, lysine, respectively.

[0062] In the present application, the term "DAR value" refers to the ratio of drug to antibody.

[0063] In the present application, the term "pharmaceutically acceptable" ingredient refers to those substances which are suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0064] In the present application, the term "camptothecin derivative" as part of another compound is a group of a camptothecin derivative, i.e. a group of a camptothecin derivative after losing H atom.

[0065] In the present application, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology 170:4854-4861). The antibody can be a murine, human, humanized, chimeric antibody or derived from another species.

[0066] In the present application, the term "antibody fragment" includes a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323); fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope- binding fragments of any of the above which bind to a cancer cell antigen, viral antigen, or microbial antigen in an immunospecific manner; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0067] In the present application, the antibody that forms the antibody-drug conjugate preferably retains its antigen binding ability as in its original wild-type state. Thus, the antibody in the present application is capable, and preferably specifically, binds to an antigen. Antigens of interest include, for example, tumor associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (as known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (as known antigens to which antibodies bind can be one or a subset of the above categories, while other subsets include other molecules / antigens of particular interest (as compared to the antigen of interest).

[0068] Antibodies for use in antibody-drug conjugates include, but are not limited to, antibodies to cell surface receptors and tumor associated antigens. Such tumor associated antigens are well known in the art and can be prepared by well known methods of antibody production and information. In an effort to develop effective cell level targets for cancer diagnosis and therapy, researchers have sought transmembrane or other tumor associated polypeptides. These targets are expressed specifically on the surface of one or more cancer cells and are expressed rarely or not at all on the surface of one or more non-cancer cells. Typically, such tumor associated polypeptides are overexpressed on the surface of cancer cells relative to non-cancer cells. Identification of such tumor associated factors can greatly enhance the specific targeting properties of antibody-based cancer therapies.

[0069] Compounds

[0070] In the present application, the compounds represented by the general formula (I), (II) and (III) can contain one or more chiral centers, and the presence of enantiomers and diastereomers. The compounds represented by the general formula (I), (II) and (III) of the present application can also contain many geometric isomers such as olefins, C=N double bonds. Unless otherwise specified, all chiral (enantiomers and diastereomers), racemates, cis geometric isomers, trans geometric isomers, cis and trans geometric isomer mixtures described above are included in the present application. For enantiomers, two enantiomers can be obtained by using general chiral resolution methods or asymmetric synthesis methods. For diastereomers, separation can be achieved by stepwise recrystallization or chromatographic separation and the like.

[0071] In the present application, the term "Y structure compound" refers to a small molecule compound with a -Boc protecting group coupled with a camptothecin drug, and its structural formula is as follows:

[0072]

[0073] Wherein, Y is selected from the following group:

[0074] In another preferred example, Y is selected from the following group:

[0075] In another preferred example, the Y structure compound is selected from the following group:

[0076]

[0077] In the present application, the LogP value of the compound of formula (I) is 1.5-3.0.

[0078] In another preferred example, the LogP value of the compound of formula (I) is 2.2-3.0, preferably 2.5-2.7.

[0079] In the present application, the linker conjugate is composed of a drug small molecule and a linker, and the linker includes a polypeptide residue L1 and a linker unit L2;

[0080] Wherein, the polypeptide residue L1 is selected from the following group: -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, preferably -Gly-Gly-Phe-Gly-;

[0081] The linker unit L2 is

[0082] In the present application, the LogP value of the compound of formula (II) is 1.8-3.2, preferably 2.65-3.10.

[0083] In the present application, the produced linker conjugate units are coupled to the antibody through the linker to produce a conjugate with partial interchain cross-linking. The antibody drug conjugate can be used to target the delivery of the drug to the target cell population, such as tumor cells. The antibody drug conjugate can specifically bind to the cell surface protein, and the produced conjugate is then endocytosed by the cell. In the cell, the drug is released in the form of an active drug to produce efficacy.

[0084] In the present application, the HIC t value of the compound of formula (III) at DAR value of 2 is 16-18 min, preferably 16.4-17.8 min. R

[0085] In another preferred embodiment, the HIC t value of the compound of formula (III) at DAR value of 3 is 16-20 min, preferably 18-20 min. R

[0086] In another preferred embodiment, the HIC t value of the compound of formula (III) at DAR value of 4 is 17-21 min, preferably 19-21 min. R

[0087] In the present application, the average DAR value of the compound of formula (III) is between 3 and 4, preferably 3.2-3.6.

[0088] In another preferred embodiment, the content of the compound of formula (III) at DAR value of 1 is 0.

[0089] In another preferred embodiment, the content of the compound of formula (III) at DAR value of 2 is 2-15%, preferably 4-8%.

[0090] In another preferred embodiment, the content of the compound of formula (III) at DAR value of 3 is 30-45%, preferably 37-43%.

[0091] In another preferred embodiment, the content of the compound of formula (III) at DAR value of 4 is 45-70%, preferably 47-60%.

[0092] In the present application, the reference compounds of the small molecule drugs Dxd and Exatecan have the following structural formulas:

[0093]

[0094] ​​​In the present application, the linker conjugates A5 and A6 can be purchased from MedChemExpress (MCE) company, which are used to synthesize the reference compounds MC-GGFG-Dxd (ADC-5) and MC-GGFG-Exatecan (ADC-6) in the present application, and the structures of A5 and A6 are shown as follows:

[0095]

[0096] Preparation method

[0097] The preparation method of the compounds of the present application represented by formula (I), (II) and (III) is described in more detail below, but these specific methods do not constitute any limitation to the present application. The compounds of the present application can also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, which can be easily performed by those skilled in the art to which the present application belongs.

[0098] Typically, the preparation process of the compounds of the present application is as follows, wherein the raw materials and reagents used are commercially available if not specifically stated.

[0099] (1) Preparation method of a small molecule drug

[0100] Specifically, the preparation method of the small molecule drug is as follows:

[0101] First step: to the DCM solution containing the compound of Y structure, add NHS and EDCI, stir and slowly drop the DMF solution containing Exatecan mesylate and TEA, stir to get white solid.

[0102] Second step: the product obtained in the first step is dissolved in DCM and TFA is added, and the reaction solution is directly purified by Prep-HPLC to obtain the small molecule drug.

[0103] (2) Preparation method of linker conjugate

[0104] Specifically, the preparation method of the linker conjugate is as follows:

[0105] First step: to the DCM solution containing the small molecule drug, add NHS and EDC, stir and slowly drop the DMF solution containing Boc-Gly-Gly-Phe-Gly-OH, stir again, spin dry the reaction solution to obtain the product, and the product is directly used for the next reaction without purification.

[0106] Second step: the product of the first step is dissolved in DCM and TFA is added, and the reaction solution is spin-dried to obtain white solid after stirring, and the product is directly used for the next reaction without purification.

[0107] Step 3: To the solution of the product of Step 2 in DCM, add NHS and EDCI, stir, and then slowly add a solution of 6-Maleimidocaproic acid in DMF until the reaction is complete to produce the linker conjugate.

[0108] (3) Preparation method of antibody-drug conjugate

[0109] Specifically, the preparation method of the antibody-drug conjugate comprises the following steps:

[0110] Step 1: At 37°C, add EDTA aqueous solution and TCEP aqueous solution to the PBS buffer solution of the antibody, mix, supplement with PBS, and then place on a metal bath shaker for shaking reaction, and then transfer to room temperature environment.

[0111] Step 2: Dissolve the linker conjugate in DMSO, and then add it to the above solution, and then place on a metal bath shaker for shaking reaction.

[0112] Step 3: Add L-cysteine to the above solution for coupling termination, and then perform ultrafiltration using a 10KDa ultrafiltration tube, with histidine-hydrochloric acid buffer as the buffer, and repeat 25 times to produce the antibody-drug conjugate (ADC), and then perform concentration detection.

[0113] Pharmaceutical composition and administration method

[0114] The pharmaceutical composition of the present application comprises a safe and effective amount of the compound of the present application or a pharmacologically acceptable salt thereof and a pharmacologically acceptable carrier. The "safe and effective amount" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably 10-1000 mg of the compound of the present application per dose. Preferably, the "dose" is a capsule or a tablet.

[0115] In the present application, the pharmaceutical composition comprises an effective amount of the ADC according to the present application as an active ingredient, and at least one pharmaceutically acceptable carrier. When prepared, the active ingredient is usually wrapped in a carrier which can exist in the form of a capsule or a sachet.

[0116] "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler substances or gel materials, which are suitable for human use and which are of sufficient purity and sufficiently low toxicity to be used in the compositions of the present application. By "compatible" is meant that the components of the composition are capable of being commingled with the compounds of the present application, and with each other, in the dosage form with no interaction that significantly affects the efficacy of the compounds. Examples of suitable pharmaceutically acceptable carriers are celluloses and their derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose sodium, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., lecithin), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0117] The pharmaceutical composition is an injection, a capsule, a tablet, a pill, a powder or a granule.

[0118] The mode of administration of the compounds or pharmaceutical compositions of the present application is not narrowly critical and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0119] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one carrier, such as sodium citrate or dicalcium phosphate, or with the following: (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) absorbents, such as kaolin and bentonite clay; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents.

[0120] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells, such as enteric coatings and other coatings and shells known in the art. They can contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients. ​

[0121] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0122] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0123] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0124] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0125] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0126] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0127] The treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.

[0128] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 50–1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0129] The main advantages of this invention are:

[0130] 1. The present application provides four compounds of formula I (P1, P2, P3, P4), which are novel camptothecin derivatives, and have good lipophilicity and membrane permeability compared with reference compounds Dxd and Exatecan.

[0131] 2. The four linker conjugates of formula II (A1, A2, A3, A4) have high lipophilicity and good membrane permeability.

[0132] 3. The present application provides four novel linker conjugates, and the antibody-drug conjugates of formula III prepared by the four conjugates have more excellent antitumor activity compared with the compounds MC-GGFG-Dxd (ADC-5) and MC-GGFG-Exatecan (ADC-6) with similar structures.

[0133] 4. The antibody-drug conjugates provided by the present application have almost zero proportion of naked antibodies and low cross-linking degree ADCs (mass spectrometry does not detect components with DAR of 0 and 1).

[0134] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The described preferred methods and materials are only examples.

[0136] In the present application, the structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR determination is carried out by using Bruker AVANCE-400 nuclear magnetic instrument. The LCMS determination is carried out by using Waters 2695 liquid chromatography-mass spectrometry (MS model: Micromass ZQ).

[0137] The HPLC determination is carried out by using Agilent 1100 high pressure liquid chromatograph (ZORBAX SP-C18 250x4.6mm chromatographic column, Eclipse Plus-C18 250x4.6mm chromatographic column).

[0138] The known starting materials of the present application can be used or synthesized according to methods known in the art, wherein A5, A6 are commercially available from MedChemExpress (MCE) company.

[0139] The reaction temperature is room temperature (20-35°C) unless otherwise specified in the examples.

[0140] The silica gel used in the thin layer chromatography (TLC) in the examples has a specification of 0.2 mm ± 0.03 mm. The prep-TLC used for purifying the compounds has a specification of 0.4-0.5 mm. The column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier. The full-automatic medium-pressure rapid purification instrument (Combi Flash Rf+UV-VIS) used has Silica Flash Column 4 g, 12 g, and 25 g. The eluent system used in the column chromatography and the developing system used in the thin layer chromatography include: A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and a small amount of ammonia water or acetic acid and the like can also be added to adjust the basicity or acidity.

[0141] The high-pressure preparative liquid chromatograph (manufacturer: Shimadzu, model: LC-20AP) used for purifying the final sample compounds in the examples has an Ultimate XB-C18 chromatographic column (150 x 30 mm, 5 μm). The mobile phase system has: A: acetonitrile and water (containing 0.1% trifluoroacetic acid) system; B: acetonitrile and water (containing 0.1% acetic acid) system.

[0142] Preparation method of P1 in Example 1

[0143] 2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-phenylacetamide

[0144]

[0145] First Step: To a solution of 1a (150 mg, 0.56 mmol) in DCM (10 mL), add NHS (68.7 mg, 0.56 mmol) and EDCI (113.28 mg, 0.56 mmol) and stir the reaction at 25 °C for 1 h, then slowly add a solution of Exatecan mesylate (260.73 mg, 0.48 mmol) and TEA (0.08 mL, 0.56 mmol) in DMF (10 mL) and stir the reaction at 25 °C for 16 h, TLC shows the reaction is complete, the reaction mixture is directly purified by silica gel column (DCM:MeOH = 3:1) to give 2a 350 mg as a white solid in 89.4% yield.

[0146] LCMS (ESI): t R = 2.745, m / z = 669.3 [M+H] + .

[0147] Second Step: Dissolve the obtained 2a (126.32 mg, 0.18 mmol) in DCM (1 mL) and add TFA (1 mL) and stir the reaction at 0 °C for 2 h, the reaction mixture is directly purified by Prep-HPLC (H2O (0.225% TFA)-ACN) to give 3a 90.7 mg as a white solid in 80.5% yield.

[0148] LCMS (ESI): t R = 1.876, t R = 2.098, m / z = 569.3 [M+H] + .

[0149] HPLC: t R = 3.88 min, 4.21 min, purity: 97%.

[0150] 1 HNMR (400 MHz, DMSO-d6) δ 8.72-8.53 (m, 1H), 7.86-7.82 (m, 1H), 7.44-7.12 (m, 6H), 6.52 (s, 1H), 5.60-5.12 (m, 5H), 4.78 (d, J = 18.96 Hz, 1H), 4.44 (s, 1H), 3.22-3.01 (m, 3H), 2.45-2.35 (m, 3H), 2.25-2.03 (m, 2H), 1.95-1.81 (m, 2H), 0.90-0.86 (m, 3H).

[0151] The chromatogram of the P1 compound is shown in Figure 1 .

[0152] Preparation method of Example 2 P2

[0153] (S)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] quinoline-1-yl)-2-phenylacetamide

[0154]

[0155] First step: To a solution of 1a (100 mg, 0.39 mmol) in DCM (3 mL), add NHS (50.38 mg, 0.429 mmol) and EDCI (83.07 mg, 0.429 mmol), stir the reaction at 20 °C for 1 h, then slowly dropwise add a solution of Exatecan mesylate (188.48 mg, 0.351 mmol) and TEA (0.061 mL, 0.429 mmol) in DMF (2 mL), stir the reaction at 25 °C for 16 h, TLC shows the reaction is complete. Then, add H2O (20 mL) and DCM (30 mL) to extract, the organic phase is washed with H2O (20 mL x 2) twice, dry over anhydrous sodium sulfate, the resulting solution is directly purified by silica gel column (DCM:EA = 1:3) to give white solid 2a 130 mg, yield 49.85%.

[0156] LCMS (ESI): t R = 1.952, m / z = 669.3 [M+H] + .

[0157] Second step: Resuspend the obtained 2a (120.0 mg, 0.18 mmol) in DCM (2 mL), and add TFA (3.10 g, 26.93 mmol), stir the reaction at 20 °C for 2 h, the reaction solution is directly purified by Prep-HPLC (H2O (0.225% TFA)-ACN) to give white solid 3a 80.0 mg, yield 78.4%.

[0158] LCMS (ESI): t R = 1.042, m / z = 569.3 [M+H] + .

[0159] HPLC: t R = 3.87 min, purity: 99%.

[0160] 1HNMR (400 MHz, DMSO-d6) δ 9.07 (d, J = 8.2 Hz, 1H), 8.75 (s, 3H), 7.78 (d, J = 10.8 Hz, 1H), 7.49 - 7.37 (m, 2H), 7.34 - 7.18 (m, 4H), 6.52 (s, 1H), 5.54 - 5.34 (m, 3H), 5.19 (d, J = 18.8 Hz, 1H), 4.89 (s, 1H), 4.39 (d, J = 19.2 Hz, 1H), 3.26 - 3.07 (m, 2H), 2.40 (d, J = 2.0 Hz, 3H), 2.36 - 2.28 (m, 1H), 2.17 - 2.06 (m, 1H), 1.94 - 1.80 (m, J = 7.2 Hz, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0161] The chromatogram of the P2 compound is shown in Figure 2

[0162] The preparation method of Example 3,

[0163] (R)-2-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-(thiophen-2-yl)acetamide

[0164]

[0165] First step: To the solution of 1a (80 mg, 0.31 mmol) in DCM (3 mL), add NHS (40.16 mg, 0.342 mmol) and EDCI (66.22 mg, 0.342 mmol), stir the reaction at 20 °C for 1 h, then slowly drop the solution of Exatecan mesylate (150.25 mg, 0.279 mmol) and TEA (34.96 mg, 0.342 mmol) in DMF (2 mL) and stir the reaction at 25 °C for 16 h, TLC shows the reaction is complete. Then, add H2O (20 mL) and DCM (30 mL) to extract, the organic phase is washed with H2O (20 mL x 2) twice, dried over anhydrous sodium sulfate, the obtained solution is directly purified by silica gel column (DCM:EA = 1:3) to give white solid 2a 50 mg, yield 23.83%.

[0166] LCMS (ESI): t R = 1.925, m / z = 675.3 [M+H] + . ​

[0167] Second step: The obtained 2a (50.0 mg, 0.074 mmol) was dissolved in DCM (0.5 mL) and TFA (0.775 g, 0.5 mL, 6.73 mmol) was added. The reaction was stirred at 20 °C for 2 h. The reaction solution was directly purified by Prep-HPLC (H20 (0.225% TFA)-ACN) to give white solid 3a 25.0 mg, yield 58.71%.

[0168] LCMS (ESI): t R = 1.014, m / z = 575.3 [M+H] + .

[0169] HPLC: t R = 3.93 min, purity: 99%.

[0170] 1H NMR (400 MHz, DMSO-d6) δ 8.80 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.36 (d, J = 5.2 Hz, 1H), 7.28 (s, 1H), 7.02 (d, J = 3.6 Hz, 1H), 6.89 (dd, J = 5.2, 3.6 Hz, 1H), 6.52 (s, 1H), 5.57 - 5.49 (m, 1H), 5.42 (s, 2H), 5.25 (d, J = 19.2 Hz, 1H), 4.93 - 4.80 (m, 2H), 3.17 (tt, J = 11.6, 6.0 Hz, 2H), 2.39 (d, J = 2.0 Hz, 3H), 2.32 - 2.21 (m, 1H), 2.10 (td, J = 11.6, 11.2, 4.2 Hz, 1H), 1.86 (hept, J = 7.2 Hz, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0171] The chromatogram of P3 compound is shown in Figure 3 .

[0172] Preparation method of Example 4 P4

[0173] (1S,3R)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)cyclopentane-1-carboxamide

[0174]

[0175] First step: To a solution of 1a (130 mg, 0.55 mmol) in DCM (10 mL), add NHS (64.59 mg, 0.55 mmol) and EDCI (106.50 mg, 0.55 mmol), stir the reaction at 20 °C for 1 h, then slowly dropwise add a solution of Exatecan mesylate (245.13 mg, 0.46 mmol) and TEA (0.08 mL, 0.55 mmol) in DMF (10 mL), stir the reaction at 25 °C for 16 h, TLC shows the reaction is complete. Then, the reaction is directly purified by silica gel column (DCM:MeOH = 3:1) to give white solid 2a 160 mg, yield 44.9%.

[0176] LCMS (ESI): t R = 2.693, m / z = 647.3 [M+H] + .

[0177] Second step: Dissolve the obtained 2a (122.45 mg, 0.18 6 mmol) in DCM (1 mL), and add TFA (1 mL), stir the reaction at 20 °C for 2 h, the reaction is directly purified by Prep-HPLC (H2O (0.225% TFA)-ACN) to give bright yellow solid 3a 93.6 mg, yield 83.4%.

[0178] LCMS (ESI): t R = 1.860, m / z = 547.3 [M+H] + .

[0179] HPLC: t R = 3.90 min, purity: 99%.

[0180] 1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 8.6 Hz, 1H), 8.37 (s, 1H), 7.79 (d, J = 10.92 Hz, 1H), 7.31 (s, 1H), 5.57-5.52 (m, 1H), 5.42 (s, 2H), 5.24-5.11 (m, 2H), 3.46-3.40 (m, 2H), 3.18-3.15 (m, 2H), 2.81-2.60 (m, 2H), 2.39-2.31 (m, 3H), 2.16-2.09 (m, 7.4 Hz, 3H), 1.95-1.37 (m, 7H), 0.87 (t, J = 14.6 Hz, 7.24 Hz, 3H).

[0181] The chromatogram of P4 compound is shown in Figure 4 .

[0182] Preparation of Example 5 A1

[0183] N-((7S)-7-benzyl-14-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-2,5,8,11,14-pentaoxo-13-phenyl-3,6,9,12-tetraazatetradecyl)-6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0184]

[0185] First Step: To a solution of 1a (86 mg, 0.15 mmol) in DCM (2 mL), was added NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stirred at 20 °C for 1 h, after which a solution of Boc-Gly-Gly-Phe-Gly-OH (69.83 mg, 0.16 mmol) in DMF (2 mL) was added dropwise slowly and stirred at 25 °C for 4 h, TLC showed the reaction was complete. The reaction mixture was concentrated to give a white solid 2a, which was used in the next step without further purification.

[0186] Second Step: 2a was dissolved in DCM (1 mL), TFA (1 mL) was added and stirred at 20 °C for 2 h, after which the reaction mixture was concentrated to give a white solid 3a, which was used in the next step without further purification.

[0187] Third Step: To a solution of 3a in DCM (2 mL), was added NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stirred at 20 °C for 1 h, after which a solution of 6-Maleimidocaproicacid (33.79 mg, 0.16 mmol) in DMF (2 mL) was added dropwise slowly and stirred at 25 °C for 4 h, TLC showed the reaction was complete. The reaction mixture was purified by Prep-HPLC (H2O (0.225% TFA)-ACN) to give a white solid 4a 72.24 mg, 42.8% yield.

[0188] LCMS (ESI): t R = 2.798, t R = 2.902, m / z = 1080.4 [M+H] + .

[0189] HPLC: tR = 5.02 min, 5.19 min, purity: 98%.

[0190] 1H NMR (400 MHz, DMSO-d6) δ 8.99 - 8.79 (m, 1H), 8.62 - 8.55 (m, 1H), 8.30 - 8.15 (m, 1H), 8.14 - 7.90 (m, 3H), 7.81 - 7.76 (m, 1H), 7.52 - 7.35 (m, 1H), 7.33 - 7.31 (m, 2H), 7.28 - 7.08 (m, 8H), 6.98 (s, 2H), 6.50 - 6.49 (m, 1H), 5.59 - 5.36 (m, 4H), 5.27 - 5.11 (m, 1H), 4.63 - 4.37 (m, 2H), 3.84 - 3.80 (m, 2H), 3.75 - 3.69 (m, 1H), 3.66 - 3.61 (m, 2H), 3.59 - 3.54 (m, 1H), 3.37 - 3.33 (m, 2H), 3.23 - 3.10 (m, 2H), 3.02 - 2.93 (m, 1H), 2.76 - 2.66 (m, 1H), 2.47 - 2.35 (m, 3H), 2.33 - 2.28 (m, 1H), 2.11 - 2.00 (m, 3H), 1.92 - 1.81 (m, 2H), 1.54 - 1.37 (m, 4H), 1.21 - 1.14 (m, 2H), 0.90 - 0.85 (m, 3H).

[0191] The chromatogram of the A1 compound is shown in Figure 5

[0192] Process for the preparation of Example 6 A2

[0193] N-((7S,13S)-7-benzyl-14-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-2,5,8,11,14-pentaoxo-13-phenyl-3,6,9,12-tetraazatetradecyl)-6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)hexanamide

[0194]

[0195] ​First step: To a solution of 1a (86 mg, 0.15 mmol) in DCM (2 mL), was added NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stirred at 20 °C for 1 h, after which a solution of Boc-Gly-Gly-Phe-Gly-OH (69.83 mg, 0.16 mmol) in DMF (2 mL) was added dropwise slowly and stirred at 25 °C for 4 h, TLC showed the completion of the reaction. The reaction mixture was concentrated to get a white solid 2a, which was used for the next step without further purification.

[0196] Second step: 2a was dissolved in DCM (1 mL), TFA (1 mL) was added and stirred at 20 °C for 2 h, after which the reaction mixture was concentrated to get a white solid 3a, which was used for the next step without further purification.

[0197] Third step: To a solution of 3a in DCM (2 mL), was added NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stirred at 20 °C for 1 h, after which a solution of 6-Maleimidocaproicacid (33.79 mg, 0.16 mmol) in DMF (2 mL) was added dropwise slowly and stirred at 25 °C for 4 h, TLC showed the completion of the reaction. The reaction mixture was directly purified by Prep-HPLC (H20 (0.225% TFA) - ACN) to get a white solid 4a 56.5 mg, yield 33.63%.

[0198] LCMS (ESI): t R = 2.812, m / z = 1080.4 [M+H] + .

[0199] HPLC: t R = 5.03 min, purity: 97%.

[0200] 1HNMR (400 MHz, DMSO-d6) δ 8.80 (d, J = 8.2 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.25 (s, 1H), 8.15 - 7.91 (m, 3H), 7.77 (d, J = 10.8 Hz, 1H), 7.22 (ddt, J = 30.0, 22.4, 11.2 Hz, 11H), 6.97 (s, 2H), 5.58 - 5.27 (m, 4H), 5.13 (d, J = 18.8 Hz, 1H), 4.61 - 4.35 (m, 2H), 3.82 - 3.63 (m, 6H), 3.35 (s, 3H), 3.18 (s, 2H), 2.95 (d, J = 13.6 Hz, 1H), 2.72 (s, 1H), 2.40 (s, 3H), 2.27 (s, 1H), 2.09 (t, J = 7.6 Hz, 3H), 1.86 (s, 2H), 1.56 - 1.37 (m, 4H), 1.18 (s, 2H), 0.89 (d, J = 7.6 Hz, 3H).

[0201] The chromatogram of the A2 compound is shown in Figure 6

[0202] Example 7 Preparation method of A3

[0203] N-((7S,13R)-7-benzyl-14-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1- yl)amino)-2,5,8,11,14-pentaoxo-13-(thiophen-2-yl)-3,6,9,12-tetraazatetradecyl)-6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexanamide

[0204]

[0205] First step: To a solution of 1a (23.51 mg, 0.043 mmol) in DCM (2 mL), add NHS (5.62 mg, 0.043 mmol) and EDCI (9.27 mg, 0.047 mmol), stir the reaction at 20 °C for 1 hour, then slowly dropwise add a solution of Boc-Gly-Gly-Phe-Gly-OH (6.98 mg, 0.043 mmol) and DMF (2 mL), stir the reaction at 25 °C for 4 hours, TLC shows the reaction is complete. The reaction is rotary evaporated to white solid 2a, the product is used directly for the next step without purification.

[0206] ​Second step: 2a was dissolved in DCM (1 mL), TFA (1 mL) was added, the reaction was stirred at 20 °C for 2 h, then the reaction solution was rotary evaporated to white solid 3a, the product was used in the next step without purification.

[0207] Third step: To the solution of 3a in DCM (2 ml), NHS (5.62 mg, 0.043 mmol) and EDCI (9.27 mg, 0.047 mmol) were added, the reaction was stirred at 20 °C for 1 h, then a solution of 6-Maleimidocaproicacid (9.5 mg, 0.045 mmol) in DMF (2 mL) was added dropwise slowly, the reaction was stirred at 25 °C for 4 h, TLC showed the reaction was completed. The reaction solution was directly purified by Prep-HPLC (H2O (0.225% TFA)-ACN) to give white solid 4a 20.13 mg, yield 42.60%.

[0208] LCMS (ESI): t R = 2.764, m / z = 1086.4 [M+H] + .

[0209] HPLC: t R = 4.99 min, purity: 99%.

[0210] 1 H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 7.6 Hz, 1H), 8.25 (t, J = 5.6 Hz, 1H), 8.11 - 8.02 (m, 2H), 7.96 (t, J = 5.6 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.34 - 7.14 (m, 7H), 6.98 (d, J = 3.6 Hz, 3H), 6.89 - 6.83 (m, 1H), 6.50 (s, 1H), 5.72 (d, J = 7.6 Hz, 1H), 5.57 - 5.48 (m, 1H), 5.40 (s, 2H), 5.27 (d, J = 19.2 Hz, 1H), 4.82 (d, J = 19.2 Hz, 1H), 4.44 (d, J = 12.4 Hz, 1H), 3.81 - 3.52 (m, 6H), 3.19 (s, 2H), 2.99 - 2.91 (m, 1H), 2.75 - 2.66 (m, 1H), 2.40 (s, 3H), 2.30 (d, J = 17.2 Hz, 1H), 2.09 (t, J = 7.2 Hz, 3H), 1.85 (p, J = 6.8 Hz, 2H), 1.53 - 1.36 (m, 4H), 1.18 (q, J = 8.0 Hz, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0211] The chromatogram of the A3 compound is shown in Figure 7

[0212] Example 8 Process for the preparation of A4

[0213] (1S,3R)-3-((S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10,13- tetraoxo-3,6,9,12-tetraazastearic acid amide)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl)cyclopentane-1-carboxamide

[0214]

[0215] First step: To a solution of 1a (82 mg, 0.15 mmol) in DCM (2 mL), add NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stir the reaction at 20 °C for 1 h, then slowly add a solution of Boc-Gly-Gly-Phe-Gly-OH (69.83 mg, 0.15 mmol) in DMF (2 mL) and stir the reaction at 25 °C for 4 h, TLC shows the reaction is complete. The reaction is concentrated to give a white solid 2a, the product is used directly in the next step without purification.

[0216] Second step: Resuspend 2a in DCM (1 mL), add TFA (1 mL) and stir the reaction at 20 °C for 2 h, then the reaction is concentrated to give a white solid 3a, the product is used directly in the next step without purification.

[0217] Third step: To a solution of 3a in DCM (2 mL), add NHS (18.04 mg, 0.15 mmol) and EDCI (29.75 mg, 0.15 mmol) and stir the reaction at 20 °C for 1 h, then slowly add a solution of 6-Maleimidocaproic acid (33.79 mg, 0.16 mmol) in DMF (2 mL) and stir the reaction at 25 °C for 4 h, TLC shows the reaction is complete. The reaction is purified directly by Prep-HPLC (H2O (0.225% TFA)-ACN) to give a white solid 4a 51.0 mg, 31.70% yield.

[0218] LCMS (ESI): t R ​= 2.625, m / z = 1058.5 [M+H] + .

[0219] HPLC: t R = 4.82 min, purity: 98%.

[0220] 1 HNMR (400 MHz, DMSO-d6) δ 8.68 (d, J = 8.7 Hz, 1H), 8.29-8.24 (m, 1H), 8.11 (d, J = 7.96 Hz, 1H), 8.05-7.97 (m, 2H), 7.91 (d, J = 7.72 Hz, 1H), 7.78 (d, J = 10.92 Hz, 1H), 7.30 (s, 1H), 7.23-7.15 (m, 4H), 7.14-7.07 (m, 1H), 6.98 (s, 2H), 6.51 (s, 1H), 5.57-5.52 (m, 1H), 5.47-5.34 (m, 2H), 5.23-5.07 (m, 2H), 4.54-4.44 (m, 1H), 4.11-4.06 (m, 1H), 3.75-3.57 (m, 6H), 3.36 (s, 2H), 3.22-3.05 (m, 3H), 2.83-2.67 (m, 2H), 2.38 (d, J = 1.92 Hz, 3H), 2.13-2.06 (m, 5H), 1.97-1.77 (m, 5H), 1.75-1.65 (m, 1H), 1.60-1.38 (m, 5H), 1.21-1.15 (m, 2H), 0.88-0.84 (m, 3H).

[0221] The chromatogram of the compound A4 is shown in Figure 8 .

[0222] Preparation method of ADC-1

[0223] Under the condition of 37°C, EDTA aqueous solution (100 mM, 20 μL), TCEP (10 mM, 15.48 μL) aqueous solution were added into the PBS buffer solution (16 mg / mL, 75 μL) of MET-EGFR bifunctional antibody (CN117659203A, SEQ ID NO: 2 and SEQ ID NO: 7), mixed, supplemented with PBS to 400 μL, and placed on a metal bath shaker, shaken at 37°C for 4 hours, and then restored to room temperature.

[0224] Compound A1 was dissolved in 100 μL of DMSO, 9 times the amount of antibody, and then added to the above solution, and placed on a metal bath shaker, shaken at 25°C for 2 hours.

[0225] To the solution, L-cysteine was added in 7 times equivalent to the antibody for stopping the coupling, and then ultrafiltration was performed using a 10 KDa ultrafiltration tube with 10 mM histidine-hydrochloric acid buffer for 25 times. Subsequently, concentration detection was performed, and the recovery rate was 66.19%.

[0226] The average value was calculated by HPLC, n = 3.56.

[0227] Example 10 Preparation method of ADC-2

[0228] To the PBS buffer solution of MET-EGFR bifunctional antibody (16 mg / mL, 75 μL) at 37°C, EDTA aqueous solution (100 mM, 20 μL) and TCEP (10 mM, 15.48 μL) aqueous solution were added, mixed, supplemented with PBS to 400 μL, and placed on a metal bath shaker and shaken at 37°C for 4 hours, and then returned to room temperature.

[0229] Compound A2 was dissolved in 100 μL of DMSO, 9 times equivalent to the antibody, and added to the above solution, and placed on a metal bath shaker and shaken at 25°C for 2 hours.

[0230] To the solution, L-cysteine was added in 7 times equivalent to the antibody for stopping the coupling, and then ultrafiltration was performed using a 10 KDa ultrafiltration tube with 10 mM histidine-hydrochloric acid buffer for 25 times. Subsequently, concentration detection was performed, and the recovery rate was 60.97%.

[0231] The average value was calculated by HPLC, n = 3.48.

[0232] Example 11 Preparation method of ADC-3

[0233] To the PBS buffer solution of MET-EGFR bifunctional antibody (16 mg / mL, 75 μL) at 37°C, EDTA aqueous solution (100 mM, 20 μL) and TCEP (10 mM, 15.48 μL) aqueous solution were added, mixed, supplemented with PBS to 400 μL, and placed on a metal bath shaker and shaken at 37°C for 4 hours, and then returned to room temperature.

[0234] Compound A3 was dissolved in 100 μL of DMSO, 9 times equivalent to the antibody, and added to the above solution, and placed on a metal bath shaker and shaken at 25°C for 2 hours.

[0235] To the solution, L-cysteine was added in 7 times equivalent to the antibody for stopping the coupling, and then ultrafiltration was performed using a 10 KDa ultrafiltration tube with 10 mM histidine-hydrochloric acid buffer for 25 times. Subsequently, concentration detection was performed, and the recovery rate was 66.75%.

[0236] HPLC calculated average n = 3.50.

[0237] Example 12 Preparation method of ADC-4

[0238] To the PBS buffer solution of MET-EGFR bifunctional antibody (16 mg / mL, 75 μL) at 37 °C, add EDTA aqueous solution (100 mM, 20 μL), TCEP (10 mM, 15.48 μL) aqueous solution, mix, supplement PBS to 400 μL, and place on a metal bath shaker, oscillate at 37 °C for 4 h, and then restore to room temperature.

[0239] Dissolve compound A4 in 100 μL of DMSO, make it 9 times the amount of antibody, and then add it to the above solution, and place on a metal bath shaker, oscillate at 25 °C for 2 h.

[0240] In the above solution, add L-cysteine 7 times the amount of antibody to stop the coupling, and then use a 10 KDa ultrafiltration tube to perform ultrafiltration, with 10 mM histidine-hydrochloric acid buffer, repeat 25 times. Then perform concentration detection, and the recovery rate is 64.73%.

[0241] HPLC calculated average n = 3.32.

[0242] Example 13 Preparation method of ADC-5

[0243] To the PBS buffer solution of MET-EGFR bifunctional antibody (16 mg / mL, 75 μL) at 37 °C, add EDTA aqueous solution (100 mM, 20 μL), TCEP (10 mM, 15.48 μL) aqueous solution, mix, supplement PBS to 400 μL, and place on a metal bath shaker, oscillate at 37 °C for 4 h, and then restore to room temperature.

[0244] Dissolve compound Deruxtecan (catalog number: HY-13631E, MedChemExpress) in 100 μL of DMSO, make it 9 times the amount of antibody, and then add it to the above solution, and place on a metal bath shaker, oscillate at 25 °C for 2 h.

[0245] In the above solution, add L-cysteine 7 times the amount of antibody to stop the coupling, and then use a 10 KDa ultrafiltration tube to perform ultrafiltration, with 10 mM histidine-hydrochloric acid buffer, repeat 25 times. Then perform concentration detection, and the recovery rate is 64.73%.

[0246] HPLC calculated average n = 3.63.

[0247] Preparation method of ADC-6

[0248] To the PBS buffer solution (16 mg / mL, 75 μL) of MET-EGFR bifunctional antibody, EDTA aqueous solution (100 mM, 20 μL) and TCEP (10 mM, 15.48 μL) aqueous solution were added at 37℃, mixed, supplemented with PBS to 400 μL, and oscillated on a metal bath oscillator at 37℃ for 4 hours, and then restored to room temperature.

[0249] Compound MC-GGFG-Exatecan (catalog number: HY-114233, MedChemExpress) was dissolved in 100 μL of DMSO, 9 times the amount of antibody, and then added to the above solution, and oscillated on a metal bath oscillator at 25℃ for 2 hours.

[0250] In the above solution, 7 times the amount of L-cysteine was added to the antibody for coupling termination, and then ultrafiltration was performed using a 10KDa ultrafiltration tube, with 10 mM histidine-hydrochloric acid buffer repeated 25 times. Then the concentration was detected, and the recovery rate was 57.58%.

[0251] The average value was calculated by HPLC n=3.62.

[0252] Example 15 DAR value detection

[0253] The experimental procedure is as follows:

[0254] 20 μg of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, and ADC-6 were injected into the HPLC HIC column, eluted with different gradient mobile phase eluent, and the chromatogram was recorded. The DAR value was calculated according to DAR = Σ (relative peak area x number of loaded drugs) / 100.

[0255] The experimental results are as follows:

[0256] The DAR values of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, and ADC-6 are shown in Table 5:

[0257] The ADC-1, ADC-2, ADC-3, and ADC-4 of the present application have no obvious advantage in DAR value compared with the known antibody-drug conjugate ADC-5 and ADC-6, and are all between 3-4.

[0258] Table 5

[0259] Number DAR1 DAR2 DAR3 DAR4 Average DAR ADC-1 0 2.66％ 38.93％ 58.41％ 3.56 ADC-2 0 4.46％ 42.64％ 52.9％ 3.48 ADC-3 0 6.4％ 37.44％ 56.1％ 3.50 ADC-4 0 13.16％ 38.3％ 47.57％ 3.32 ADC-5 0 0 34.21％ 65.11％ 3.63 ADC-6 0 0 35.88％ 63.59％ 3.62

[0260] Evaluation of cytotoxicity of compounds P1, P2, P3, P4

[0261] To detect the inhibitory activity of compounds on the in vitro proliferation of HCT116 cells and HT29 cells. After treating the cells with different concentrations of compounds for six days, the proliferation of the cells was detected using the CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent, and the in vitro activity of the compound was evaluated according to the IC 50 value.

[0262] The experimental procedure is as follows:

[0263] 1. HCT116 / / HT-29 cells were cultured with 10% FBS RPMI-1640 medium

[0264] 2. Take HCT116 / / HT-29 cells in the logarithmic growth phase, wash once with PBS, add 2-3 mL trypsin and digest for 2-3 min, add 15 mL complete culture medium after the cells are well digested, blow and centrifuge, discard the supernatant, and add 10 mL complete culture medium to prepare a single cell suspension.

[0265] 3. HCT116 / / HT-29 cells were evenly spread in a black-walled transparent-bottom 96-well plate, 90 μL per well, and the cell number was 1000. The culture plate was incubated in an incubator for 18 hours.

[0266] 4. The compound was dissolved in DMSO to prepare a 10 mM stock solution.

[0267] 5. The compound was diluted to 5 concentration gradients: 1000, 200, 40, 8, and 1.6 nM.

[0268] 6. Add 10 μL of the prepared compound of different concentrations to the culture plate, two duplicate wells for each concentration. Incubate the culture plate in an incubator for 6 days.

[0269] 7. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and measure the chemiluminescence using a microplate reader.

[0270] 8. Process and analyze the data using Microsoft Excel and Graphpad Prism 7.

[0271] The experimental results are as follows:

[0272] IC of compounds P1, P2, P3, P4 on the in vitro proliferation of HCT-116 and HT-29 cells 50The values are shown in Table 6, and the effective killing of HCT-116 (Figure A) and HT-29 (Figure B) cells is shown Figure 9 .

[0273] Table 6

[0274] Number HCT116 HT-29 P1 16.09 8.53 P2 171.4 - P3 11.36 8.24 P4 849.5 111 Exatecan 9.83 6.08

[0275] * The numerical value is nM

[0276] The experimental results show that the compounds P1, P2, P3 and P4 in the application have obvious proliferation inhibition activity on HCT-116 and HT-29 cells.

[0277] Example 17 Detection of hydrophilicity and hydrophobicity of antibody-drug conjugates

[0278] The experimental steps are as follows:

[0279] 20 μg of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5 and ADC-6 were injected into an HPLC HIC chromatographic column, eluted by different gradient mobile phase eluent, and the retention time (t R ) was recorded, and the hydrophilicity and hydrophobicity of the antibody-drug conjugates were evaluated according to the length of the retention time.

[0280] The experimental results are as follows:

[0281] The HIC t R values of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5 and ADC-6 are shown in Table 7.

[0282] Among them, the retention time of ADC-3 is 19.48 min when the DAR value is 4, which is greater than that of ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan), indicating that the hydrophobicity of A3 molecules is higher than that of A5 (MC-GGFG-Dxd) and A6 (MC-GGFG-Exatecan), which has better membrane permeability and can improve the efficacy of ADC drugs. Secondly, the retention time of ADC-1, ADC-2 and ADC-3 when the DAR value is 4 is also greater than that of ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan), indicating that they also have better hydrophobicity.

[0283] Table 7

[0284] Number DAR1 DAR2 DAR3 DAR4 ADC-1 0 16.68 19.44 20.05 ADC-2 0 16.67 19.27 20.03 ADC-3 0 16.45 18.77 19.48 ADC-4 0 17.64 18.45 19.33 ADC-5 0 0 16.91 17.51 ADC-6 0 0 17.34 18.05

[0285] * The numerical value is min

[0286] The experimental results show that the antibody-drug conjugates (ADC-1, ADC-2, ADC-3 and ADC-4) have a longer retention time, indicating that the four ADC drugs have good hydrophobicity.

[0287] LogP calculation of compounds P1, P2, P3, P4, A1, A2, A3 and A4

[0288] The experimental steps are as follows:

[0289] The compound calculation simulation software is used to calculate the LogP values of compounds P1, P2, P3, P4, A1, A2, A3 and A4.

[0290] The experimental results are as follows:

[0291] The calculated LogP values of A1, A2, A3, A4, A5 and A6 are shown in Table 8, and the calculated LogP values of P1, P2, P3 and P4 are shown in Table 9.

[0292] Among them,

[0293] The Consensus LogP of P3 molecule is 3.08, which is significantly higher than the Consensus LogP of Dxd and Exatecan, so it has higher lipophilicity and better membrane penetration. In addition, other molecules also have higher lipophilicity.

[0294] Table 8

[0295]

[0296] Table 9

[0297]

[0298] The experimental results show that the compounds (P1, P2, P3, P4, A1, A2, A3 and A4) in the application have higher Consensus LogP values and higher lipophilicity.

[0299] Example 19 Evaluation of the cytotoxicity of antibody-drug conjugates on colorectal cancer tumor cells

[0300] In order to detect the in vitro proliferation inhibition activity of antibody-drug conjugates on HT29, HCT116, RKO and SW480 cells, the cells were treated with different concentrations of antibody-drug conjugates, and after six days of culture, the proliferation of the cells was detected using CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent, and the in vitro activity of the drug was evaluated according to the cell survival rate (%).

[0301] The experimental steps are as follows:

[0302] 1. HT29, HCT116, RKO, SW480 cells were cultured with 10% FBS RPMI-1640 / DMEM medium.

[0303] 2. Take the logarithmic growth period of HT29, HCT116, RKO, SW480 cells, wash once with PBS, add 2-3 mL trypsin and digest for 2-3 min, add 15 mL complete culture medium after the cell digestion is completed, blow and centrifuge, discard the supernatant, and add 10 mL complete culture medium to prepare a single cell suspension.

[0304] 3. HT29, HCT116, RKO, SW480 cells were evenly spread in a black wall transparent bottom 96-well plate, 90 μL per well, and the cell number was 1000. The culture plate was incubated in an incubator for 18 hours.

[0305] 4. The antibody-drug conjugate was diluted to 6 concentration gradients: 50, 10, 2, 0.4, 0.08, 0.016 μg / mL.

[0306] 5. Add 10 μL of different concentrations of antibody-drug conjugates to be tested to the culture plate, two duplicate wells for each concentration. The culture plate was incubated in an incubator for 6 days.

[0307] 6. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and use an enzyme label to measure the chemiluminescence. Use Microsoft Excel and Graphpad Prism 7 to process and analyze the data.

[0308] The experimental results are as follows:

[0309] The effective killing of HCT116 (Figure A), HT29 (Figure B), RKO (Figure C), and SW480 (Figure D) cells by ADC-1, ADC-2, ADC-3, and ADC-4 is shown in Figure 10 ;

[0310] Among them, as shown in HCT-116 (Figure A), ADC-3 and ADC-2 have obvious proliferation inhibition activity at high, medium and low concentrations, and are better than ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan).

[0311] As shown in HT-29 (Figure B), ADC-3 and ADC-2 have obvious proliferation inhibition activity at high and medium concentrations, and are better than ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan).

[0312] As shown in RKO (Figure C), ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan) only have obvious proliferation inhibition activity at high concentration, and the inhibition is weak; while ADC-3 has obvious proliferation inhibition activity at high and medium concentrations, and is superior to ADC-5 and ADC-6.

[0313] As shown in SW480 (Figure D), ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan) only have obvious proliferation inhibition activity at high concentration, and the inhibition is weak; while ADC-3 has obvious proliferation inhibition activity at high and medium concentrations, and is superior to ADC-5 and ADC-6.

[0314] The experimental results show that the antibody-drug conjugate has obvious proliferation inhibition activity on colorectal cancer tumor cells.

[0315] Example 20 Evaluation of the cytotoxicity of antibody-drug conjugate on pancreatic cancer tumor cells

[0316] In order to detect the in-vitro proliferation inhibition activity of the antibody-drug conjugate on BxPC-3 cells, the cells are treated with the antibody-drug conjugate at different concentrations, and after six days of culture, the proliferation of the cells is detected using CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent, and the in-vitro activity of the drug is evaluated according to the cell survival rate (%).

[0317] The experimental steps are as follows:

[0318] 1. BxPC-3 cells are cultured with 10% FBS RPMI-1640 medium.

[0319] 2. Take the BxPC3 cells in the logarithmic growth phase, wash them once with PBS, add 2-3 mL of trypsin for digestion for 2-3 min, add 15 mL of complete culture medium after the cells are well digested, blow and centrifuge, discard the supernatant, and then add 10 mL of complete culture medium to prepare a single cell suspension.

[0320] 3. The BxPC3 cells are evenly spread in a black-walled transparent-bottom 96-well plate, 90 μL per well, and the cell number is 1000. The culture plate is cultured in a culture box for 18 hours.

[0321] 4. The antibody-drug conjugate is diluted to 6 concentration gradients, which are 50, 10, 2, 0.4, 0.08, and 0.016 μg / mL.

[0322] 5. Add 10 μL of the prepared antibody-drug conjugate at different concentrations to the culture plate, with two replicates for each concentration. Incubate the culture plate in an incubator for 6 days.

[0323] 6. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and measure the chemiluminescence using a microplate reader. Process and analyze the data using Microsoft Excel and Graphpad Prism 7.

[0324] The experimental results are as follows:

[0325] The effective killing effects of ADC-1, ADC-2, ADC-3, and ADC-4 on BxPC-3 cells are shown in the figure. Figure 11 ;

[0326] in,

[0327] In BxPC-3 cells, ADC-3 exhibited significant inhibitory activity on proliferation at high, medium, and low concentrations, which was significantly superior to ADC-5 (MC-GGFG-Dxd).

[0328] Experimental results show that the antibody-drug conjugate in this invention has significant inhibitory activity against the proliferation of pancreatic cancer cells.

[0329] Example 21 Evaluation of the cytotoxicity of antibody-drug conjugates against cervical cancer tumor cells

[0330] To detect the inhibitory activity of antibody-drug conjugates on the in vitro proliferation of SiHa cells, cells were treated with different concentrations of the antibody-drug conjugates and cultured for six days. Cell proliferation was then assessed using the CTG (Cell Titer-Luminescent Cell Viability Assay, Promega) reagent, and the in vitro activity of the drug was evaluated based on cell viability (%).

[0331] The experimental steps are as follows:

[0332] 1. SiHa cells were cultured in 10% FBS RPMI-1640 medium.

[0333] 2. Take SiHa cells in the logarithmic growth phase, wash them once with PBS, add 2-3 mL of trypsin to digest them for 2-3 min, and after digestion with cells, add 15 mL of complete culture medium, pipette, centrifuge, discard the supernatant, and add another 10 mL of complete culture medium to prepare a single-cell suspension.

[0334] 3. Spread SiHa cells evenly into 96-well plates with black walls and a transparent bottom, 90 μL per well, with a cell count of 1000. Incubate the plates in an incubator for 18 hours.

[0335] The antibody-drug conjugate was diluted to 6 concentration gradients, i.e. 50, 10, 2, 0.4, 0.08, 0.016 μg / mL.

[0336] 4. 10 μL of the prepared antibody-drug conjugate of different concentrations was added to the culture plate, and two duplicate wells were set for each concentration. The culture plate was cultured in an incubator for 6 days.

[0337] 5. 100 μL of CTG reagent was added to the culture plate, and the plate was incubated at room temperature for 10 minutes. Chemiluminescence was measured using a microplate reader. The data were analyzed by Microsoft Excel and Graphpad Prism 7.

[0338] The experimental results are as follows:

[0339] The effective killing of SiHa cells by ADC-1, ADC-2, ADC-3 and ADC-4 is shown in Figure 12 ;

[0340] Among them,

[0341] ADC-3 has obvious proliferation inhibition on SiHa cells at a high concentration, while ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan) have no obvious proliferation inhibition activity on SiHa cells, and ADC-3 is significantly better than ADC-5 and ADC-6.

[0342] The experimental results show that the antibody-drug conjugate has obvious proliferation inhibition activity on cervical cancer tumor cells.

[0343] Example 22 Evaluation of the toxicity of antibody-drug conjugate on brain cancer tumor cells

[0344] In order to detect the in-vitro proliferation inhibition activity of the antibody-drug conjugate on U-87MG and U-118MG cells, the cells were treated with the antibody-drug conjugate at different concentrations, and after six days of culture, the proliferation of the cells was detected using the CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent. The in-vitro activity of the drug was evaluated according to the cell survival rate (%).

[0345] The experimental steps are as follows:

[0346] 1. U-87MG and U-118MG cells were cultured with 10% FBS RPMI-1640 / DMEM medium.

[0347] Logarithmic growth phase U-87MG, U-118MG cells, PBS wash once, add 2-3 mL trypsin digestion 2-3 min, take the cell digestion, add 15 mL of complete medium, blow, centrifugal, discard the supernatant, add 10 mL of complete medium, and prepare a single cell suspension.

[0348] 2. U-87MG, U-118MG cells are evenly spread in a black wall transparent bottom 96-well plate, 90 μL per well, and the cell number is 1000. The culture plate is incubated in the incubator for 18 hours.

[0349] 3. The antibody-drug conjugate is diluted to 6 concentration gradients: 50, 10, 2, 0.4, 0.08, 0.016 μg / mL.

[0350] 4. Add 10 μL of the prepared antibody-drug conjugate of different concentrations to the culture plate, two duplicate wells for each concentration. The culture plate is incubated in the incubator for 6 days.

[0351] 5. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and use the enzyme label meter to measure the chemiluminescence. The data are processed and analyzed using Microsoft Excel and Graphpad Prism 7.

[0352] The experimental results are as follows:

[0353] The effective killing of U-87MG (Figure A) and U-118MG (Figure B) cells by ADC-1, ADC-2, ADC-3 and ADC-4 is shown in Figure 13 ;

[0354] Among them,

[0355] As shown in U-87MG (Figure A), ADC-3 has obvious proliferation inhibition activity at high and medium concentrations, while ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan) only have proliferation inhibition activity at high concentrations, and ADC-3 is significantly better than ADC-5 and ADC-6.

[0356] As shown in U-118MG (Figure B), ADC-3 has obvious proliferation inhibition activity at high and medium concentrations, while ADC-5 (MC-GGFG-Dxd) and ADC-6 (MC-GGFG-Exatecan) only have proliferation inhibition activity at high concentrations, and ADC-3 is significantly better than ADC-5 and ADC-6.

[0357] The experimental results show that the antibody-drug conjugate disclosed by the application has obvious proliferation inhibition activity on brain tumor cells.

[0358] Example 23 Evaluation of antibody-drug conjugate cytotoxicity on lung cancer tumor cells

[0359] To detect the inhibitory activity of antibody-drug conjugate on the in vitro proliferation of HCC827, EBC-1 cells. Cells were treated with different concentrations of antibody-drug conjugate, and after six days of culture, the proliferation of cells was detected using CTG (CellTiter-Luminescent Cell Viability Assay, Promega) reagent, and the in vitro activity of the drug was evaluated according to the cell survival rate (%).

[0360] The experimental procedure is as follows:

[0361] 1. HCC827, EBC-1 cells were cultured with 10% FBS RPMI-1640 / DMEM medium.

[0362] 2. Take the logarithmic growth period of HCC827, EBC-1 cells, wash once with PBS, add 2-3 mL trypsin and digest for 2-3 min, add 15 mL complete culture medium after the cell digestion is complete, blow and centrifuge, discard the supernatant, and add 10 mL complete culture medium to prepare a single cell suspension.

[0363] 3. HCC827, EBC-1 cells were evenly spread in black wall transparent bottom 96-well plates, 90 μL per well, and the cell number was 1000. The culture plate was incubated in an incubator for 18 hours.

[0364] 4. Dilute the antibody-drug conjugate to 6 concentration gradients: 50, 10, 2, 0.4, 0.08, 0.016 μg / mL.

[0365] 5. Add 10 μL of the prepared antibody-drug conjugate of different concentrations to the culture plate, two duplicate wells for each concentration. Incubate the culture plate in an incubator for 6 days.

[0366] 6. Add 100 μL of CTG reagent to the culture plate, incubate at room temperature for 10 minutes, and use a microplate reader to measure chemiluminescence. Use Microsoft Excel, Graphpad Prism 7 to process and analyze the data.

[0367] The experimental results are as follows:

[0368] The effective killing of ADC-1, ADC-2, ADC-3, ADC-4 on HCC827 (Figure A), EBC-1 (Figure B) cells is shown in Figure 14 ;

[0369] Among them, HCC827 (Figure A) shows that ADC-3, ADC-2 has obvious proliferation inhibition activity at high, medium and low concentrations.

[0370] EBC-1 (Figure B) shows that ADC-3, ADC-2 has obvious proliferation inhibition activity at high, medium and low concentrations.

[0371] The experimental results show that the antibody-drug conjugate in the application is better than ADC-5 and ADC-6 at low concentration, and the proliferation inhibition activity at medium and high concentrations is equivalent to that of the two.

[0372] Example 24 In-vivo pharmacodynamic evaluation of antibody-drug conjugate

[0373] In order to detect the in-vivo tumor inhibition activity of the antibody-drug conjugate, human colorectal cancer cells HT29 cells (5x10 6 ) were inoculated subcutaneously on the right flank of Balb / c nude mice, and the mice were grouped on day 0, 5 in each group, and the average tumor volume was about 178.91mm 3 .

[0374] The experimental steps are as follows:

[0375] 1. Tail vein injection, a total of 2 times of administration, respectively on day 1 and day 7.

[0376] 2. The tumor volume and body weight were measured once every two days, and the data were recorded.

[0377] 3. The tumor volume (V) calculation formula is: V=1 / 2*L 长 *L 宽 2 ;

[0378] Relative volume: RTV=VT / V0;

[0379] Tumor inhibition rate (%)=(CRTV-TRTV) / CRTV (%);

[0380] The data were processed and analyzed by the above formula, and the experimental data are shown in Table 10.

[0381] Table 10

[0382]

[0383] The experimental results are as follows:

[0384] The significant in-vivo tumor inhibition activity of ADC-3 on colorectal cancer cells HT29 cells is shown in Figure 15 ;

[0385] As shown in Figures A and B, the tumor inhibition rate of ADC-3 is 62%, which is significantly higher than 28.9% of ADC-5 and 3% of ADC-6.

[0386] The experimental results show that the antibody-drug conjugate ADC-3 in the present application has significantly better tumor inhibition activity than the control ADC-5 and ADC-6 in the human colorectal cancer HT29 tumor-bearing mouse model.

[0387] Discussion:

[0388] (1) Compared with Exatecan, the in-vitro proliferation inhibition IC50 values of the first two, P2 and P3, on HCT-16 and HT-29 cells are higher than that of the latter, but ADC-2 and ADC-3 formed by conjugating P2 and P3 with antibodies have stronger antitumor activity on HCT-116, HT-29, RKO, SiHa, U-87MG and U-118MG cells than ADC-6. The experimental results show that ADC-2 and ADC-3 formed by conjugating P2 and P3 with antibodies have unexpected effects.

[0389] (2) ADC-3 is more sensitive to SiHa cells than other ADC drugs of the present application, and has obvious proliferation inhibition activity on the cells at high concentrations when other conjugates have no obvious proliferation inhibition activity.

[0390] (3) ADC-2 and ADC-3 can effectively inhibit more than 50% of BxPC-3 cells at a concentration of about 10 nM, and have significantly stronger proliferation inhibition activity than ADC-5.

[0391] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is individually incorporated by reference. In addition, it should be understood that those skilled in the art can make various modifications or improvements to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. A compound of formula (I), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein, Y is selected from the group consisting of:

2. The compound of claim 1, wherein the compound is selected from the group consisting of: 。 3.A linker-conjugate of formula (II), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, characterized in that, wherein Y is as defined in claim 1; L 1 is a polypeptide residue selected from the group consisting of -Phe-Lys-, -Val-Cit-, -Val-Ala-, -Phe-Cit-, -Gly-Val-, -Ala-Lys-, -Ala-Lys-, -Ala-Ala-Ala-, -Glu-Val-Ala-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, preferably -Gly-Gly-Phe-Gly-; L2 is a linker unit for attachment to an antibody, and is 4. The linker conjugate of claim 3, wherein, the linker-conjugate is selected from the group consisting of: 。 5.An antibody-drug conjugate of formula (III), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, characterized in that, wherein Ab is an antibody or an antibody fragment; n is 2-8; Y, L 1, L 2 are as defined in claim 3.

6. The antibody-drug conjugate of claim 5, wherein, the antibody-drug conjugate is selected from the group consisting of:

7. The use of a linker conjugate of Formula II, characterized in that, for preparing an antibody-drug conjugate.

8. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises: (i) the antibody-drug conjugate of claim 5, and (ii) a pharmaceutically acceptable carrier.

9. Use of the antibody-drug conjugate of claim 5 or the pharmaceutical composition of claim 8, wherein the antibody-drug conjugate or the pharmaceutical composition is for treating cancer. for preparing a medicament for treating a tumor.

10. Use according to claim 9, characterized in that, the tumor is selected from the group consisting of colorectal cancer, pancreatic cancer, cervical cancer, brain cancer, lung cancer, head and neck cancer, cholangiocarcinoma, gastric cancer.

Images