CN109810039A - A kind of disubstituted maleic amide class connexon and its preparation method and application for antibody-drug conjugate - Google Patents

Abstract

The present invention provides a kind of and antibody coupling disubstituted maleic amide class connexons and its preparation method and application, and specifically, strong cell toxicity agents and large biological molecule are coupled by the present invention by a new class of connexon.Such connexon alternative acts on simultaneously with disulfide bond, to greatly improve the substance homogeneity and stability of conjugate.Conjugate prepared by connexon of the invention has high inhibitory activity for the cell strain for expressing corresponding antigens.The present invention also provides the preparation method of above-mentioned conjugate and purposes.

Description

A kind of disubstituted maleic amide class connexon and its system for antibody-drug conjugate Preparation Method and purposes

Technical field

The present invention relates to a kind of novel disulfide bond bridge joint cross-linking reagent, macromolecular, treatment conjugate and its synthetic methods. More particularly it relates to by based on replace the disulfide bond of maleic amide bridge cross-linking reagent by cytotoxic drug and Conjugate obtained from macromolecular is crosslinked and its preparation method and application.

Background technique

As novel target therapeutic agent, antibody-drug conjugates (Antibody Drug Conjguate, ADC) are opened Created new era of tumor therapeuticing method, fundamental design idea be derived from earliest Borrow's Ehrlich (Paul Ehrlich) in " magic bullet " (Magic bullet) and targeted drug delivery (Drug targeting) concept being put forward for the first time for 1913, i.e., By carrier appropriate by targeted drug delivery to illness position.However, being limited by antibody and high-activity fine cytotoxic drugs skill The restriction of art, until 2000 first for treating the antibody-drug conjugates of acute myeloid leukemia (AML) (Mylotarg^TM) just ratified to list by FDA.What recent Seattle Genetics Inc. (Seattle Genetics) developed is used to treat Hodgkin lymphoma (HL)/recurrent primary cutaneous type (ALCL) new drug Adcetris^TM(2011) Ji Jiantai section The new drug Kadcyla for being used to treat breast cancer that biotech company (Genentech) develops^TM(2013) FDA batches are passed in succession through Quasi- listing then indicates that antibody-drug conjugates enter Rapid development stage in the application of therapeutic field of tumor.

Antibody-drug conjugates generally consist of three parts: antibody or antibody class ligand, small-molecule drug, and by ligand The connexon to get up with drug coupling.Enter in the antibody-drug conjugates structure of clinical test at present, the cell toxicant of high activity Property drug be usually pass through connexon be connected to ligand surface lysine residue or antibody hinge region cysteine it is residual On base (being obtained by interchain disulfide bond partial reduction), optimal drug/ligand ratio (DAR) is 2-4.Antibody surface largely relies Histidine residue (more than 80) and coupling reaction is non-selective, leads to the uncertainty for being coupled number and site, and then lead Cause the inhomogeneity of the antibody-drug conjugates generated.For example, the DAR Distribution value of T-DM1 (average DAR value is 3.5) is 0-8. Equally, it although the interchain disulfide bond of antibody hinge region only has four pairs, for the requirement for reaching best averagely DAR value (2-4), needs Partial reduction interchain disulfide bond.Since existing reducing agent (DTT, TCEP etc.) can not selectively restore interchain disulfide bond, because This conjugate generated is also not uniform product, is made of various ingredients, and the DAR value of main component is 0,2,4,6,8, and And the component of each corresponding specific DAR value all exists due to the isomers that connection site is different and is formed.Antibody-drug conjugate The inhomogeneity of produce product can lead to pharmacokinetic property between each member's component, the inhomogeneity of potency and toxicity.Example Such as, the component with higher DAR value is removed faster in vivo, and leads to higher toxicity.

In order to solve the problems, such as that antibody-drug conjugates homogeneity, site-directed coupling technology have obtained more favors recently, this One technology controls the coupling between antibody drug in terms of site and quantity two.Although these technologies can realize coupling medicine Level point and quantity it is controllable, applied antibody/albumen is obtained by way of genetic recombination.Gene recombination technology It needs largely to work and exquisite design, to find suitable site for drug coupling or polyethyleneglycol modified, and existing base Because antibody/albumen expression quantity of the obtained pointed decoration of recombinant technique is lower, therefore in large scale preparation production very Time-consuming and research and development and final industrialization expense cost are very high.And it is yet needed further by improved antibody/albumen Verify the factors such as internal drug effect and the safety of its own.

The above coupling technology there are aiming at the problem that, by simple chemical method to existing antibody realize site-directed coupling mesh , a large amount of manpower, material resources and financial resources can be saved, therefore more attractive.Wherein having relevant research includes: Pohle Thailand A kind of coupling technology CN200480019814.4 of Li Kesi Co., Ltd report；Igenica Biotherapeutics company The WO2014197871A2 of application；The CN201380025774.3 of Sorento medical treatment Co., Ltd application；Shanghai new concept biology The patent documents such as the CN201310025021.4 of Pharmaceutical Technology Co., Ltd's application.However there are coupling reagent synthesis for above-mentioned technology Route is longer, coupling reagent chemical stability is bad, antibody coupling matter electrophoretogram is more mixed and disorderly, prompts to deposit in coupling process The problems such as sulfydryl during side reaction, existing scheme and unresolved body-internal-circulation exchanges (inverse Michael addition reaction).

Therefore, there is an urgent need in the art to provide efficient, simple, practical chemical conjugation methods, and reach site-directed coupling Purpose, while also needing to take into account the properties such as stability, safety for improving antibody-drug conjugates.

Summary of the invention

The object of the present invention is to provide the connexons that one kind can be simply coupled with most of antibody.

The first aspect of the present invention provides a kind of substitution maleic amide class connexon segment, and structure is shown in Formulas I a:

Wherein, R is X or ArS-,

X is selected from the group: halogen, preferably bromine or iodine；

Ar is selected from the group: substituted C6-C10 aryl, substituted or unsubstituted 5-12 unit's heteroaryl；

Ar ' is selected from the group: substituted C6-C10 arlydene；Substituted or unsubstituted 5-12 member inferior heteroaryl；

L₁For-O (the CH being connected on Ar ' group₂CH₂O)_n, wherein n any integer in 1-20.

One of preferred embodiment, Ar are selected from phenyl, halogeno-benzene, C1-C4 alkyl phenyl, C1-C4 alkoxy benzene Base, 2- pyridyl group, 2- pyrimidine radicals, 1- methylimidazole -2- base,Wherein W is the amido R connecting with carbonyl¹, R¹Selected from- NH₂、 Deng；Wherein: C1-C4 alkyl phenyl is more preferably 4- aminomethyl phenyl；C1-C4 alkoxyl phenyl is more preferably 4- methoxy Base phenyl.

One of preferred embodiment, Ar ' are selected from the phenylene or pyridyl group replaced, wherein the substitution refers to base Hydrogen atom in group is replaced one or more substituent groups selected from the group below: halogen, C1-C4 alkyl, C1-C4 alkoxy, three Methyl fluoride, itrile group, amide groups etc..

One of preferred embodiment, the n are any integer in 1-10.

In another preferred example, the connection sub-piece has structure selected from the group below:

The second aspect of the present invention provides a kind of containing Formulas I a connexon segment as described in the first aspect of the invention Replace maleic amide class connexon-drug conjugate, its pharmaceutically acceptable salt or its solvent object, structure such as general formula Ib institute Show:

Wherein, R, Ar ', L₁It is defined as above；

L₂For chemical bond or AA-PAB structure；Wherein, AA is that (i.e. 2-3 amino acid passes through peptide bond for dipeptides or tripeptides segment Connect the segment formed), preferably include Val-Cit (valine-citrulline), Val-Ala (valine-glycine), Phe-Lys (Phe-Lys), Ala-Ala-Asn (Gly-Gly-asparagine), D-Ala-Phe-Lys (the sweet ammonia of D type Acid-Phe-Lys) etc., PAB is p- aminobenzyl carbamyl；

CTD is to be bonded to L by amido bond₂Cytotoxicity micromolecular drug and/or treatment autoimmune disease and/ Or the drug of anti-inflammatory.

In another preferred example, the Formulas I b compound is selected from the group:

The third aspect of the present invention, provides a kind of antibody-drug conjugates, and the antibody-drug conjugates are to use Formulas I b as described in respect of the second aspect of the invention replaces maleic amide class connexon-drug conjugate and antibody to carry out coupling formation 's.

In another preferred example, conjugate covalent linkage has one or more drug components.

In another preferred example, in the conjugate, antibody and drug are by covalent manner (as by covalent respectively It is connected on connexon) it is coupled.

Another aspect of the present invention, provides a kind of antibody-drug conjugates, and the antibody-drug conjugates have The structure of general formula Ic and/or Id；

Wherein, Ar ', L₁、L₂, CTD is defined as above；

M=1.0~5.0, preferably 3.0-4.2；

Ab is selected from the group: for protein, enzyme, antibody, antibody fragment, polypeptide.

In another preferred example, the general formula Id is product after N- phenyl maleimide open loop in general formula Ic.

In another preferred example, the antibody or Ab are selected from the group: monoclonal antibody, bispecific antibody, inosculating antibody Body, humanized antibody, antibody fragment (preferably Fab fragments).

In another preferred example, in the antibody-drug conjugates, pass through the antibody or antibody fragment hinge area Disulfide bond reduction generates a pair of of cysteine residues, and passes through sulfydryl in the cysteine residues and the virtue in the general formula Ib Substitution reaction occurs for base thioether, so that the compounds of general formula Ib is connected on the antibody or antibody fragment.

In another preferred example, the CTD be cytotoxicity micromolecular drug, preferably Antitubulin, Topoisomerase enzyme inhibitor or DNA bonding agent.

In another further preferred example, the Antitubulin is derivative selected from maytansine (maytansine) Object, Monomethyl auristatin E (MMAE), Monomethylauristatin F (MMAF), Monomethyl Dolastatin 10, Tubulysin analog derivative, Cryptophycin analog derivative, Taltobulin.

In another further preferred example, the DNA bonding agent is selected from PBD analog derivative, duocarmycin analog derivative.

In another further preferred example, the topoisomerase enzyme inhibitor is selected from adriamycin (Doxorubicin) generation Thank product PNU-159682 derivative, Irinotecan (irinotecan, CPT-11) metabolite SN38 derivative.

In another preferred example, the CTD has the molecular structure selected from D1-D13:

In another preferred example, the antibody be can be with the antibody in conjunction with tumor associated antigen selected from the group below:

In another preferred example, the antibody is HER2 antibody, further preferably Herceptin (Trastuzumab) or handkerchief trastuzumab (Pertuzumab).

In another preferred example, the antibody is EGFR antibody, further preferably Erbitux or Vectibix.

In another preferred example, the antibody is Tissue factor (TF) antibody.

The fourth aspect of the present invention, provides a kind of pharmaceutical composition, and aforementioned pharmaceutical compositions include: (a) such as present invention Antibody-drug conjugates described in the third aspect；(b) pharmaceutically acceptable diluent, carrier or excipient.

The fifth aspect of the present invention provides a kind of antibody-drug conjugates as described in third aspect present invention and exists It is used to prepare the purposes of the drug for the treatment of tumour.

In another preferred example, the tumour is selected from the group: breast cancer, oophoroma, non-Hodgkin lymphoma, Huo Qijin Lymthoma, acute lymphatic leukemia, primary cutaneous type, Huppert's disease, prostate cancer, non-small cell Lung cancer, Small Cell Lung Cancer, malignant mela noma, squamous cell carcinoma, glioblastoma, clear-cell carcinoma, gastroenteric tumor, pancreas Cancer, prostate cancer, straight colon, gastric cancer, glioma, celiothelioma.

Another aspect of the present invention provides a kind of method for treating tumour, and the method is comprising steps of to needs Object applies antibody-drug conjugates described in the third aspect present invention of therapeutically effective amount.

In another preferred example, the object is mammal, is preferably people.

The sixth aspect of the present invention provides a kind of system of antibody-drug conjugates as described in the third aspect of the present invention Preparation Method, the method includes the steps:

(1) it is reacted in buffer with antibody with go back original reagent, obtains antibody after carrying out reduction；

(2) antibody after carrying out reduction obtained with connexon-drug conjugate with step (1) has in buffer with a certain amount of It is crosslinked in solvent mixed liquor, obtains antibody-drug conjugates.

Antibody described in step (1) is restored through go back original reagent, so that antibody interchain disulfide bond be made to be reduced, generates sulfydryl base Group.

In another preferred example, the go back original reagent is three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP), beta- sulfydryl second Alcohol, beta- mercaptoethylamine hydrochloride or dithiothreitol (DTT) (DTT).

In another preferred example, the buffer is selected from the group: potassium dihydrogen phosphate-sodium hydroxide (KH₂PO₄-NaOH)/ Sodium chloride (NaCl)/diethyl pentetic acid (DTPA) buffer, disodium hydrogen phosphate-citric acid/sodium chloride (NaCl)/diethyl Base pentaacetic acid (DTPA), boric acid-borax/sodium chloride (NaCl)/diethyl pentetic acid (DTPA), histidine-hydrogen-oxygen Change sodium/sodium chloride (NaCl)/diethyl pentetic acid (DTPA) and PBS/ diethyl pentetic acid (DTPA).

In another preferred example, in the step (2), volume accounting of the organic solvent in reaction solution is no more than 15%.

In another preferred example, the organic solvent in the step (2) is selected from the group: acetonitrile (ACN), dimethyl formyl Amine (DMF), dimethyl acetamide (DMA), dimethyl sulfoxide (DMSO).

In another preferred example, in the step (2), the coupling reaction carries out at 0-37 DEG C.

In another preferred example, if the step (1) using beta- mercaptoethanol, beta- mercaptoethylamine hydrochloride or DTT reduction, between the step (1) and step (2) further include: after the completion of reduction reaction, carried out desalination to product Column or ultrafiltration are to remove reducing agent.

In another preferred example, the reaction route of the method is as follows:

Wherein R, Ar ', L1, L2 be defined as above.

In another preferred example, it the described method comprises the following steps:

1) it restores: antibody stoste being diluted to 2-10mg/mL with reaction buffer, 140-200 times of excess molar ratio is added Dithiothreitol (DTT) (DTT), or be added 6.0-20 times of excess molar ratio three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP), reaction solution In 10-35 DEG C agitation 2-48 hours；

2) it is coupled: above-mentioned reaction solution is cooled to 0-10 DEG C, add and replace maleic amide class compound, stirred at 0-37 DEG C 2-5 hours.

Further include the step in another further preferred example: when step 1) is restored using DTT, being restored in step 1) Reaction solution is crossed into desalting column after the reaction was completed or ultrafiltration removes excessive DTT；

In another further preferred example, the substitution maleic amide class compound can be previously dissolved in organic solvent, Organic solvent is preferably selected from: acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA)；Further preferably, replace maleic amide class compound and organic solvent to dissolve by 10mg/ml, and guarantee organic solvent Volume accounting is no more than the 15% of reaction solution.

It further include the step in another further preferred example: by reaction mixture after the completion of step 2) coupling reaction It is gel-filtration purified with sodium succinate/NaCl buffer or histidine-acetic acid/sucrose, it is collected out according to UV280 ultraviolet absorption value Peak sample.

It further include the step in another further preferred example: by reaction mixture after the completion of step 2) coupling reaction Ultrafiltration, then filtration sterilization, products therefrom cryo-conservation；Further preferred storage temperature is -100~60 DEG C；Further preferably, The device aperture that the ultrafiltration uses is 0.15~0.3 micron.

In another further preferred example, the step 1) is restored using TCEP.Excessive TCEP can not be removed.

In another further preferred example, reaction buffer described in step 1) may is that 50mM potassium dihydrogen phosphate-hydrogen-oxygen Change sodium (KH₂PO₄- NaOH)/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), pH 6-9；50mM phosphoric acid Disodium hydrogen-citric acid/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), pH 6-9；50mM boric acid-boron Sand/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), pH 6-9；50mM histidine-sodium hydroxide/ 150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), pH 6-9 and PBS//1mM diethyl pentetic acid (DTPA), pH 6-9.

The drug antibody coupling ratio (DAR) of gained antibody-drug conjugates is more uniform, using it is heretofore described not With the antibody-drug conjugates for replacing maleic amide connexon that product uniformity can also be had to certain difference, such as need to obtain equal The one better sample of property, can further using but be not limited to following methods and isolated and purified: hydrophobic interaction chromatography method (HIC), molecular exclusion chromatography (SEC), ion-exchange chromatography (IEC).

The seventh aspect of the present invention, providing substitution maleic amide class connexon described in first aspect, (Formulas I a's is preferred Example E) preparation method:

By intermediate C and 2,3- dibromomaleic acid acid anhydride cyclization reaction obtains intermediate D, then replace instead with aryl thiophenol Should after obtain connexon fragmental molecule E, reaction equation is as follows:

Wherein R, n are same as above, and X represents halogen, preferably Br, Cl；U, V respectively independently represents N or C.

In another preferred example, the C can be restored by B, and reaction equation is as follows:

Wherein R, n, U, V are same as described above.

In another further preferred example, the B can be obtained by A and Fluoronitrobenzene substitution reaction, and reaction equation is as follows:

Wherein R, n, U, V are same as described above.

In another further preferred example, the B can be prepared by following formula:

Wherein R, n, U, V are same as described above.

In another still more preferably example, A is reacted with the halogenated acetic acids tert-butyl ester by n glycol and is obtained.Reaction equation is as follows:

Wherein n, X are same as described above.

The eighth aspect of the present invention provides substitution maleic amide class connexon-drug conjugate described in second aspect The preparation method of (the preference F1 or F ' 1 of Formulas I b): replace maleic amide class connexon (the preference Ea of Formulas I a) and have two Peptide/tripeptides-PAB cell toxicity medicament CTD is condensed, and F1 or F ' 1 is respectively obtained.

Reaction route is as follows:

Wherein for R with the definition of Formulas I a, Rx represents halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, itrile group or acyl Amido, Ry represent H or alkyl.

The present inventor is after extensive and in-depth study, it was found that one kind connection minor structure, which can whole/portion Divide the light-heavy chain and heavy chain-heavy chain of cross-coupling antibody, and the antibody-drug conjugates obtained using such coupling method, Compared with conventional antibodies-drug conjugates, it is distributed with narrower drug/Antibody ratio (DAR).Based on above-mentioned discovery, invention People completes the present invention.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Term

Herein, except place is illustrated, term " C1-C4 alkyl " refers to the linear chain or branched chain with 1-4 carbon atom Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl group, sec-butyl, tert-butyl or similar group.

Term " C1-C4 alkoxy " refers to the straight or branched alkoxyl with 1-4 carbon atom, such as methoxyl group, ethoxy Base, propoxyl group, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy or similar group.

Term " halogen " refers to F, Cl, Br and I.

Term " C6-C10 aryl " refers to aryl with 6-10 carbon atom, such as phenyl, naphthalene etc., and the aryl can To be substituted or unsubstituted.

Term " C6-C10 aryl " refers to aryl with 6-10 carbon atom, such as phenyl, naphthalene etc., and the aryl can To be substituted or unsubstituted.

Term " 5-12 unit's heteroaryl ", " 5-12 member inferior heteroaryl " refer to (excellent with 5-12 carbon atom and one or more Select 1-3) it is selected from the heteroatomic heteroaryl or inferior heteroaryl of O, S and/or N, preferably 5-8 unit's heteroaryl or inferior heteroaryl.Institute The heteroaryl or inferior heteroaryl stated can be substituted or unsubstituted.

In the present invention, term " pharmaceutically acceptable " ingredient refers to suitable for people and/or animal and without excessive bad pair It reacts (such as toxicity, stimulation and allergy), that is, has the substance of reasonable benefit/risk ratio.

In the present invention, amount or table that term " effective quantity " refers to therapeutic agent treatment, alleviates or prevent target disease or situation Reveal the detectable amount for treating or preventing effect.The figure of the object is depended on for the accurate effective quantity of certain an object and is good for The combination of therapeutic agent and/or therapeutic agent that health situation, the property and degree of illness and selection are given.Therefore, standard is preassigned True effective quantity is useless.However, can determine the effective quantity with routine experiment for the situation that Mr. Yu gives, face Bed doctor can judge.

Unless stated otherwise, in the present invention, the compound occurred is intended to including all possible optical isomer, Such as the compound of single chiral or the mixture (i.e. racemic modification) of various different chipal compounds.All chemical combination of the invention Among object, each asymmetric carbon atom can be optionally the mixture of R configuration or S configuration or R configuration and S configuration.

As used herein, term " the compounds of this invention " refers to Formulas I compound represented.The term further includes and Formulas I chemical combination Various crystalline forms, pharmaceutically acceptable salt, hydrate or the solvate of object.

As used herein, term " pharmaceutically acceptable salt " refers to that the compounds of this invention and acid or alkali are formed by suitable use Make the salt of drug.Pharmaceutically acceptable salt includes inorganic salts and organic salt.A kind of preferred salt is the compounds of this invention and acid The salt of formation.The acid for suitably forming salt includes but is not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid etc. are inorganic Acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, Picric acid, methanesulfonic acid, benzene methanesulfonic acid, the organic acids such as benzene sulfonic acid；And the acidic amino acids such as aspartic acid, glutamic acid.

Unless stated otherwise, " amino acid " used herein is intended to include any conventional amino acid, as aspartic acid, Glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine (methionine), color Propylhomoserin, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, arginine.

When trade name used herein, which is intended to include trade name product formulation, its corresponding imitation medicine, with And the active medicine component of trade name product.

The term " antibody " of this paper is used with its broadest sense and especially covers monoclonal antibody, Anti-TNF-α Body, dimer, polymer, multi-specificity antibody (such as bispecific antibody) and antibody fragment, as long as needed for they show Bioactivity (Miller etc. (2003) Journal of Immunology 170:4854-4861).Antibody can for mouse, People, humanization, chimeric antibody derive from other species.Antibody is by that can identify the siberian crabapple with binding specificity antigen Unite generate protein (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5thEd.,Garland Publishing,NewYork).Target antigen generally has by a large amount of knots of the CDRs identification of Multiple Antibodies Coincidence point, also referred to as epitope.Each antibody for specifically binding different epitopes has different structures.Therefore, a kind of antigen can be with With more than one corresponding antibody.Antibody includes the immune of complete-long immunoglobulin molecules or complete-long immunoglobulin molecules Active part, the i.e. molecule containing the antigen for specifically binding target of interest or part thereof, this kind of target include, but are not limited to Cancer cell or the cell for generating autoimmune antibody relevant to autoimmune disease.Immunoglobulin disclosed herein can be with With immunoglobulin molecules any type (such as IgG, IgE, IgM, IgD and IgA), classification (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.Immunoglobulin can derive from arbitrary species.However, in an aspect, Immunoglobulin derives from people, mouse or rabbit.

" antibody fragment " includes a part of full length antibody, generally its antigen binding domain or variable region.Antibody fragment Example includes: Fab, Fab ', F (ab ') 2 and Fv segment；Double antibody；Linear antibodies；Miniantibody (minibody) (Olafsen etc. (2004)Protein Eng.Design&Sel.17(4):315-323)；The segment of Fab expression library preparation；Anti- idiotype (anti-Id) antibody；CDR (complementary determining region)；With with immune specificity pattern combination cancer cell antigen, viral antigen or microorganism The above-mentioned arbitrary epitope-binding fragment of antigen；Mono- chain antibody molecule；With the multi-specificity antibody formed by antibody fragment.

The antigen binding energy when antibody of antibody-drug conjugates is preferably maintained in its original wild state is formed in the present invention Power.Therefore, the present invention in antibody can, preferably in specific manner, with antigen binding.The antigen being related to includes, for example, tumour phase It closes antigen (TAA), cell surface receptor protein and other cell surface molecules, cell survival regulatory factor, cell Proliferation is adjusted The factor, and tissue growth and the relevant molecule of differentiation (as known or precognition with functional), lymphokine, cell because Son participates in the molecule that cell cycle is adjusted, and participates in the molecule of angiogenesis, and (such as known with the molecule of associated angiogenesis The antigen that antibody combines can be one or a subset in above-mentioned classification, and other subsets then include other with special Molecule/antigen of property (compared with target antigen).

The antibody in antibody-drug conjugates is applied to include, but are not limited to, for cell surface receptor and tumour phase Close the antibody of antigen.Such tumor associated antigen be it is known in the industry, can pass through in the industry known to preparation method for antibody It is prepared with information.In order to develop the effective cellular level object that can be used for cancer diagnosis and treatment, researcher tries hard to Look for cross-film or other tumor relative polypeptides.These objects can be expressed specifically in one or more cancer cell tables Face, and one or more non-cancerous cells surface expressions seldom or do not express.In general, for non-cancerous cells surface, this The tumor relative polypeptide of sample is more over-expressed in cancer cell surfaces.Confirm such tumor related genes, is greatly improved base In the single-minded targeting characteristic of antibodies for treating cancer.

Tumor associated antigen includes, but are not limited to, tumor associated antigen (1)-(53) being listed below.It rises for convenience See, it is as follows for antigen relevant information mark known in the industry, including title, other titles, Genbank accession number.With tumour phase It closes the corresponding nucleic acid of antigen and protein sequence can be found in public database, such as Genbank.The corresponding tumour of antibody target is related Antigen includes all amino acid sequence mutation and of the same race, has at least 70%, 80% with the sequence confirmed in bibliography, 85%, 90% or 95% homology, or have with the tumor associated antigen sequence in citation with completely the same Biological property and feature.

(1) HER2 (Gene ID:2064, ErbB-2 (English: human epidermal Growth factor receptor 2, is abbreviated as HER2, also known as Neu, ErbB-2, CD340 (differentiation group 340) or p185) It is a kind of protein encoded by ERBB2 gene.HER2 be member in EGF-R ELISA (EGFR/ErbB) family it One)；(2) (Gene ID:2065, skin factor receptor 3 (ErbB3/HER3) are epidermal growth factor transmembrane receptor families to HER3 One of member.The treatment of the morbidity of confirmation ErbB3/HER3 and breast cancer, relapse and metastasis, chemotherapy and endocrine therapy in recent years It imitates closely related, it has also become very promising treatment candidate targets)；(3)CD19(Gene ID:930)；(4)CD20(Gene ID:931)；(5)CD22(Gene ID:933)；(6)CD30(Gene ID:943)；(7)CD33(Gene ID:945)；(8) CD37(Gene ID:951)；(9)CD45(Gene ID:5788)；(10)CD56(Gene ID:4684)；(11)CD66e(Gene ID:1048)；(12)CD70(Gene ID:970)；(13)CD74(Gene ID:972)；(14)CD79b(Gene ID:974)； (15)CD138(Gene ID:6382)；(16)CD147(Gene ID:682)；(17)CD223(Gene ID:3902)；(18) EpCAM(Gene ID:4072)；(19)Mucin 1(Gene ID:4582)；(20)STEAP1(Gene ID:26872)；(21) GPNMB(Gene ID:10457)；(22)FGF2(Gene ID:2247)；(23)FOLR1(Gene ID:2348)；(24)EGFR (Gene ID:1956)；(25)EGFRvIII(GenBank:GM832119.1)；(26)Tissue factor(TF)(Gene ID:2152)；(27)c-MET(Gene ID:4233)；(28)Nectin 4(Gene ID:81607)；(29)AGS-16；(30) Guanylyl cyclase C(Gene ID:2984)；(31)Mesothelin(Gene ID:10232)；(32)SLC44A4 (Gene ID:80736)；(33)PSMA(Gene ID:2346)；(34)EphA2(Gene ID:1969)；(35)AGS-5；(36) GPC-3(Gene ID:2719)；(37)c-KIT(Gene ID:3815)；(38)RoR1(Gene ID:4919)；(39)PD-L1 (Gene ID:29126)；(40)CD27L(Gene ID:970)；(41)5T4(Gene ID:7162)；(42)Mucin 16 (Gene ID:94025)；(43)NaPi2b(Gene ID:10568)；(44)STEAP(Gene ID:26872)；(45) SLITRK6(Gene ID:84189)；(46)ETBR(Gene ID:1910)；(47)BCMA(Gene ID:608)；(48)Trop- 2(Gene ID:4070)；(49)CEACAM5(Gene ID:1048)；(50)SC-16；(51)SLC39A6(Gene ID: 25800)；(52)Delta-like protein3(DLL3)(Gene ID:10683)；(53)Claudin 18.2(Gene ID: 51208)。

As used herein, " drug " refers to any with desired bioactivity, and has reactive functional groups to make The compound of standby conjugate of the present invention.Desired bioactivity includes, and diagnoses, and cures, and alleviates, treatment, prevent people or its The disease of its animal.Therefore, as long as having required reactive functional groups, the compound that term " drug " is related to includes formal state Family's pharmacopeia and, for example, United States Non-Provisional homeopathy pharmacopeia, the medicine of the formal whole nation confirmations such as formulary or its any enlarged edition Object.Typical drug is listed in orange paper of doctor's desk medication with reference to (PDR) and U.S. Food and Drug Administration (FDA). As newtype drug is constantly found and develops, this patent provides that these drugs should be also included in coupling drug of the present invention " drug ".

Preferably, the drug refers to: cytotoxic drug use for cancer treatment, or with desired bioactivity Albumen or polypeptide, such as a kind of toxin, such as abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin；Other Suitable albumen includes tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth because Son, the molten protogrowth factor of tectotype fibre enzyme and biological respinse adjust preparation, such as lymphokine, interleukin 1 (IL- 1), interleukin 2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), Granulocyte colony stimulating factor or other growth factors.

A kind of preferred drug of the present invention is maytansine or class maytansine.Maytansine compound is by inhibiting tubulin Micro-pipe forms to inhibit cell Proliferation.Class maytansine is the derivative of maytansine.Maytansine and class maytansine all have efficiently Cytotoxicity, but they have significant limitation in the clinical application for the treatment of of cancer, this stems primarily from such molecule To the low selectivity of tumour.But this high cell toxicity promotes them to become the choice drug portion of antibody-drug conjugates Point.It is listed below the structure of deacetylate maytansine.

Another preferred drug of the present invention is ear chalone peptide medicament.Ear chalone peptide medicament is aplysiatoxin 10 (Dolastatin10) analog, and the latter be separated out of sea mollusk sea hare body it is biologically active Polypeptide.Aplysiatoxin 10 is by combining tubulin (bond area same as vincristine) to inhibit tubulin polymerization. Aplysiatoxin 10, ear chalone peptide PE, ear chalone peptide E is linear polypeptide, containing there are four amino acid, (wherein three amino acid are seas Rabbit endotoxin compound is exclusive) and the end C- amide group.Two representative ear chalone peptides, the suppression of monomethyl ear Plain peptide E (MMAE) and monomethyl ear chalone peptide F (MMAF), are the choice drugs of antibody-drug conjugates.

Another preferred drug of the present invention is tubulysin (Tubulysin).Tubulysin first by research group from It is separated in slime bacteria culture, is very effective cytostatic agent, by inhibiting tubulin polymerization and thereby luring Guided cell apoptosis and work.Tubulysin D in tubulysin be it is most effective, having is more than other most of tubulins The activity of 10 to 100 times of regulator (including Epothilones, vincaleukoblastinum and taxol).Taxol and vincaleukoblastinum are currently used for a variety of The treatment of cancer, and epothilone derivatives carry out activity rating just in clinical test.The synthesis of derivatives of tubulysin D will Necessary information related with inhibition and crucial binding interactions is provided, and can have superior property as anticancer agent, The anticancer agent is as isolated entity or as the chemical warhead on targeting antibodies or ligand.Tubulysin D is the four of complexity Peptide, can be divided into four regions, Mep (D-N- methyl piperidine formic acid), Ile (isoleucine), Tuv (tubular type valine, Tubuvaline) and Tup (tubular type phenylalanine, tubuphenylalanine), it is shown below:

Another preferred drug of the present invention is the microbe-derived cryptophycin derivative that can inhibit microtubule polymerization Object.Cryptophycin is isolated a kind of new antitumoral activity that micro-pipe can be inhibited to generate from the culture of cyanobacteria Substance, it is active to kinds of tumors.Cryptophycin is a fat-soluble compound, contains 2 peptide bonds and 2 ester bonds, there is 5 A optical activity center and 1 epoxy group.Double peptide dibasic acid esters keys are all in a macrocyclic structure.Cryptophycin derivative CP1 and CP2 structure is shown below:

Another preferred drug of the present invention is novel anti-micro-pipe agent Taltobulin (HTI-286, SPA-110). Taltobulin inhibits the multimerization of purifying micro-pipe, and interference intracellular canaliculus tissue, induced mitogenesis block and induction is thin Born of the same parents' apoptosis.Taltobulin is cell Proliferation potent inhibitor, and the average IC50 to 18 kinds of human tumor cell lines is 2.5nM.With The anti-micro-pipe agent applied at present is compared, and Taltobulin is not the suitable substrate of p- glycoprotein, and wherein Taltobulin is tied Structure is shown below.

On the one hand, drug is camptothecine derivative Exatecan.It is the conjunction of topoisomerase I inhibitor camptothecine At analog, activity is stronger compared with SN-38, the inhibition of DNA topoisomerase I can be caused most strong, dose dependent and Time Dependent Inhibit the synthesis of DNA to property, and causes frequent DNA single-strand break.Wherein Exatecan structure is shown below.

Another preferred drug of the present invention is goose cream Tan bases drug (alpha-Amanitin), structure such as following formula institute Show.Alpha-Amanitin is a kind of mycotoxin for coming from poisonous mushroom Phallus goose gill fungus (Amanitaphalloides), two rings eight Peptide can inhibit eukaryotic RNA Polymerase II and RNA polymerase III to transcribe.

Another preferred drug of the present invention be benzodipyrrole class antibiotic (duocarmycins, CC-1065 etc.) and Other cyclopropyl pyrroles indoles -4- ketone (cyclopropapyrroloind-4-one, CPI) derivatives.This kind of compound is Effective DNA minor groove binding-alkylating reagent.Cyclopropyl benzindole -4- ketone (cyclopropabenzindol-4-one, CBI) The chemical structure of analog is more stable, and bioactivity is higher, and contains the paternal chemical combination of natural CPI alkylation subunit with them Object is synthesized compared to more easily.One representative CBI derivative is phenolic hydroxyl group protection derivative CBI, has the preceding poisoning of drug of reduction The water solubility (wherein CBI-seco structural general formula is shown below) of property and enhancing:

Another preferred drug of the present invention is pyrrolo- Benzodiazepines (pyrrolo [2,1-c] [1,4] benzodi- Azepines, PBDs) or PBD dimer class (PBD dimers).PBD is a kind of natural products generated by streptomycete, Unique property is definitely can be to form the covalent adduction of non-distorted at xanthine-guanine-purine sequence in DNA ditch Object.Locking DNA sequence dna is targeted as part small molecule strategy using PBD and is caused as novel anticancer and antibacterials More and more interest.The hydroxyl group of the C8/C8 ' of two PBD units, resulting dimer are connected using a flexible carbochain Bioactivity with enhancing.PBD dimer is considered as the DNA damage that can produce into sequence selectivity, such as the 5 '-of inverted order Pu-GATC-Py-3 ' interchain linkage, so as to cause its bioactivity.These compounds have proved to be efficient cytotoxicity medicine Object can be used as the drug candidate of antibody-drug conjugates.

Another preferred drug of the present invention is PNU-159682 derivative, and PNU-159682 is Nemorubicin in people Major active metabolite product in hepatomicrosome, compared with MMDX and adriamycin, activity improves 3000 times.

On the other hand, drug is not limited only to classification mentioned above, further include it is all can be used for antibody-drug idol Join the drug of object.And especially those can be coordinated by the amido bond with connector, such as by having alkaline amido (level-one Amine or secondary amine) Lai Peiwei cytotoxin, such as above shown in cytotoxin D1-D13 structure.

According to the mechanism of drug release in the cell, " connexon " or " connexons of antibody-drug conjugates " can be divided For two classes: connexon can not be broken and connexon can be broken.

For containing the antibody-drug conjugates that can not be broken connexon, mechanisms for drug release are as follows: conjugate and antigen In conjunction with and by after cell endocytic, antibody is digested in lysosome, is released by small-molecule drug, connexon and antibody amino groups The bioactive molecule that sour residue collectively constitutes.Thus bring drug molecular structure, which changes, does not weaken its cytotoxicity, but due to Bioactive molecule is electrically charged (amino acid residue), cannot penetrate into adjacent cells so as to cause it.Therefore, such active medicine is not Neighbouring tumour cell (bystander effect, the bystander for not expressing targeting antigen (antigen negative cells) can be killed effect)。

It can be broken connexon, as its name suggests, can be broken in target cell and release active medicine (small-molecule drug Itself).Connexon, which can be broken, can be divided into two main classifications: chemically unstable connexon and the unstable connexon of enzyme.Chemistry Unstable connexon can the fracture of selectivity due to the difference of blood plasma and cytoplasm property.Such property includes pH value, Glutathione concentrations etc..To the connexon of pH sensitive, also commonly known as acid fracture connexon.Such connexon is in blood (pH7.3-7.5) relatively stable under neutral environment, but in weakly acidic endosome (pH5.0-6.5) and lysosome (pH4.5- 5.0) it will be hydrolyzed in.The antibody-drug conjugates of the first generation apply this kind of connexon, such as hydrazone, carbonic ester, contracting mostly Aldehyde, ketal class.Since acid is broken the limited plasma stability of connexon, the antibody-drug conjugates based on such connexon are logical Often with there is shorter half-life period (2-3 days).This shorter half-life period limits pH sensitive linker new to a certain extent Application in generation antibody-drug conjugates.

For the connexon of glutathione sensitivity, also known as disulfide bond connexon.Drug release is based on intracellular gluathione In the high concentration (mM range) and blood of peptide caused by relatively low glutathione concentrations (micro-molar range) difference.It is right Especially true for tumour cell, low oxygen content leads to the increased activity of reductase, thus leads to higher glutathione Concentration.Disulfide bond has thermodynamic stability, therefore has preferable stability in blood plasma.

The unstable connexon of enzyme being capable of preferably Drug controlled release such as peptide connexon.Peptide connexon can be by lysosome Interior protease has such as cathepsin (CathepsinB) or fibrinolysin (this fermentoid content increases in some tumor tissues) The cutting of effect ground.This peptide connection is considered highly stable in plasma circulation, this is because extracellular inapt pH value and blood Albumosease inhibitor causes protease usually not having activity.In view of higher plasma stability and good intracellular disconnected Split selectivity and validity, what the unstable connexon of enzyme was used as antibody-drug conjugates extensively is broken connexon.Typically The unstable connexon of enzyme includes Val-Cit (VC), Phe-Lys etc..

It is generally entrenched in and can be broken between connexon and active medicine from release connexon, or inherently can the company of fracture Connect a part of son.Mechanism of action from release connexon is: after can be broken connexon and be broken under conditions of suitable, releasing certainly Structural rearrangement can spontaneously be carried out by putting connexon, and then discharge the active medicine being attached thereto.Common suicide connexon Including to aminobenzyl alcohol class (PAB) and beta-glucuronidase class (β-Glucuronide) etc..

Connexon

Connexon or coupling reagent of the invention includes two arylthio maleic amide units and a coupling group.Two virtues Sulfenyl maleic amide unit is used for the mercapto groups (after reduction) of cross-linking antibody interchain, and coupling group is used for and small-molecule drug Or drug-linker unit coupling.Due to-half Guang ammonia of opening cysteine in the two arylthios maleic amide unit and antibody Two teeth of two sulphur atoms of sour disulfide bond combine (bidentate binding), therefore these ADC are homogeneous and ratio contains The ADC of monodentate connector has stronger stability.Therefore they will have the Half-life in vivo increased, reduce systemic release Cytotoxic amount, and the ADC more safe drugs property than having monodentate connector.

On the other hand, generated drug-linker unit passes through the connexon and antibody coupling, generating portion chain Between the conjugate that is crosslinked.Compared with traditional antibody-drug conjugates, using the antibody-drug conjugate of the method for the present invention preparation Drug/Antibody ratio (DAR) distribution of object is narrower, to greatly improve product homogeneity and pharmacological characteristics homogeneity.

The antibody-drug conjugates can be used for targeting conveying drug and reach target cell population, such as tumour cell.It is anti- Body-drug conjugates can specificity in conjunction with cell surface protein, generated conjugate is immediately by cell endocytic.Thin Intracellular, drug releases generation effect in a manner of active medicine.Antibody includes chimeric antibody, humanized antibody, human antibody； It can be with the antibody fragment of antigen binding；Or antibody Fc fusion protein；Or albumen." drug " is high-activity drug, at certain In the case of, drug can be polyethylene glycol.

Antibody-drug conjugates

Antibody-drug conjugates provided by the invention are made of antibody, connexon, connexon and drug, the connexon It is that can be broken connection sub-portfolio or can not be broken connexon.

Antibody is globular preteins, can be used for coupling drug-connexon containing a series of amino acid sites.Due to its three-level and Quaternary structure, only solvent can and amino acid for coupling.In fact, the coupling of high yield usually occurs in lysine residue Epsilon-amino group or cysteine residues mercapto groups on.

A large amount of lysine side-chains on antibody protein surface cause a large amount of site for drug coupling, so as to cause generation Antibody-drug conjugates are mixtures, contain different drug coupling quantity (drug/Antibody ratio, DAR) and conjugation sites.

Coupling product provided by the invention, although being still mixture, the antibody-drug being coupled with traditional approach Conjugate is compared, and DAR distribution is very narrow.Its average DAR value is averaged DAR close to 4 close to optimum antibody-drug conjugates It is worth (2-4) range.In addition, coupling product does not contain naked anti-(DAR=0) seldom, this component does not work to cell poisoning.Together When, coupling product does not contain severe coupling product (DAR=8) yet, the removing speed of this component in vivo quickly, relative to low For the component of DAR.Therefore, antibody-drug conjugates product heterogencity provided by the invention is very significantly improved.

Pharmaceutical composition and method of administration

Due to antibody-drug conjugates provided by the invention, it can target and aim at special cell colony, with cell surface Differential protein (antigen) combines, to be penetrated by conjugate endocytosis or drug so that drug is discharged into cell in an active Interior, therefore, antibody-drug conjugates of the invention can be used for therapeutic purpose disease, antibody-drug conjugates above-mentioned Subject (such as people) can be given by suitable approach with therapeutically effective amount.Subject in need for the treatment of can be wind Danger, or suspect with the activity of specific antigen or expression quantity in relation to the patient of illness.Such patient can pass through conventional body Inspection is to identify.

Conventional method, it is known that medical domain those of ordinary skill, can be used for applying pharmaceutical composition to subject, This depends on the position of the type or disease to be treated of disease.This composition can also be by other conventional routes, for example, mouth Clothes, parenteral administration is spraying by sucking, part, rectum, intranasal, oral cavity, vagina or is administered by implantation.This paper institute Term " parenteral " is including subcutaneous, intradermal, intravenously, intramuscular, intra-articular, intra-arterial, intrasynovial, intrathecal in breastbone, disease In stove and intracranial injection or infusion techniques.In addition, it can be applied to by apply injectable reservoir approach, such as using The theme of 1-, 3- or 6 months reservoir injectables or biodegradable material and method.

Injectable composition can contain various carriers such as vegetable oil, dimethyl acetamide (dimethylactamide), two Methylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethyl alcohol, polyalcohol (glycerol, propylene glycol, the poly- second of liquid Glycol, etc.).For intravenous injection, thus water-soluble antibody can be contained antibody and can physiologically be connect by drop method The pharmaceutical preparation administered by infusion for the excipient received.Physiologically acceptable excipient may include, for example, 5% glucose, 0.9% salt water, Ringer's solution or other suitable excipient.Intramuscular formulations, for example, a suitable soluble salt of antibody Sterile preparation, the pharmaceutical excipient such as water that can be dissolved and apply changes injection, and 0.9% salt water or 5% glucose are molten Liquid.

When being treated with antibody-drug conjugates of the invention, can be delivered by the method for this field routine.Example Such as, it can be by using liposome, hydrogel, cyclodextrin, biodegradable Nano capsule or bioadhesive microballoon quilt It is introduced into cell.Alternatively, the nucleic acid or carrier can be delivered local by direct injection or by using infusion pump.It is other Method includes by using conjugate and Biodegradable polymeric using various transports and carrier system.

The antibody-drug conjugates of the invention and pharmaceutically may be used that pharmaceutical composition of the invention contains safe and effective amount The carrier of receiving.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof. Usual pharmaceutical preparation should match with administration mode, and pharmaceutical composition of the invention can be made into solution form, such as with Physiological saline or aqueous solution containing glucose and other adjuvants are prepared by conventional method.The pharmaceutical composition is suitable Aseptically manufacture.The dosage of active constituent is therapeutically effective amount.

The effective quantity of antibody-drug conjugates of the present invention can with administration mode and disease to be treated it is serious Degree etc. and change.Preferred a effective amount of selection can determine (example depending on various factors by those of ordinary skill in the art Such as pass through clinical test).The factor includes but is not limited to: the pharmacokinetic parameter of the bifunctional antibody conjugate Such as bioavailability, metabolism, half-life period etc.；Patient the severity of disease to be treated, the weight of patient, patient exempt from Epidemic disease situation, approach of administration etc..In general, when antibody-drug conjugates of the invention are dynamic with about 0.0001mg-50mg/kg daily The dosage of object weight (preferable 0.001mg-10mg/kg the weight of animals) is given, and satisfactory effect can be obtained.For example, by An urgent demand of situation is treated, dosage separated several times can be given once daily, or dosage is reduced pari passu.

The dosage form of the compounds of this invention for local administration includes ointment, powder, patch, stock solution and inhalant. Active constituent aseptically with physiologically acceptable carrier and any preservative, buffer, or when necessary may need Propellant be mixed together.

The compounds of this invention can be administered alone, or be administered with other pharmaceutically acceptable therapeutic agents.

It is the mammal that the compounds of this invention of safe and effective amount is applicable to treatment when using pharmaceutical composition (such as people), wherein dosage is the effective dosage pharmaceutically thought when application, for the people of 60kg weight, day is to medicament Amount is usually 1~2000mg, preferably 5~500mg.Certainly, specific dosage be also contemplated that administration route, patient health situation etc. because Element.

Experiment shows that main advantages of the present invention are:

1, novel connexon provided by the invention, can be by simple chemical method and antibody coupling, with traditional coupling Mode is compared, and the conjugate DAR Distribution value obtained using this connexon is very narrow, therefore the product homogeneity generated is high, obtains 80% or more component (DAR 4) accounting of the single distribution of cross-linking agent obtained.

2, antibody-drug conjugates provided by the invention, naked anti-and low crosslinking degree almost nil (the mass spectrum inspection of ADC accounting The component that DAR is 0 and 1 is not measured).

3, applicant proves through a large number of experiments, antibody-drug conjugates provided by the invention, in terms for the treatment of tumour With certain safety and validity.The hydrophily that ethylene glycol is assigned after coupling can be used to adjust biomolecule characteristic；It hands over Join the more traditional mcVC-PAB crosslinked bio activity of tumor cell in vitro proliferation inhibition activity, drug metabolism stability, peace of object The patent medicine properties such as full property are increased or are kept.

4, coupling method provided by the invention is suitable for most of antibody, so as to avoid carrying out each antibody Cumbersome modified recombinant is with a wide range of applications with introducing site-directed coupling site.

5, coupling method provided by the invention is compared with existing coupling method, the disulfide bond of the invention based on maleic amide The advantages of bridging cross-linking reagent includes: with faster crosslinking rate, and cross-linking reaction time can be anti-usually within 2-4 hours It should finish.

6, the present invention is based on the disulfide bond of maleic amide bridge joints to have better stability, is not susceptible to the friendship of mercapto ether in vivo It changes, while secondary in the cyclization that the position Ar ' the introducing more unsubstituted phenyl of substituent group can slow down significantly after maleic amide open loop Hydrolysis further enhances the stability of antibody-drug conjugates in vitro and in vivo.

Detailed description of the invention

Fig. 1-1: hydrophobic interaction chromatography (HIC) map of handkerchief trastuzumab (Pertuzumab)；

Fig. 1-2: handkerchief trastuzumab-drug conjugates ADC-I hydrophobic interaction chromatography (HIC) map；

Fig. 1-3: handkerchief trastuzumab-drug conjugates ADC-II hydrophobic interaction chromatography (HIC) map；

Fig. 1-4: handkerchief trastuzumab-drug conjugates ADC-III hydrophobic interaction chromatography (HIC) map；

Fig. 1-5: handkerchief trastuzumab-drug conjugates ADC-IV hydrophobic interaction chromatography (HIC) map；

Fig. 1-6: handkerchief trastuzumab-drug conjugates ADC-V hydrophobic interaction chromatography (HIC) map；

Fig. 1-7: handkerchief trastuzumab-drug conjugates ADC-VI hydrophobic interaction chromatography (HIC) map；

Fig. 1-8: handkerchief trastuzumab-drug conjugates ADC-VII hydrophobic interaction chromatography (HIC) map；

Fig. 2-1: hydrophobic interaction chromatography (HIC) map of Herceptin (Trastuzumab)；

Fig. 2-2: Herceptin-drug conjugates ADC-VIII hydrophobic interaction chromatography (HIC) map；

Fig. 3-1: the mass-spectrogram of handkerchief trastuzumab (Pertuzumab)；

Fig. 3-2: handkerchief trastuzumab-drug conjugates ADC-I mass-spectrogram；

Fig. 3-3: handkerchief trastuzumab-drug conjugates ADC-II mass-spectrogram；

Fig. 3-4: handkerchief trastuzumab-drug conjugates ADC-III mass-spectrogram；

Fig. 3-5: handkerchief trastuzumab-drug conjugates ADC-IV mass-spectrogram；

Fig. 3-6: handkerchief trastuzumab-drug conjugates ADC-V mass-spectrogram；

Fig. 3-7: handkerchief trastuzumab-drug conjugates ADC-VI mass-spectrogram；

Fig. 3-8: handkerchief trastuzumab-drug conjugates ADC-VII mass-spectrogram；

Fig. 4-1: the mass-spectrogram of Herceptin (Trastuzumab)；

Fig. 4-2: Herceptin-drug conjugates ADC-VIII mass-spectrogram；

Fig. 5: it shows and each ADC control, ADC-I, ADC-II, ADC-VII was measured at 0-7 days using LC-MS (Q-TOF) Corresponding secondary hydrolysate generates trend chart at room temperature；

Fig. 6-1: show control ADC at 0 day corresponding HIC map at room temperature；

Fig. 6-2: show control ADC at 2 days corresponding HIC map at room temperature；

Fig. 6-3: show control ADC at 4 days corresponding HIC map at room temperature；

Fig. 6-4: show control ADC at 7 days corresponding HIC map at room temperature；

Fig. 7-1: show ADC-I at 0 day corresponding HIC map at room temperature；

Fig. 7-2: show ADC-I at 2 days corresponding HIC map at room temperature；

Fig. 7-3: show ADC-I at 4 days corresponding HIC map at room temperature；

Fig. 7-4: show ADC-I at 7 days corresponding HIC map at room temperature；

Fig. 8-1: show ADC-II at 0 day corresponding HIC map at room temperature；

Fig. 8-2: show ADC-II at 2 days corresponding HIC map at room temperature；

Fig. 8-3: show ADC-II at 4 days corresponding HIC map at room temperature；

Fig. 8-4: show ADC-II at 7 days corresponding HIC map at room temperature；

Fig. 9-1: show ADC-VII at 0 day corresponding HIC map at room temperature；

Fig. 9-2: show ADC-VII at 2 days corresponding HIC map at room temperature；

Fig. 9-3: show ADC-VII at 4 days corresponding HIC map at room temperature；

Fig. 9-4: show ADC-VII at 7 days corresponding HIC map at room temperature；

Figure 10: ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, Pertuzumab (Perjeta) to the proliferation inhibition test result figure of gastric carcinoma cells NCI-N87；

Figure 11: ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, Pertuzumab (Perjeta) to the proliferation inhibition test result figure of human breast cancer cell BT-474；

Figure 12: ADC-VIII, proliferation inhibition test of the Trastuzumab (Herceptin) to gastric carcinoma cells NCI-N87 Result figure；

Figure 13: ADC-VIII, Trastuzumab (Herceptin) is real to the Proliferation Ability of human breast cancer cell BT-474 Test result figure；

Figure 14: P-mcVC-MMAE (1.0mg/kg), control ADC (0.5,1.0mg/kg), ADC-I (1.0mg/kg), ADC- IV (1.0mg/kg), ADC-V (1.0mg/kg), ADC-VI (1.0mg/kg), ADC-VII (0.5,1.0mg/kg) inhibit human gastric cancer The activity research figure of NCI-N87 nude mouse subcutaneous transplantation tumor.

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Embodiment group one, compound synthesis and preparation method

The synthesis of 1.1 compound E-1 (Formulas I a-1)

1.1.1 (the step a) of intermediate A -1

Triethylene glycol (92g, 613mmol) is dissolved in tBuOH (200ml).Under ice bath be added KOtBu (22.91g, It 204mmol) stirs half an hour, under argon gas protection, bromo-acetic acid tert-butyl (39.8g, 204mmol) is added dropwise tBuOH's (40ml) Solution is stirred overnight at room temperature.Second day, TLC detection reaction terminated.After revolving removes the tert-butyl alcohol, 400ml dichloro is added in residue Methane, organic phase are washed with 400ml, and water phase 300ml methylene chloride extracts once, use saturated salt solution after merging organic phase It washes once, anhydrous sodium sulfate is dry, and revolving is evaporated.Crude product is through petroleum ether: ethyl acetate=3:1-- > 1:1 column chromatography, obtains Mesosome A-1 (24g, 44.5%yield) is yellow oil.

1.1.2 (the step b) of intermediate B -1

By intermediate A -1 (7.8g, 29.5mmol), the fluoro- 2- nitro-trifluoromethyl toluene (9.26g, 44.3mmol) of 5-, K₂CO₃ (6.12g, 44.3mmol) powder is heated to 80 DEG C and stirs 48 hours in 250mL round bottom reaction flask under nitrogen protection, TLC prison It surveys, only a small amount of starting material left.:

It is down to room temperature to be extracted with 500 methylene chloride, 400ml 1N dilute hydrochloric acid is washed once, and 400ml washing is primary, and 400ml is full Primary with salt washing, anhydrous sodium sulfate is dry, and revolving is evaporated.Column chromatographs (200 mesh~300 mesh silica gel) purifying, petroleum ether: second Acetoacetic ester 30:1-10:1 elution, obtains intermediate B -1 (7.5g, 56.1%yield), is yellow oil.

1.1.3 intermediate C-1

Intermediate B -1 (6g, 13.23mmol) is dissolved in 100 milliliters of dehydrated alcohols and by solution, is added and is equipped with 10% In the reaction flask of Pd-C1.2g.Hydrogenation reaction 6 hours (1atm, 38 DEG C), TLC detected fully reacting.Diatomite filtering reacting liquid, Filter cake ethanol rinse, filtrate are evaporated, and obtaining intermediate C-1 (5g, 89%yield) is yellow oil.

1.1.4 compound E-1

Intermediate C-1 (0.8g, 1.889mmol) is weighed in parallel reaction pipe, AcOH (3ml) is added under nitrogen protection, Stirring and dissolving.After be slowly added into 3,4- dibromomaleic acid acid anhydride (0.483g, 1.889mmol).110 DEG C are heated under nitrogen protection to stir It mixes overnight.TLC detection reaction.After reaction solution is cooled to room temperature, solvent evaporated is rotated, and toluene is added and is evaporated twice, Obtain brown oil compound E-1.The next step is directly used in without purifying.

Synthesis (the step e) of 1.2 compound E-2 (Formulas I a-2)

It is anhydrous that 30ml is added in Weigh Compound E-1 (2.0g, 1.35mmol) in 100 milliliters of round-bottomed bottles, under nitrogen protection Methylene chloride stirring and dissolving.It weighs and is added in reaction solution under 297mg benzenethiol nitrogen protection, is slowly added dropwise under ice bath after dissolution DIPEA (0.44ml, 2.70mmol), after stir 5 minutes, remove ice bath.2 hours are stirred at room temperature under nitrogen protection, TLC Detection reaction terminates.

After evaporated under reduced pressure solvent, column chromatography (200 mesh~300 mesh silica gel) is isolated and purified, and methylene chloride fills column and elution, so Afterwards slowly increase from 2% to 10% methanol of polarity elution, collect solvent evaporated obtain crocus oil product E-2 (0.92g, 79% yield)。LC-MS(M⁺) theoretical value: 595.13, measured value: 596.15 (ESI, M+H⁺)。

The synthesis of 1.3 compound E-3 (Formulas I a-3)

The synthesis of compound E-3 is identical as the synthesis step of compound E-2 in example 1.2, only by the benzenethiol in step e It is changed to 2- mercaptopyridine, obtaining product E-3 is crocus grease.

The synthesis of 1.4 compound E-4 (Formulas I a-4)

The synthesis of compound E-4 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to 2- methoxyl group -4- fluoronitrobenzene, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzene sulphur Phenol, obtaining product E-4 is crocus grease.

The synthesis of 1.5 compound E-5 (Formulas I a-5)

The synthesis of compound E-5 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 2- methoxyl group -4- nitrobenzene of 1-, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) Benzenethiol, obtaining product E-5 is crocus grease.

The synthesis of 1.6 compound E-6 (Formulas I a-6)

The synthesis of compound E-6 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 2- nitrobenzonitrile of 5-, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzene sulphur Phenol, obtaining product E-6 is crocus grease.

The synthesis of 1.7 compound E-7 (Formulas I a-7)

The synthesis of compound E-7 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 5- nitrobenzonitrile of 2-, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzene sulphur Phenol, obtaining product E-7 is crocus grease.

The synthesis of 1.8 compound E-8 (Formulas I a-8)

The synthesis of compound E-8 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 2- nitrobenzamide of 5-, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzene sulphur Phenol, obtaining product E-8 is crocus grease.

The synthesis of 1.9 compound E-9 (Formulas I a-9)

The synthesis of compound E-9 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 1- nitro -2- trifluoromethylbenzene of 4-, and the benzenethiol in step e is changed to 4- (N- morpholine formyl Amine) benzenethiol, obtaining product E-9 is crocus grease.

The synthesis of 1.10 compound E-10 (Formulas I a-10)

The synthesis of compound E-10 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 4- nitro -2- trifluoromethylbenzene of 1-, and the benzenethiol in step e is changed to 4- (N- morpholine formyl Amine) benzenethiol, obtaining product E-10 is crocus grease.

The synthesis of 1.11 compound E-11 (Formulas I a-11)

1.11.1 intermediate F-11 (step f)

By intermediate A -1 (4g, 15.13mmol), triethylamine (2.53ml, 18.16mmol) and two in 250ml round-bottomed bottle Picolilamine (0.370g, 3.03mmol), which is dissolved in 100 milliliters of molecular sieve dry methylene chlorides, to be stirred, and is added portionwise under ice bath P-methyl benzene sulfonic chloride (3.17g, 16.65mmol) moves to the protection of room temperature argon gas and is stirred overnight.

The extraction of 100ml methylene chloride is added in reaction system, is washed once with 200ml 1N dilute hydrochloric acid, 200ml is washed twice, The washing of 200ml saturated salt is primary, and anhydrous sodium sulfate is dry, is evaporated organic phase.Column, PE:EA=are filled with 200-300 mesh silica gel 5:1-2:1 elution carries out column chromatography for separation.It is evaporated to obtain intermediate F-11 (2.8g, yield 44.2%).

1.11.2 (the step g) of intermediate B -11

By intermediate F-11 (1g, 2.389mmol), 2,6- bis- fluoro-4-nitrophenols (0.315g, 1.797mmol) are dissolved in In 20ml DMF, K is added₂CO₃(0.497g, 3.59mmol) is heated to 100 degree and stirs 5 hours.Solvent evaporated is rotated, is added 200ml 1N dilute hydrochloric acid is used in the dissolution of 200ml methylene chloride, extraction respectively, and 200ml water and the washing of 200ml saturated salt are respectively washed once, Anhydrous sodium sulfate is dry, and revolving is evaporated, and fills column with 200-300 mesh silica gel, PE:EA=5:1-3:1 elutes column chromatographic purifying, revolving It is evaporated to obtain intermediate B -11 (600mg, yield 79%)

1.11.3 intermediate C-11

Intermediate B -11 (600mg, 1.42mmol) is dissolved in 100 milliliters of dehydrated alcohols and by solution, addition is equipped with

In the reaction flask of 10%Pd-C 120mg.Hydrogenation reaction 6 hours (1atm, 38 DEG C), TLC detected fully reacting.Silicon Diatomaceous earth filtering reacting liquid, filter cake ethanol rinse, filtrate are evaporated, and obtaining intermediate C-11 (450mg, yield 81%) is yellow Grease.

1.11.4 intermediate D-11

Intermediate C-11 (0.40g, 1.02mmol) is weighed in parallel reaction pipe, AcOH (3ml) is added under nitrogen protection, Stirring and dissolving.After be slowly added into 3,4- dibromomaleic acid acid anhydride (0.261g, 1.02mmol).110 DEG C are heated under nitrogen protection to stir It mixes overnight.TLC detection reaction.After reaction solution is cooled to room temperature, solvent evaporated is rotated, and toluene is added and is evaporated twice, Obtain brown oil compound D-11.The next step is directly used in without purifying.

1.11.5 intermediate E -11

Weigh Compound D-11 (600mg, 0.95mmol) in 100 milliliters of round-bottomed bottles, under nitrogen protection be added 30ml without Water methylene chloride stirring and dissolving.It weighs and is added instead under 425mg (1.91mmol) 4- (N- morpholine formamide) benzenethiol nitrogen protection Answer in liquid, be slowly added dropwise under ice bath after dissolution DIPEA (0.36ml, 1.91mmol), after stir 5 minutes, remove ice bath. It is stirred at room temperature under nitrogen protection 2 hours, TLC detection reaction terminates.

After evaporated under reduced pressure solvent, column chromatography (200 mesh~300 mesh silica gel) is isolated and purified, and methylene chloride fills column and elution, so Afterwards slowly increase from 2% to 10% methanol of polarity elution, collect solvent evaporated obtain crocus oil product E-11 (0.62g, 76% yield)。LC-MS(M⁺) theoretical value: 857.21, measured value: 858.23 (ESI, M+H⁺)。

The synthesis of 1.12 compound E-12 (Formulas I a-12)

The synthesis of compound E-12 is identical as the synthesis step of compound E-11 in example 1.11, only by 2 in step g, Bis- fluoro-4-nitrophenol of 6- is changed to 3- fluoro-4-nitrophenol, and obtaining product E-12 is crocus grease.

The synthesis of 1.13 compound E-13 (Formulas I a-13)

The synthesis of compound E-13 is identical as the synthesis step of compound E-11 in example 1.11, only by 2 in step g, Bis- fluoro-4-nitrophenol of 6- is changed to 2,5-, bis- fluoro-4-nitrophenol, and obtaining product E-13 is crocus grease.

The synthesis of 1.14 compound E-14 (Formulas I a-14)

The synthesis of compound E-14 is identical as the synthesis step of compound E-11 in example 1.11, only by three in step a Glycol is changed to diethylene glycol (DEG), and obtaining product E-14 is crocus grease.

The synthesis of 1.15 compound E-15 (Formulas I a-15)

The synthesis of compound E-15 is identical as the synthesis step of compound E-11 in example 1.11, only by three in step a Glycol is changed to tetraethylene glycol, and obtaining product E-15 is crocus grease.

The synthesis of 1.16 compound E-16 (Formulas I a-16)

The synthesis of compound E-16 is identical as the synthesis step of compound E-11 in example 1.11, only by three in step a Glycol is changed to five glycol, and obtaining product E-16 is crocus grease.

The synthesis of 1.17 compound E-17 (Formulas I a-17)

The synthesis of compound E-17 is identical as the synthesis step of compound E-11 in example 1.11, only by three in step a Glycol is changed to hexaethylene glycol, and obtaining product E-17 is crocus grease.

The synthesis of 1.18 compound E-18 (Formulas I a-18)

The synthesis of compound E-18 is identical as the synthesis step of compound E-11 in example 1.11, only by three in step a Glycol is changed to ten diethylene glycol (DEG)s, and obtaining product E-18 is crocus grease.

The synthesis of 1.19 compound E-19 (Formulas I a-19)

The synthesis of compound E-19 is identical as the synthesis step of compound E-11 in example 1.11, only by the 4- in step e (N- morpholine formamide) benzenethiol is changed to 1,1- titanium dioxide thiomorpholine, and obtaining product E-19 is crocus grease.

The synthesis of 1.20 compound E-20 (Formulas I a-20)

The synthesis of compound E-20 is identical as the synthesis step of compound E-11 in example 1.11, only by the 4- in step e (N- morpholine formamide) benzenethiol is changed to 4- (N-METHYLFORMAMIDE) benzenethiol, and obtaining product E-20 is crocus grease.

The synthesis of 1.21 compound E-21 (Formulas I a-21)

The synthesis of compound E-21 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to 2- nitro -5- fluorine pyridine, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzenethiol, Obtaining product E-21 is crocus grease.

The synthesis of 1.22 compound E-22 (Formulas I a-22)

The synthesis of compound E-21 is identical as the synthesis step of compound E-2 in example 1.2, only that the 5- in step b is fluoro- 2- nitro-trifluoromethyl toluene is changed to the fluoro- 5- nitropyridine of 2-, and the benzenethiol in step e is changed to 4- (N- morpholine formamide) benzenethiol, Obtaining product E-22 is crocus grease.

Embodiment group 2: the synthesis and preparation of Formulas I b-1~Ib-24

The synthesis of 2.1 compound F1-1 (Formulas I b-1)

It is weighed into compound E1-9 (300mg, 0.337mmol), is added under nitrogen protection anhydrous into 100 milliliters of round-bottomed bottles After DMF (20mL) makes it completely dissolved, HATU (154mg, 0.404mmol) and DIEA (0.11ml, 0.674mmol) are successively weighed It is added in bottle.Compound D1-1 (219mg, 0.337mmol) is added after being stirred at room temperature 15 minutes, was stirred at room temperature under nitrogen protection Night.TLC reacts overnight with HPLC tracking, and raw material E9 disappears.Evaporated under reduced pressure solvent, does quantitative analysis, purifies by reversed-phase HPLC, Obtaining product is Yellow amorphous powder F1-1 (0.350g, 0.230mmol, 68.2%yield).LC-MS(M⁺) theoretical value: 1520.48, measured value: 1521.51 (ESI, M+H⁺)。

The synthesis of 2.2 compound F1-2 (Formulas I b-2)

The synthesis of compound F1-2 is identical as the synthesis step of compound F1-1 in example 2.1, only wherein being changed to of D1-1 Object D1-2 is closed, obtaining product F1-2 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1993.91, measured value: 1994.93 (ESI,M+H⁺)。

The synthesis of 2.3 compound F1-3 (Formulas I b-3)

The synthesis of compound F1-3 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-3, obtaining product F1-3 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 2007.89, measured value: 2008.91(ESI,M+H⁺)。

The synthesis of 2.4 compound F1-4 (Formulas I b-4)

The synthesis of compound F1-4 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-4, obtaining product F1-4 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 2046.88, measured value: 2047.86(ESI,M+H⁺)。

The synthesis of 2.5 compound F1-5 (Formulas I b-5)

The synthesis of compound F1-5 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-5, obtaining product F1-5 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1800.67, measured value: 1801.65(ESI,M+H⁺)。

The synthesis of 2.6 compound F1-6 (Formulas I b-6)

The synthesis of compound F1-6 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-6, obtaining product F1-6 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1989.79, measured value: 1990.80(ESI,M+H⁺)。

The synthesis of 2.7 compound F1-7 (Formulas I b-7)

The synthesis of compound F1-7 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein compound D1- 1 is changed to compound D1-7, and obtaining product F1-7 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1973.72, measured value: 1974.72(ESI,M+H⁺)。

The synthesis of 2.8 compound F1-8 (Formulas I b-8)

The synthesis of compound F1-8 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-8, obtaining product F1-8 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1973.72, measured value: 1974.72(ESI,M+H⁺)。

The synthesis of 2.9 compound F1-9 (Formulas I b-9)

The synthesis of compound F1-9 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 and D1- 1 is changed to compound E1-17 and D1-9 respectively, and obtaining product F1-9 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 2015.72, measured value: 2016.73 (ESI, M+H⁺)。

The synthesis of 2.10 compound F1-10 (Formulas I b-10)

The synthesis of compound F1-10 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-10, obtaining product F1-10 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1912.63, measured value: 1913.65(ESI,M+H⁺)。

The synthesis of 2.11 compound F1-11 (Formulas I b-11)

The synthesis of compound F1-11 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-11, obtaining product F1-11 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1916.63, measured value: 1917.61(ESI,M+H⁺)。

The synthesis of 2.12 compound F1-12 (Formulas I b-12)

The synthesis of compound F1-12 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-12, obtaining product F1-12 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 2031.70, measured value: 2032.71(ESI,M+H⁺)。

The synthesis of 2.13 compound F1-13 (Formulas I b-13)

The synthesis of compound F1-13 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-13, obtaining product F1-13 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1711.57, measured value: 1712.55(ESI,M+H⁺)。

The synthesis of 2.14 compound F1-14 (Formulas I b-14)

The synthesis of compound F1-14 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein D1-1 be changed to Compound D1-2, obtaining product F1-14 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1767.82, measured value: 1768.83(ESI,M+H⁺)。

The synthesis of 2.15 compound F1-15 (Formulas I b-15)

The synthesis of compound F1-15 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E-19 and D1-2 respectively, and obtaining product F1-15 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 2057.84, measured value: 2058.87 (ESI, M+H⁺)。

The synthesis of 2.16 compound F1-16 (Formulas I b-16)

The synthesis of compound F1-16 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-20 and D1-2 respectively, and obtaining product F1-16 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1849.85, measured value: 1850.83 (ESI, M+H⁺)。

The synthesis of 2.17 compound F1-17 (Formulas I b-17)

The synthesis of compound F1-17 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-10 and D1-2 respectively, and obtaining product F1-17 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1993.91, measured value: 1994.90 (ESI, M+H⁺)。

The synthesis of 2.18 compound F1-18 (Formulas I b-18)

The synthesis of compound F1-18 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-11 and D1-2 respectively, and obtaining product F1-18 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1961.90, measured value: 1962.91 (ESI, M+H⁺)。

The synthesis of 2.19 compound F1-19 (Formulas I b-19)

The synthesis of compound F1-19 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-5 and D1-2 respectively, and obtaining product F1-19 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1955.93, measured value: 1956.95 (ESI, M+H⁺)。

The synthesis of 2.20 compound F1-20 (Formulas I b-20)

The synthesis of compound F1-20 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-4 and D1-2 respectively, and obtaining product F1-20 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1955.93, measured value: 1956.95 (ESI, M+H⁺)。

The synthesis of 2.21 compound F1-21 (Formulas I b-21)

The synthesis of compound F1-21 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-12 and D1-2 respectively, and obtaining product F1-21 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1943.91, measured value: 1944.90 (ESI, M+H⁺)。

The synthesis of 2.22 compound F1-22 (Formulas I b-22)

The synthesis of compound F1-22 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-6 and D1-2 respectively, and obtaining product F1-22 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1950.92, measured value: 1951.93 (ESI, M+H⁺)。

The synthesis of 2.23 compound F1-23 (Formulas I b-23)

The synthesis of compound F1-23 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-21 and D1-2 respectively, and obtaining product F1-23 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1926.92, measured value: 1927.93 (ESI, M+H⁺)。

The synthesis of 2.24 compound F1-24 (Formulas I b-24)

The synthesis of compound F1-24 is identical as the synthesis step of compound F1-1 in example 2.1, only will wherein E1-9 with D1-1 is changed to compound E1-22 and D1-2 respectively, and obtaining product F1-24 is Yellow amorphous powder.LC-MS(M⁺) theoretical value: 1926.92, measured value: 1927.93 (ESI, M+H⁺)。

The preparation of embodiment group two, antibody coupling matter

1, the preparation of ADC-I

By pa appropriate pearl antibody stoste 50mM potassium dihydrogen phosphate-sodium hydroxide (KH₂PO₄- NaOH)/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), 7 reaction buffer of pH are diluted to 2mg/mL, and 6.0 times of molar excess are added Three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP) of ratio, reaction solution stir 2.5 hours in 35 DEG C.

Above-mentioned reaction solution is cooled to 8 DEG C, it is not purified that suitable dimethyl sulfoxide (DMSO) is added, it adds 6 times of excess and rubs The compound F1-17 (10mg/ml is dissolved in DMSO in advance) of your ratio guarantees that the volume accounting of DMSO in reaction system is no more than 15%, it stirs 3 hours and is coupled in 37 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.15 micron pore size, -60 DEG C of preservations.

2, the preparation of ADC-II

By pa appropriate pearl antibody stoste 50mM potassium dihydrogen phosphate-sodium hydroxide (KH₂PO₄- NaOH)/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), 6 reaction buffer of pH are diluted to 5mg/mL, and 10 times of molar excess are added Three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP) of ratio, reaction solution stir 40 hours in 10 DEG C.

Above-mentioned reaction solution is cooled to 5 DEG C, it is not purified that suitable diethyl acetamide (DMA) is added, add 6 times of excess The compound F1-2 (10mg/ml is molten in advance in the dma) of molar ratio, guarantees that the volume accounting of DMA in reaction system is no more than 10%, it stirs 2.5 hours and is coupled in 25 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.22 micron pore size, -80 DEG C of preservations.

3, the preparation of ADC-III

By pa appropriate pearl antibody stoste PBS//1mM diethyl pentetic acid (DTPA), the dilution of 7.4 reaction buffer of pH To 5mg/mL, three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP) of 20 times of excess molar ratios are added, reaction solution is small in 15 DEG C of agitations 2 When.

Above-mentioned reaction solution is cooled to 10 DEG C, it is not purified that suitable acetonitrile (ACN) is added, add 6 times of excess molar ratios Compound F1-20 (10mg/ml is dissolved in ACN in advance), guarantee reaction system in ACN volume accounting be no more than 10%, in 10 DEG C of agitations are coupled for 4 hours.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 8.0, according to UV280 ultraviolet absorption value collects peak sample, then filtration sterilization, products therefrom cryo-conservation；Such as via 0.20 micron pore size Filter device degerming, -90 DEG C preservation.

4, the preparation of ADC-IV

By pa appropriate pearl antibody stoste 50mM potassium dihydrogen phosphate-sodium hydroxide (KH₂PO₄- NaOH)/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), 7 reaction buffer of pH are diluted to 8mg/mL, and 8 times of excess molar ratios are added Three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP), reaction solution in 25 DEG C stir 25 hours.

Above-mentioned reaction solution is cooled to 5 DEG C, it is not purified that suitable dimethylformamide (DMF) is added, add 6 times of excess The compound F1-19 (10mg/ml is dissolved in DMF in advance) of molar ratio guarantees that the volume accounting of DMF in reaction system is no more than 8%, it stirs 2 hours and is coupled in 0 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.3 micron pore size, -80 DEG C of preservations.

5, the preparation of ADC-V

By pa appropriate pearl antibody stoste 50mM histidine-sodium hydroxide/150mM sodium chloride (NaCl)/1mM diethyl triamine Pentaacetic acid (DTPA), 7.4 reaction buffer of pH are diluted to 6mg/mL, and three (2- carboxyethyl) phosphonium salts of 8 times of excess molar ratios are added Hydrochlorate (TCEP), reaction solution stir 15 hours in 35 DEG C.

Above-mentioned reaction solution is cooled to 10 DEG C, it is not purified that suitable dimethylformamide (DMF) is added, add 6 times of mistakes The compound F1-22 (10mg/ml is dissolved in DMF in advance) of molar ratio is measured, guarantees that the volume accounting of DMF in reaction system is no more than 8%, it stirs 5 hours and is coupled in 0 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.15 micron pore size, -100 DEG C of preservations.

6, the preparation of ADC-VI

By pa appropriate pearl antibody stoste 50mM boric acid-borax/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), 9 reaction buffer of pH is diluted to 10mg/mL, and three (2- carboxyethyl) phosphonium salt hydrochlorates of 8 times of excess molar ratios are added (TCEP), reaction solution stirs 10 hours in 25 DEG C.

Above-mentioned reaction solution is cooled to 10 DEG C, it is not purified that suitable dimethylformamide (DMF) is added, add 6 times of mistakes The compound F1-21 (10mg/ml is dissolved in DMF in advance) of molar ratio is measured, guarantees that the volume accounting of DMF in reaction system is no more than 8%, it stirs 4 hours and is coupled in 0 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.2 micron pore size, -60 DEG C of preservations.

7, the preparation of ADC-VII

By pa appropriate pearl antibody stoste 50mM potassium dihydrogen phosphate-sodium hydroxide (KH₂PO₄- NaOH)/150mM sodium chloride (NaCl)/1mM diethyl pentetic acid (DTPA), pH8 reaction buffer are diluted to 3mg/mL, and 8 times of excess molar ratios are added Three (2- carboxyethyl) phosphonium salt hydrochlorates (TCEP), reaction solution in 15 DEG C stir 48 hours.

Above-mentioned reaction solution is cooled to 0 DEG C, it is not purified that suitable dimethylformamide (DMF) is added, add 6 times of excess The compound F1-18 (10mg/ml is dissolved in DMF in advance) of molar ratio guarantees that the volume accounting of DMF in reaction system is no more than 8%, it stirs 3 hours and is coupled in 0 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.3 metre hole diameter, -70 DEG C of preservations.

8, the preparation of ADC-VIII

By toltrazuril antibody stoste 50mM disodium hydrogen phosphate-citric acid/150mM sodium chloride (NaCl)/1mM diethyl three Triamine pentaacetic acid (DTPA), 7.4 reaction buffer of pH are diluted to 5mg/mL, and three (2- carboxyethyl) phosphines of 8 times of excess molar ratios are added Hydrochloride (TCEP), reaction solution stir 5 hours in 25 DEG C.

Above-mentioned reaction solution is cooled to 0 DEG C, it is not purified that suitable dimethylformamide (DMF) is added, add 6 times of excess The compound F1-2 (10mg/ml is dissolved in DMF in advance) of molar ratio guarantees that the volume accounting of DMF in reaction system is no more than 8%, it stirs 2 hours and is coupled in 0 DEG C.

It is using desalting column that coupling reaction mixture is gel-filtration purified with histidine-acetic acid/sucrose of pH 6.0, according to UV280 ultraviolet absorption value collects peak sample.Then via the filter device degerming of 0.3 metre hole diameter, -80 DEG C of preservations.

Embodiment group three, the detection of antibody coupling matter and stability study

Conjugation sites number and location can be obtained by carrying out hydrophobic interaction chromatograph HIC analysis for antibody coupling drug With the important informations such as drug antibody coupling ratio (drug to antibody ratio, DAR).We are based on the following conditions with regard to above-mentioned ADC product carries out HIC analysis, and analysis map is shown in Fig. 1-1~1-8 and Fig. 2-1~2-2.

Agilent 1290Infinity

Chromatographic column: Waters Protein-Pak Hi Res HIC (4.6*100mm, 2.5 μm)

Mobile phase: 2.5M ammonium sulfate (phosphate buffer containing 125mM): 125mM phosphate buffer: isopropanol

Flow velocity: 0.7mL/min, column temperature: 25 DEG C

In addition, LC-MS technology, has been used for ADC medicines structure and composition analysis, the stabilization of ADC drug connector is evaluated Property, analysis measures the relative scale etc. of different DAR components.We are based on the following conditions and carry out lcms analysis with regard to above-mentioned ADC product. Analysis map is shown in Fig. 3-1~3-8 and Fig. 4-1~4-2.

Instrument: 6520 Q-TOF of Agilent

Chromatographic column: Polyhydroxyethyl-A (PHEA) (PolyLC, Columbia, MD) 2.1mm*200mm；5μm

particles withpores

Mobile phase: 200mM ammonium acetate

Flow velocity: 0.1mL/min；

Column temperature: 25 DEG C

The present invention is based on the disulfide bond of maleic amide bridge joints to have better stability, is not susceptible to the friendship of mercapto ether in vivo It changes, in order to further confirm after the position Ar ' the introducing more unsubstituted phenyl of substituent group can slow down significantly maleic amide open loop The secondary hydrolysis of cyclization, while the stability of antibody-drug conjugates can be strengthened.We are prepared for pair in this experiment According to ADC, it is only the substituted benzene ring compound of Isosorbide-5-Nitrae-(such as following formula) by the appropriate pearl antibody of pa and Ar ', is coupled, coupling method is same The preparation of ADC-I.

And ADC-I is picked, ADC-II, ADC-VII are compared with control ADC, respectively by same protein concentration (10mg/mL) is stored in the ADC sample in preparation buffer, is placed in 25 DEG C, is measured by sampling respectively at 0,2,4,7 day.

Corresponding secondary hydrolysate in each antibody-drug conjugates (ADC) is measured using LC-MS (Q-TOF), is extracted The mass spectral characteristic peak of secondary hydrolysate, obtains its peak area.The situation of change for comparing 0-7 days peak areas obtains ADC bis- times hydrolysis The trend of product, is detailed in following data and Fig. 5.It can be seen that control bis- hydrolysates of ADC are apparently higher than ADC- from data Secondary hydrolysate in I, ADC-II, ADC-VII sample.

In addition, the situation of change for also using HIC method to measure 0,2,4,7 day using each ADC sample, from Fig. 6-1~6-4 As can be seen that there are impurity peaks in 6.904 position of retention time when compareing ADC sample at 7 days, and ADC-I, ADC-II, From 0 to 7 day HIC map substantially changes unobvious in ADC-VII sample, respectively referring to Fig. 7-1~7-4, Fig. 8-1~8-4, figure 9-1~9-4.

Embodiment group four, the Biological Detection of antibody coupling matter

1. in vitro cell proliferation assay biological activity test

Experimental material used in experiment derives from below: DMEM culture medium, DMEM/F12K culture medium, RPMI 1640 are trained It supports base, 0.25% trypsase-EDTA, fetal calf serum, 100 × Sodium Pyruvate, 100 × mycillin and is purchased from Gibco company.Sulphur Acyl rhodamine B (Sulforhodamine B, SRB) is purchased from Sigma company.NCI-N87 gastric carcinoma cells, BT-474 human breast carcinoma Cell comes from Chinese Academy of Sciences Kunming cell bank.Other reagents are that analysis is pure.96 hole flat-bottomed polystyrene (Corning, products Catalog number (Cat.No.) 3599).2 microplate reader of Synergy (Bio-Tek).

In the present embodiment, ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII are had studied, The effect that ADC-VIII is proliferated tumor cell line.

The present embodiment evaluates the antiproliferative effect of pharmaceutical composition using Sulforhodamine B (SRB) colorimetric method.SRB is one Kind of pink anionic dye, soluble easily in water, alkaline ammonia that in acid condition can specifically with intracellular constitutive protein matter Base acid combines；Absorption peak, light absorption value and the linear positive correlation of cell concentration are generated under 510nm wavelength, therefore can be used as cell number Quantitative detection.The cell line of the present embodiment selection has: BT-474 human breast cancer cell, NCI-N87 gastric carcinoma cells.

BT-474, NCI-N87 cell are in 1640 culture medium of RPMI containing 10% fetal calf serum, and in 37 DEG C, 5%CO2 is trained It is to logarithmic growth phase, the above-mentioned cell in logarithmic growth phase is every with 2 × 103~9 × 103 cells respectively to support culture in case The density in hole is seeded to 96 well culture plates, and after culture 24 hours the drug effect of various concentration is added 5 days in every 100 μ L of hole, point Do not prepare 9 concentration with 3,4 or 5- times of dilution, each concentration sets multiple holes, and set respective concentration Vehicle controls and cell-free training Support datum hole.After drug effect, incline culture solution, the 100 μ l of solution of trichloroacetic acid (30%, w/v) of 4 DEG C of pre-coolings is added, in 4 DEG C 1 hour is fixed, then rinsed 5 times with deionized water, after drying at room temperature, the SRB dye liquor of 0.4% (w/v) is added in every hole (Sigma, 1% glacial acetic acid are prepared) 100 μ L are rinsed 4 times after being incubated for dyeing 30min at room temperature with 1% glacial acetic acid, and removal is not tied The dyestuff of conjunction, room temperature are dried.100 μ L of 10mM Tris solution is added in every hole, after being incubated for dyeing 15min at room temperature, with 1% ice second Acid, which rinses, washes away unbonded SRB for five times, after drying at room temperature, every hole be added 10mM Tris buffer (pH=10.5) dissolution with The dyestuff that cell protein combines, using measurement absorbance value at 2 microplate reader of Synergy (Bio-Tek) wavelength 510nm and 690nm (OD value), and obtain A=OD₅₁₀-OD₆₉₀。

Inhibiting rate (%)=(A control-A administration)/A control × 100%.

This experiment has used ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, ADC-VIII The research of Cell culture invitro proliferation function has been carried out to the highly expressed tumor cell line of Her2.As shown in the table, relative to naked Anti- Perjeta and Herceptin, corresponding ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII with ADC-VIII handles the highly expressed NCI-N87 gastric carcinoma cells of Her2, BT-474 human breast cancer cell, can obviously inhibit tumour Cell Proliferation.Corresponding Proliferation Ability curve is shown in Figure 10-13.

2. antitumor efficacy measurement experiment in body

The effect of combination of the invention can be measured in vivo, is implanted into the allogeneic of cancer cell that is, in rodent Graft or xenograft, and with the combined treatment tumour.By test mice drug or control treatment, and monitor several weeks Or the longer time is to measure the time for reaching tumour multiplication, log cell killing and tumor suppression.

1) experimental animal

BALB/cA-nude nude mouse 6-7 weeks, ♀, is purchased from Shanghai Ling Chang Biotechnology Co., Ltd.Production licence Number: SCXK (Shanghai) 2013-0018；Animal certificate number 2013001815683.Feeding environment: SPF grades.

2) experimental procedure

Nude mouse inoculates 6 106 human gastric cancer NCI-N87 cells, after tumour growth to 100-200mm3, according to swollen Knurl is long-pending by animal packet (D0).Mouse mainline (IV)；Administered volume 10mL/kg；Solvent group gives the " molten of same volume Agent " (0.1%BSA physiological saline)；Specific dosage and dosage regimen see the table below.2 gross tumor volumes are surveyed weekly, claim Mice Body Weight records data.

Experimental index is the influence for investigating drug to tumour growth, and specific targets are T/C% or tumour inhibiting rate TGI (%).

It is secondary weekly to use vernier caliper measurement diameter of tumor, gross tumor volume (V) calculation formula are as follows:

Wherein a, b respectively indicate length and width to V=1/2 × a × b2.

T/C (%)=(T-T0)/(C-C0) x100 wherein T, C be experiment at the end of gross tumor volume；T0, C0 are that experiment is opened The gross tumor volume when beginning.

Tumour inhibiting rate (TGI) (%)=100-T/C (%).

When tumour subsides, tumour inhibiting rate (TGI) (%)=100- (T-T0)/T0x 100

If being defined as tumor partial regression (PR) when tumour is than initial volume diminution, i.e. T < T0 or C < C0；If swollen Tumor completely disappears, that is, is defined as completed tumor regression (CR).

Experiment terminates (D21), reaches experimental endpoints or gross tumor volume reaches 1500mm³, CO2 anesthesia execution animal, then Solution takes tumor and takes pictures.

3) experimental result

Drug see the table below to the curative effect of HER2 positive human gastric cancer NCI-N87 nude mouse subcutaneous transplantation tumor and Figure 14；Lotus knurl is small Mouse can preferably be resistant to the above drug, not have the symptoms such as weight loss.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can be with The present invention is made various changes or modifications, these equivalent forms also fall within the scope of the appended claims of the present application.

Claims (20)

1. a kind of substitution maleic amide class connexon segment, structure is shown in Formulas I a:

Wherein, R is X or ArS-,

X is selected from the group: halogen；Preferably bromine or iodine；

Ar is selected from the group: substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-12 unit's heteroaryl；

Ar ' is selected from the group: substituted C6-C10 arlydene；Substituted or unsubstituted 5-12 member inferior heteroaryl；

L₁For-O (the CH being connected on Ar ' group₂CH₂O)_n, wherein n any integer in 1-20.

2. replacing maleic amide class connexon segment as described in claim 1, which is characterized in that Ar ' is selected from the sub- benzene replaced Base or pyridyl group, the substitution refer to the hydrogen atom on group replaced one or more substituent groups selected from the group below: halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, itrile group, amide groups.

3. replacing maleic amide class connexon segment as described in claim 1, which is characterized in that Ar is selected from the group: phenyl, halogen For benzene, C1-C4 alkyl phenyl, C1-C4 alkoxyl phenyl, 2- pyridyl group, 2- pyrimidine radicals, 1- methylimidazole -2- base, Wherein W is the amido R connecting with carbonyl¹, R¹Selected from-NH₂、

4. replacing maleic amide class connexon segment as described in claim 1, which is characterized in that the connection sub-piece has Structure selected from the group below:

5. a kind of substitution maleic amide class connexon-drug conjugate and its pharmaceutically acceptable salt or solvent object, structure is such as Shown in general formula Ib:

Wherein, R, Ar ', L₁Definition as claimed in claim 1；

L₂For chemical bond or AA-PAB structure；Wherein, AA is dipeptides or tripeptides segment, and PAB is p- aminobenzyl carbamyl；

CTD is to be bonded to L by amido bond₂Cytotoxicity micromolecular drug and/or treatment autoimmune disease and/or anti- The drug of inflammation.

6. replacing maleic amide class connexon-drug conjugate as claimed in claim 5, which is characterized in that the AA choosing From the following group: Val-Cit, Val-Ala, Phe-Lys.

7. replacing maleic amide class connexon-drug conjugate as claimed in claim 5, which is characterized in that the CTD choosing From the following group: Antitubulin, topoisomerase enzyme inhibitor, DNA bonding agent.

8. replacing maleic amide class connexon-drug conjugate as claimed in claim 7, which is characterized in that the micro-pipe Protein inhibitor is selected from the group: maytansine derivative, Monomethyl auristatin E, Monomethylauristatin F, Monomethyl Dolastatin 10, Tubulysin analog derivative, Cryptophycin analog derivative, Taltobulin.

9. replacing maleic amide class connexon-drug conjugate as claimed in claim 7, which is characterized in that the DNA knot Mixture is selected from the group: PBD analog derivative, duocarmycin analog derivative.

10. replacing maleic amide class connexon-drug conjugate as claimed in claim 7, which is characterized in that the topology Isomerase inhibitors are selected from the group: adriamycin metabolite PNU-159682 derivative, exatecan.

11. replacing maleic amide class connexon-drug conjugate as claimed in claim 5, which is characterized in that the CTD With selected from flowering structure:

12. as claimed in claim 5 replace maleic amide class connexon-drug conjugate, its pharmaceutically acceptable salt or Its solvent object, which is characterized in that the general formula Ib is selected from the group:

13. a kind of antibody-drug conjugates, which is characterized in that the conjugate is with such as claim 5 to claim 12 It is described in any item that maleic amide class connexon-drug conjugate and antibody is replaced to carry out coupling formation.

14. antibody-drug conjugates as claimed in claim 13, which is characterized in that the conjugate have general formula Ic and/ Or the structure of Id；

Wherein, Ar ', L₁、L₂, CTD it is as defined in claim 5；

M=1.0~5.0；

Ab is selected from the group: protein, enzyme, antibody, antibody fragment, polypeptide.

15. antibody-drug conjugates as claimed in claim 13, which is characterized in that the antibody is selected from the group: monoclonal Antibody, bispecific antibody, chimeric antibody, humanized antibody, antibody fragment.

16. antibody-drug conjugates as claimed in claim 13, which is characterized in that the antibody be can be selected from the group Tumor associated antigen combine antibody: HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e、CD70、CD74、CD79b、CD138、CD147、CD223、EpCAM、Mucin 1、STEAP1、GPNMB、FGF2、 FOLR1、EGFR、EGFRvIII、Tissuefactor、c-MET、FGFR、Nectin 4、AGS-16、Guanylyl cyclase C、Mesothelin、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、 Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta- like protein3、Claudin 18.2。

17. antibody-drug conjugates as claimed in claim 16, which is characterized in that the HER2 antibody is selected from the group: bent Trastuzumab, handkerchief trastuzumab.

18. a kind of pharmaceutical composition characterized by comprising the antibody-medicine of (a) as described in any one of claim 13-17 Object conjugate；(b) pharmaceutically acceptable diluent, carrier or excipient.

19. the antibody-drug conjugates as described in any one of claim 13-17 are the drug for being used to prepare treatment tumour Purposes.

20. the preparation method of the antibody-drug conjugates as described in any one of claim 13-17, which is characterized in that including Step:

(1) it is reacted in buffer with antibody with go back original reagent, obtains antibody after carrying out reduction；

(2) it is mixed in buffer with organic solvent with connexon-drug conjugate with the antibody after carrying out reduction that step (1) obtains It is crosslinked in liquid, obtains antibody-drug conjugates.