CN115429890B - Conjugate and antibody drug conjugate prepared by using same - Google Patents

Conjugate and antibody drug conjugate prepared by using same Download PDF

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CN115429890B
CN115429890B CN202210934549.2A CN202210934549A CN115429890B CN 115429890 B CN115429890 B CN 115429890B CN 202210934549 A CN202210934549 A CN 202210934549A CN 115429890 B CN115429890 B CN 115429890B
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antibody
compound
cycloalkyl
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CN115429890A (en
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郝婧
闫胜勇
王庆彬
冯丛然
杨宇
郭军
赵宣
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Liaoning Kai Kai Technology Co ltd
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Abstract

The invention discloses a novel conjugate of beta-glucuronidase cleavable linker containing PEG and cytotoxic drug by taking DBCO as a connector, which can realize the fixed-point coupling with the modified azide group on an antibody through simple SPAAC reaction to obtain the antibody coupling drug with high uniformity; meanwhile, PEG groups are introduced into the linker, so that the aim of improving the hydrophilicity of the linker can be fulfilled, the water solubility of the linker-drug conjugate is increased, and the problem of poor solubility of the linker-drug conjugate is solved.

Description

Conjugate and antibody drug conjugate prepared by using same
Technical Field
The invention relates to the field of medicines, in particular to the field of drug conjugates, and particularly relates to a linker-drug conjugate with a DBCO connector and application thereof.
Background
The antibody drug conjugate (Antibody drug conjugate, ADC) is a novel targeted therapeutic drug, is formed by coupling a monoclonal antibody targeting specific antigen and a small-molecule cytotoxic drug through a linker, combines the advantages of the antibody and the cytotoxic drug, and has the characteristics of strong targeting, high cytotoxicity, long degradation half-life, low toxic and side effects and the like compared with the traditional small-molecule antitumor drug. Along withAnd/>12 Drugs, etc. are approved by the FDA and marketed, and ADC drugs have been rapidly developed in recent years, and about 110 ADC drugs are currently in clinical trials.
ADC is generally composed of three parts, an antibody (anti), a small molecule cytotoxin (cytotoxin) and a linker (linker). The design of the linker has important significance for ADC drugs, which must be stable in the blood circulation system and allow for rapid and efficient release of active toxins after reaching the target tissue. There are a number of important considerations in developing ADC linkers, including the conjugation site of the antibody, the average number of conjugated cytotoxins per molecule of antibody (drug to antibody ratio, DAR), the cleavable nature and hydrophilicity of the linker, and the like. The linker can be classified into two types, namely, a cleavable linker and a non-cleavable linker, and the cleavable linker is classified into a pH-sensitive type and an enzyme-sensitive type according to the cleavage mechanism. Currently, enzyme-sensitive linkers have become the dominant choice for ADCs. The more mature enzyme-sensitive linker was studied as a dipeptide linker dependent on cleavage by cathepsin B, such as Valine-citrulline (Valine-CITRILINE). Most ADCs today share common structural features, such as being connected by maleimide linkers. The disclosed linker of ADC drugs has the problems of single design, poor water solubility and the like.
Regarding the coupling mode of toxins and antibodies, currently marketed ADC drugs are mostly randomly coupled by lysine or cysteine, and the generated antibody drug conjugates are non-uniform due to the uncertainty of the coupling number and the site, so that the non-uniformity of the pharmacokinetic properties, the titers and the toxicity among the components of the products is caused. In order to solve the problem of ADC uniformity, more preference has been given to site-directed coupling techniques in recent years, such as GlycoConnect techniques based on natural glycosylation sites, in which the Fc region N297 glycosylation site of an antibody is enzymatically cleaved from the sugar chain and attached to an azide group, which is then reacted with cyclooctyne on the linker via sparc (strain-promoted copper-FREE CLICK) to form a uniform DAR conjugate with a value of 2.
Patent document CN108743968a discloses a cysteine engineered antibody-toxin conjugate, the antibody is a cysteine site-directed insertion antibody, the cysteine insertion site comprises two classes of kappa/lambda light chain constant region light chain and IgG antibody heavy chain constant region heavy chain.
However, the uncertainty of the drug is introduced into the antibody-toxin conjugate through genetic engineering, the antibody is required to be reduced by using a reducing agent, shielding on the modified cysteine residue on the antibody is removed, DTT and the shielding are removed through cation exchange chromatography or ultrafiltration liquid exchange and other modes, and then the antibody is oxidized by using an oxidizing agent, so that inter-chain disulfide bonds of the antibody are reconnected, and the complex and complex process limits the use of the invention.
Patent document CN103083680B discloses a pharmaceutical conjugate having the general structural formula of polyethylene glycol-amino acid oligopeptide-enotecan. In the conjugate, each polyethylene glycol end group can be connected with a plurality of enotheran through amino acid oligopeptide, so that the drug loading rate is greatly improved. The modification of the hydrophilic polymer can protect enotiac, improve drug absorption, prolong action time, enhance curative effect, reduce administration dosage and avoid toxic and side effects.
However, the uniformity of the antibody drug conjugate is not high, so that the pharmacokinetic property, potency and toxicity among the components of the product are not uniform, the repeatability of the product batch is poor, and the therapeutic index is low.
Aiming at the defects of poor uniformity, single linker design, poor water solubility and the like of the antibody coupling drugs in the prior art, the development of a novel linker technology is urgently needed, the aim of site-directed coupling is achieved while the hydrophilicity of the linker is increased, and the uniformity of the antibody drug conjugate is improved.
Disclosure of Invention
In a first aspect, the present invention provides a conjugate having the structure of formula (I):
Q-X-L1-L2-D (I)
wherein, Q is selected from: one of, preferably, Q is
R 5 and R 6 each independently have the structure-X 1—Q1, Q 1 is selected from the group consisting of-H, -F, -Cl, -Br, -I linear/branched SO 2、-NO2、C1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aryl, X 1 is selected from the group consisting of single bond, -O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl,/>Or combinations thereof, preferably, R 5 and R 6 are H.
X is a linking group, X isWherein m 1、m2 is each independently selected from integers from 0 to 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), xa, X b are each independently selected from: -O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl,/>
Wherein R 9 is selected from: -H, C 1-10 straight chain/branched alkyl groups.
Preferably, m 1、m2 is each independently selected from integers from 0 to 6, more preferably, m 1、m2 is each independently selected from integers from 0 to 3.
In one embodiment of the invention, m 1 is 0.
In one embodiment of the invention, m 2 is 2.
Preferably, xa is selected from: More preferably Xa is/>
Preferably, X b is selected from: More preferably, X b is/>
Preferably, R 9 is-H.
L 1 is selected from: one of linear, Y-type and multi-branched polyethylene glycol residues;
When L 1 is a Y-type and multi-branched polyethylene glycol residue, it may have one or more branches and Are connected;
L 1 includes, but is not limited to, linear double ended PEG, Y-type PEG, 4-arm branched PEG, 6-arm branched PEG, or 8-arm branched PEG, preferably L 1 is a linear polyethylene glycol residue having a structure represented by general formula (III):
Wherein n 1 is independently selected from integers from 1 to 30, preferably from 1 to 25, more preferably from 4 to 24 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and particularly preferably 8.
L 2 is a linking group, which isWherein a is glucuronide or a glucuronide derivative, said a being selected from the group consisting of:Wherein R 12 is selected from: one of C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, and C 6-12 heteroaryl, wherein said C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, C 6-12 heteroaryl may be substituted with at least one C 1-18 straight-chain/branched alkyl, halogen, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-10 alkoxy, aryl.
Preferably, A is
In one embodiment of the present invention, A is
And B is selected from the following components:
Wherein each Y 1、Y2 is independently selected from: -H, halogen, -OC 1-10 alkyl, C 1-10 straight/branched alkyl, C 3-10 cycloalkyl, -OH, Preferably, Y 1、Y2 is-H.
Y 3、Y4 are each independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl, Preferably, Y 3、Y4 are each independently selected from: -H, C 1-10 straight/branched alkyl; more preferably, Y 3、Y4 is-CH 3.
In one embodiment of the invention, each of said Y 3、Y4 is independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl,
X 2 is selected from the group consisting of-O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, />Or a combination thereof, wherein R 9 is selected from the group consisting of: -H, C 1-10 linear/branched alkyl, preferably R 9 is-H;
preferably, X 2 is selected from the group consisting of C 1-12 straight/branched alkyl groups A group of groups.
In one embodiment of the present invention, X 2 is
In one embodiment of the present invention, the X 2 is:
R 10 is selected from: -H, C 1-12 straight-chain/branched alkyl, C 1-12 alkoxy, -NO 2、-CN、-F、-Cl、-Br、-I、-SO2、C3-12 cycloalkyl, C 6-12 aryl, and, Preferably, R 10 is selected from: -H, C 1-12 straight chain/branched alkyl, -NO 2, more preferably R 10 is selected from: -H, -CH 3、-NO2, still more preferably R 10 is-H.
R 11 is selected from: -H, C 1-10 straight-chain/branched alkyl groups,
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
in one embodiment of the present invention, B is
Further, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is
Further, D is a drug molecule.
Preferably, the conjugate has a structure of formula (ii):
Wherein the DBCO (dibenzocyclooctyne) linker in formula (ii) is used for site-directed coupling to an azide group of a modified antibody; l 1 is selected from: one of the linear, Y-shaped and multi-branched polyethylene glycol residues, L 1 includes, but is not limited to, linear double ended PEG, Y-shaped PEG, 4 arm branched PEG, 6 arm branched PEG or 8 arm branched PEG, preferably L 1 is a linear polyethylene glycol residue having a structure represented by general formula (iii):
Wherein n 1 is independently selected from integers from 1 to 30, preferably from 1 to 25, more preferably from 4 to 24 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and particularly preferably 8.
Further, the drug molecule is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, carbohydrates, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoid phenols, steroids, and one of glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from the group consisting of: amanita, auristatin, calicheamicin, camptothecins derivatives and metabolites (e.g.: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptosporin, daunorubicin, dolastatin, doxorubicin, docarubicin, epothilone, espamycin, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, microtubulolysin, vindesine, toxoid, irinotecan, topotecan, belotecan, isatecan, lu Tuoti, difluotecan, ginitecan, cinirudine, doxorubicin, epirubicin, morpholino doxorubicin, cyano morpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, MMAD, PBD and its derivatives, TPL, taxane, loop-forming, bleomycin B and its derivatives, mesitzeocin, spinosyn, 5-acetyl-1, 5-dioxetine, 5-acetyl-1-spinosyn, 5-acetyl-1, 5-acetyl-5-spinosyn.
In one embodiment of the invention, the drug molecule is selected from the group consisting of: amanitamycin, auristatin, calicheamicin, camptothecine derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, docarubicin, epothilone, espartomycin, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing.
In a specific embodiment of the invention, the drug molecule is selected from the group consisting of: SN38 and its derivatives, dxd, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:
One of them.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:
In one embodiment of the invention, the drug molecule is
The DBCO linker-drug conjugate is selected from the group consisting of:
/>
/>
in one embodiment of the invention, the DBCO linker-drug conjugate is
In a second aspect, the present invention provides the use of a conjugate of formula (II) in a medicament for the prophylaxis and/or treatment of disease.
In particular, the disease is cancer, a pathogenic organism infection or an autoimmune disease.
In particular, the cancer is a hematopoietic tumor, carcinoma, sarcoma, melanoma, or glioma.
In particular, the pathogenic organism is selected from the group consisting of: human Immunodeficiency Virus (HIV), mycobacterium tuberculosis, streptococcus agalactiae, methicillin-resistant staphylococcus aureus, pneumophilia legionella, streptococcus pyogenes, escherichia coli, neisseria gonorrhoeae, neisseria meningitidis, pneumococcus, haemophilus influenzae type B, treponema pallidum, lyme disease spirochete, west nile virus, pseudomonas aeruginosa, mycobacterium leprae, abortus, rabies virus, influenza virus, cytomegalovirus, type i herpes simplex virus, type ii herpes simplex virus, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, measles virus, adenovirus, human T cell leukemia virus, epstein-barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitis virus, wart virus, sendai virus, feline leukemia virus, reovirus, polio virus, simian tumor virus 40, murine mammary tumor virus, dengue virus, plasmodium falciparum, plasmodium, and plasmodium falciparum.
In particular, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sienchohmmer's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyadenopathy syndrome, bullous pemphigoid, diabetes, henry-sjogren's purpura, post-streptococcal nephritis, erythema nodosum, takayasu's arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, forced spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary strain, hashimoto thyroiditis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord inflammation/polymyalgia, pernicious anemia, glomerulonephritis, fibrosis nephritis and juvenile diabetes.
In a third aspect, the invention provides an antibody drug conjugate of formula (IV).
Wherein Ab is an antibody, which includes monoclonal antibodies, polyclonal antibodies, preferably monoclonal antibodies, more preferably internalizing monoclonal antibodies.
In the present invention, the antibody may be in the form of, for example: chimeric antibodies, humanized antibodies, human antibodies, antibody fragments (Fab, fab', F (ab) 2) that bind to an antigen, subfragments (single chain constructs), or antibody Fc fusion proteins, and the like, preferably, the monoclonal antibodies are reactive with an antigen or epitope thereof associated with a cancer, malignant cell, infectious organism, or autoimmune disease.
In one embodiment of the present invention, preferably, the monoclonal antibody is selected from the group consisting of: anti-HER 2 antibody, anti-EGFR antibody, anti-PMSA antibody, anti-VEGFR antibody, anti-CD 20 antibody, anti-CD 30 antibody, anti-froma antibody, anti-CD 22 antibody, anti-CD 56 antibody, anti-CD 29 antibody, anti-GPNMB antibody, anti-CD 138 antibody, anti-CD 74 antibody, anti-ENPP 3 antibody, anti-Nectin-4 antibody, anti-EGFR viii antibody, anti-SLC 44A4 antibody, anti-mesothelin antibody (anti-mesothelin antibody), anti-ET 8R antibody, anti-CD 37 antibody, anti-CEACAM 5 antibody, anti-CD 70 antibody, anti-MUC 16 antibody, anti-CD 79b antibody, anti-MUC 16 antibody, and anti-MUC 1 antibody.
In a specific embodiment of the present invention, preferably, the antigen is selected from the group consisting of: HER-2/neu, EGFR, FR α, nectin-4, carbonic anhydrase Ⅸ、B7、CCCL19、CCCL21、CSAp、BrE3、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD67、CD70、CD74、CD79a、CD80、CD83、CD95、CD126、CD133、CD138、CD147、CD154、CEACAM5、CEACAM-6、 Alpha Fetoprotein (AFP), VEGF, ED-B fibronectin, EGP-1, EGP-2, EGF receptor (ErbB 1), erbB2, erbB3, factor H, FHL-1, flt-3, folate receptor, ga733, GROB, HMGB-1, hypoxia-inducible factor (HIF), HM1.24, insulin-like growth factor (ILGF)、IFN-γ、IFN-α、IFN-β、IL-2R、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-25、IP-10、IGF-1R、Ia、HM1.24、 ganglioside, HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage Migration Inhibitory Factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, placental growth factor (PIGF), PSA, PSMA, PSMA dimer, PAM4 antigen, NCA-95, NCA-90, A3, a33, ep-CAM, KS-1, le (y), mesothelin, S100, tenascin, TAC, tn antigen, thomas-friedeh antigen, tumor necrosis factor C3, tumor cell R2, tumor cell necrosis factor C101, C3, C2, C5, C3, and the like.
L is selected from integers of 1-50 (e.g 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50).
L 3 isWherein Z 1 is an integer from 1 to 10 (e.g., 1,2, 3, 4, 5, 6,7, 8,9, 10), preferably Z 1 is an integer from 1 to 5.
In one embodiment of the invention, Z 1 is 1.
In one embodiment of the invention, Z 1 is 2.
L 4 is selected fromN 2 is selected from integers from 1 to 30 (such as 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30), preferably from 1 to 25, more preferably from 4 to 24.
Q' is selected from One of the following; /(I)
Preferably, Q' is selected fromOne of the following;
more preferably, Q' is selected from
R 5 and R 6 each independently have the structure-X 1—Q1, Q 1 is selected from the group consisting of-H, -F, -Cl, -Br, -I linear/branched SO 2、-NO2、C1-12 alkyl, C 3-12 cycloalkyl, C 6-12 aryl, X 1 is selected from the group consisting of single bond, -O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl,/>Or combinations thereof, preferably, R 5 and R 6 are H.
X is a linking group, X isWherein m 1、m2 is each independently selected from integers from 0 to 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), xa, X b are each independently selected from: -O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl,/> Wherein R 9 is selected from: -H, C 1-10 straight chain/branched alkyl groups.
Preferably, m 1、m2 is each independently selected from integers from 0 to 6, more preferably, m 1、m2 is each independently selected from integers from 0 to 3.
In one embodiment of the invention, m 1 is 0.
In one embodiment of the invention, m 2 is 2.
Preferably, xa is selected from: More preferably Xa is/>
Preferably, X b is selected from:/> More preferably, X b is/>
Preferably, R 9 is-H.
L 1 is selected from: one of linear, Y-type and multi-branched polyethylene glycol residues; when L 1 is a Y-type and multi-branched polyethylene glycol residue, it may have one or more branches andAre connected;
L 1 includes, but is not limited to, linear double ended PEG, Y-type PEG, 4-arm branched PEG, 6-arm branched PEG, or 8-arm branched PEG, preferably L 1 is a linear polyethylene glycol residue having a structure represented by general formula (III):
Wherein n 1 is independently selected from integers from 1 to 30, preferably from 1 to 25, more preferably from 4 to 24 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and particularly preferably 8.
L 2 is a linking group, which isWherein a is glucuronide or a glucuronide derivative, said a being selected from the group consisting of:Wherein R 12 is selected from: one of C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, and C 6-12 heteroaryl, wherein said C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, C 6-12 heteroaryl may be substituted with at least one C 1-18 straight-chain/branched alkyl, halogen, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-10 alkoxy, aryl.
Preferably, A is
In one embodiment of the present invention, A is/>
And B is selected from the following components:
Wherein each Y 1、Y2 is independently selected from: -H, halogen, -OC 1-10 alkyl, C 1-10 straight/branched alkyl, C 3-10 cycloalkyl, -OH, Preferably, Y 1、Y2 is-H.
Y 3、Y4 are each independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl, Preferably, Y 3、Y4 are each independently selected from: -H, C 1-10 straight/branched alkyl; more preferably, Y 3、Y4 is-CH 3.
In one embodiment of the invention, each of said Y 3、Y4 is independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl,
X 2 is selected from the group consisting of-O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, />Or a combination thereof, wherein R 9 is selected from the group consisting of: -H, C 1-10 linear/branched alkyl, preferably R 9 is-H; /(I)
Preferably, X 2 is selected from the group consisting of C 1-12 straight/branched alkyl groupsA group of groups.
In one embodiment of the present invention, X 2 is
In one embodiment of the present invention, the X 2 is:
R 10 is selected from: -H, C 1-12 straight-chain/branched alkyl, C 1-12 alkoxy, -NO 2、-CN、-F、-Cl、-Br、-I、-SO2、C3-12 cycloalkyl, C 6-12 aryl, and, Preferably, R 10 is selected from: -H, C 1-12 straight chain/branched alkyl, -NO 2, more preferably R 10 is selected from: -H, -CH 3、-NO2, still more preferably R 10 is-H.
R 11 is selected from: -H, C 1-10 straight-chain/branched alkyl groups,
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from: />
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
in one embodiment of the present invention, B is
Further, L 2 is selected from: />
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is />
Further, D is a drug molecule.
Further, the drug molecule is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, carbohydrates, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoid phenols, steroids, and one of glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from the group consisting of: amanita, auristatin, calicheamicin, camptothecins derivatives and metabolites (e.g.: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptosporin, daunorubicin, dolastatin, doxorubicin, docarubicin, epothilone, espamycin, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, microtubulolysin, vindesine, toxoid, irinotecan, topotecan, belotecan, isatecan, lu Tuoti, difluotecan, ginitecan, cinirudine, doxorubicin, epirubicin, morpholino doxorubicin, cyano morpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, MMAD, PBD and its derivatives, TPL, taxane, loop-forming, bleomycin B and its derivatives, mesitzeocin, spinosyn, 5-acetyl-1, 5-dioxetine, 5-acetyl-1-spinosyn, 5-acetyl-1, 5-acetyl-5-spinosyn.
In one embodiment of the invention, the drug molecule is selected from the group consisting of: amanitamycin, auristatin, calicheamicin, camptothecine derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, docarubicin, epothilone, espartomycin, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing.
In a specific embodiment of the invention, the drug molecule is selected from the group consisting of: SN38 and its derivatives, dxd, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:/> One of them.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:/>
In one embodiment of the invention, the drug molecule is
Preferably, the antibody drug conjugate has a structure represented by formula (V):
Wherein i is selected from integers of 1 to 20 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
In one embodiment of the invention, i is 12.
Preferably, the antibody drug conjugate has a structure represented by formula (VI):
The invention also provides pharmaceutical compositions comprising the antibody drug conjugates of the invention and their use in combination with a pharmaceutically acceptable carrier or excipient.
In particular, the pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants and the like, for example: citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.
Specifically, the pharmaceutical composition is in the form of tablet, capsule, pill, granule, powder, suppository, injection, solution, suspension, ointment, patch, lotion, drop, liniment, spray and the like.
In particular, the conjugates of the invention may be administered in the form of a pure compound or in the form of a suitable pharmaceutical composition, using any acceptable mode of administration or agent for similar use. Thus, the mode of administration employed may be selected from oral, intranasal, parenteral, topical, transdermal or rectal modes of administration in the form of solid, semi-solid or liquid pharmaceutical forms, for example, tablets, suppositories, pills, soft and hard gelatin capsules, powders, solutions, suspensions, injections and the like, preferably in unit dosage forms suitable for simple administration of precise doses.
In particular, pharmaceutical compositions which may be administered in liquid form may be prepared, for example, by dissolving, dispersing or the like the conjugate of the invention (about 0.5 to about 20%) and optionally pharmaceutically acceptable adjuvants in a carrier, examples of which are water, saline, aqueous dextrose, glycerol, ethanol or the like, to form a solution or suspension.
In a fourth aspect, the present invention provides a method of preparing an antibody drug conjugate of formula (V).
First, the antibody is reacted with a PEG derivative containing an N3 functional group and an NHS functional group, so that the antibody is labeled with the N3 functional group. Then the N3 functional group on the antibody and the DBCO functional group of the linker undergo a click reaction to obtain the antibody coupling drug.
The synthetic route of the preparation method is schematically shown as follows:
Further, ab is an antibody;
Wherein i is selected from integers of 1 to 20 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).
In one embodiment of the invention, i is 12.
L 1 is selected from: one of the linear, Y-type and multi-branched polyethylene glycol residues, preferably, the L 1 is a linear polyethylene glycol residue having the structure shown below:
Wherein n 1 is independently selected from integers from 1 to 30, preferably from 1 to 25, more preferably from 4 to 24;
L 2 is a linking group, which is Wherein a is glucuronide or a glucuronide derivative, said a being selected from the group consisting of:Wherein R 12 is selected from: one of C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, and C 6-12 heteroaryl, wherein said C 1-12 straight-chain/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, C 3-12 heterocycloalkyl, C 6-12 heteroaryl may be substituted with at least one C 1-18 straight-chain/branched alkyl, halogen, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-10 alkoxy, aryl.
Preferably, A is
In one embodiment of the present invention, A is
And B is selected from the following components: />
Wherein each Y 1、Y2 is independently selected from: -H, halogen, -OC 1-10 alkyl, C 1-10 straight/branched alkyl, C 3-10 cycloalkyl, -OH, Preferably, Y 1、Y2 is-H.
Y 3、Y4 are each independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl, Preferably, Y 3、Y4 are each independently selected from: -H, C 1-10 straight/branched alkyl; more preferably, Y 3、Y4 is-CH 3.
In one embodiment of the invention, each of said Y 3、Y4 is independently selected from: -H, C 1-10 straight-chain/branched alkyl, C 3-10 cycloalkyl,
X 2 is selected from the group consisting of-O-, -S-, C 1-12 straight/branched alkyl, C 3-12 cycloalkyl, C 6-12 aryl, />Or a combination thereof, wherein R 9 is selected from the group consisting of: -H, C 1-10 linear/branched alkyl, preferably R 9 is-H;
preferably, X 2 is selected from the group consisting of C 1-12 straight/branched alkyl groups A group of groups.
In one embodiment of the present invention, X 2 is
In one embodiment of the present invention, the X 2 is:
R 10 is selected from: -H, C 1-12 straight-chain/branched alkyl, C 1-12 alkoxy, -NO 2、-CN、-F、-Cl、-Br、-I、-SO2、C3-12 cycloalkyl, C 6-12 aryl, and, Preferably, R 10 is selected from: -H, C 1-12 straight chain/branched alkyl, -NO 2, more preferably R 10 is selected from: -H, -CH 3、-NO2, still more preferably R 10 is-H. /(I)
R 11 is selected from: -H, C 1-10 straight-chain/branched alkyl groups,
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from:
In one embodiment of the present invention, said B is selected from: />
in one embodiment of the present invention, B is
Further, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is selected from: />
In one embodiment of the present invention, L 2 is selected from:
In one embodiment of the present invention, L 2 is
Further, D is a drug molecule.
Further, the drug molecule is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, carbohydrates, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoid phenols, steroids, and one of glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from the group consisting of: amanita, auristatin, calicheamicin, camptothecins derivatives and metabolites (e.g.: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptosporin, daunorubicin, dolastatin, doxorubicin, docarubicin, epothilone, espamycin, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, microtubulolysin, vindesine, toxoid, irinotecan, topotecan, belotecan, isatecan, lu Tuoti, difluotecan, ginitecan, cinirudine, doxorubicin, epirubicin, morpholino doxorubicin, cyano morpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, MMAD, PBD and its derivatives, TPL, taxane, loop-forming, bleomycin B and its derivatives, mesitzeocin, spinosyn, 5-acetyl-1, 5-dioxetine, 5-acetyl-1-spinosyn, 5-acetyl-1, 5-acetyl-5-spinosyn.
In one embodiment of the invention, the drug molecule is selected from the group consisting of: amanitamycin, auristatin, calicheamicin, camptothecine derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, docarubicin, epothilone, espartomycin, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobiacin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing.
In a specific embodiment of the invention, the drug molecule is selected from the group consisting of: SN38 and its derivatives, dxd, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
In one embodiment of the invention, the drug molecule is selected from the group consisting of: /> One of them.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:
In one embodiment of the invention, the drug molecule is
In a fifth aspect, the present invention provides an antibody drug conjugate of general formula (iv) or a salt thereof and the use of a pharmaceutical composition in a medicament for the prophylaxis and/or treatment of a disease.
Further, the disease is cancer, pathogenic organism infection or autoimmune disease.
Further, the cancer is a hematopoietic tumor, carcinoma, sarcoma, melanoma, or glioma.
Further, the pathogenic organism is selected from the group consisting of: human Immunodeficiency Virus (HIV), mycobacterium tuberculosis, streptococcus agalactiae, methicillin-resistant staphylococcus aureus, pneumophilia legionella, streptococcus pyogenes, escherichia coli, neisseria gonorrhoeae, neisseria meningitidis, pneumococcus, haemophilus influenzae type B, treponema pallidum, lyme disease spirochete, west nile virus, pseudomonas aeruginosa, mycobacterium leprae, abortus, rabies virus, influenza virus, cytomegalovirus, type i herpes simplex virus, type ii herpes simplex virus, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, measles virus, adenovirus, human T cell leukemia virus, epstein-barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitis virus, wart virus, sendai virus, feline leukemia virus, reovirus, polio virus, simian tumor virus 40, murine mammary tumor virus, dengue virus, plasmodium falciparum, plasmodium, and plasmodium falciparum.
Further, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sienchohmmer's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyadenopathy syndrome, bullous pemphigoid, diabetes, henry-sjogren's purpura, post-streptococcal nephritis, erythema nodosum, takayasu's arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, forced spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary strain, hashimoto thyroiditis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord inflammation/polymyalgia, pernicious anemia, glomerulonephritis, fibrosis nephritis and juvenile diabetes.
In a sixth aspect, the invention provides a method of treatment of a disease comprising administering to a subject an antibody drug conjugate of the invention.
Further, the antibody drug conjugate is administered in combination with one or more therapeutic methods selected from the group consisting of: unconjugated antibodies, radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugated antibodies, gene therapy, chemotherapy, therapeutic peptides, oligonucleotides, localized radiation therapy, surgery, and interfering RNA therapy.
Further, the disease is cancer, pathogenic organism infection or autoimmune disease.
Further, the cancer is a hematopoietic tumor, carcinoma, sarcoma, melanoma, or glioma.
Further, the pathogenic organism is selected from the group consisting of: human Immunodeficiency Virus (HIV), mycobacterium tuberculosis, streptococcus agalactiae, methicillin-resistant staphylococcus aureus, pneumophilia legionella, streptococcus pyogenes, escherichia coli, neisseria gonorrhoeae, neisseria meningitidis, pneumococcus, haemophilus influenzae type B, treponema pallidum, lyme disease spirochete, west nile virus, pseudomonas aeruginosa, mycobacterium leprae, abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus type i, herpes simplex virus type ii, human serum parvovirus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, measles virus, adenovirus, human T cell leukemia virus, epstein barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitis virus, wart virus, myxoviridae virus, feline leukemia virus, reovirus, polio virus, simian tumor virus 40, murine mammary tumor virus, dengue virus, varicella, plasmodium falciparum, plasmodium falciparum, etc
Further, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sienchohmmer's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyadenopathy syndrome, bullous pemphigoid, diabetes, henry-sjogren's purpura, post-streptococcal nephritis, erythema nodosum, takayasu's arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, forced spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary strain, hashimoto thyroiditis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord inflammation/polymyalgia, pernicious anemia, glomerulonephritis, fibrosis nephritis and juvenile diabetes.
The conjugate of the beta-glucuronidase cleavable linker containing PEG and the cytotoxic drug, which takes DBCO as a joint, can realize the fixed-point coupling with the modified azide group on the antibody through a simple SPAAC reaction, thus obtaining the antibody coupling drug with higher uniformity; meanwhile, PEG groups are introduced into the linker, so that the aim of improving the hydrophilicity of the linker can be fulfilled, the water solubility of the linker-drug conjugate is increased, and the problem of poor solubility of the linker-drug conjugate is solved.
The term C 1-8 linear/branched alkyl as used herein includes methyl, ethyl, C 3 linear/branched alkyl, C 4 linear/branched alkyl, C 5 linear/branched alkyl, C 6 linear/branched alkyl, C 7 linear/branched alkyl, C 8 linear/branched alkyl.
The term C 1-10 linear/branched alkyl as used herein includes methyl, ethyl, C 3 linear/branched alkyl, C 4 linear/branched alkyl, C 5 linear/branched alkyl, C 6 linear/branched alkyl, C 7 linear/branched alkyl, C 8 linear/branched alkyl, C 9 linear/branched alkyl, C 10 linear/branched alkyl.
The term C 1-5 straight-chain/branched-chain alkyl as used herein includes methyl, ethyl, C 3 straight-chain/branched-chain alkyl, C 4 straight-chain/branched-chain alkyl, C 5 straight-chain/branched-chain alkyl. The term C 3-12 cycloalkyl as described in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl, C 9 cycloalkyl, C 10 cycloalkyl, C 11 cycloalkyl, C 12 cycloalkyl.
The term C 1-12 straight-chain/branched alkyl as used herein includes methyl, ethyl, C 3 straight-chain/branched alkyl, C 4 straight-chain/branched alkyl, C 5 straight-chain/branched alkyl, C 6 straight-chain/branched alkyl, C 7 straight-chain/branched alkyl, C 8 straight-chain/branched alkyl, C 9 straight-chain/branched alkyl, C 10 straight-chain/branched alkyl, C 11 straight-chain/branched alkyl, C 12 straight-chain/branched alkyl.
The term C 3-12 cycloalkyl as described in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl, C 9 cycloalkyl, C 10 cycloalkyl, C 11 cycloalkyl, C 12 cycloalkyl.
The term C 3-10 cycloalkyl as described in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl, C 9 cycloalkyl, C 10 cycloalkyl.
The term C 6-12 aromatic ring group as used in the present invention includes C 6 aromatic ring group (phenyl group), C 7 aromatic ring group, C 8 aromatic ring group, C 9 aromatic ring group, C 10 aromatic ring group, C 11 aromatic ring group, and C 12 aromatic ring group.
The term C 3-12 heterocycloalkyl as used herein includes C 3 heterocycloalkyl, C 4 heterocycloalkyl, C 5 heterocycloalkyl, C 6 heterocycloalkyl, C 7 heterocycloalkyl, C 8 heterocycloalkyl, C 9 heterocycloalkyl, C 10 heterocycloalkyl, C 11 heterocycloalkyl, C 12 heterocycloalkyl.
The term C 6-12 heteroaryl as described in the present invention includes C 6 heteroaryl, C 7 heteroaryl, C 8 heteroaryl, C 9 heteroaryl, C 10 heteroaryl, C 11 heteroaryl, C 12 heteroaryl.
The term C 3-8 cycloalkyl as described in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkyl.
The term C 3-8 cycloalkoxy as described in the present invention includes, C 3 cycloalkoxy, C 4 cycloalkoxy, C 5 cycloalkoxy, C 6 cycloalkoxy, C 7 cycloalkoxy, C 8 cycloalkoxy.
The term C 1-12 alkoxy as described in the present invention includes methoxy, ethoxy, C 3 alkoxy, C 4 alkoxy, C 5 alkoxy, C 6 alkoxy, C 7 alkoxy, C 8 alkoxy, C 9 alkoxy, C 10 alkoxy, C 11 alkoxy, C 12 alkoxy.
The term C 1-10 alkoxy as described in the present invention includes methoxy, ethoxy, C 3 alkoxy, C 4 alkoxy, C 5 alkoxy, C 6 alkoxy, C 7 alkoxy, C 8 alkoxy, C 9 alkoxy, C 10 alkoxy.
The term MMAE as used in the present invention refers to monomethyl auristatin E.
The term SN38 as used herein refers to 7-ethyl-10-hydroxycamptothecin.
The term MMAF as used herein refers to monomethyl auristatin F.
The term MMAD as described in the present invention refers to monomethyl auristatin D.
The term PBD as used in the present invention refers to a pyrrolobenzodiazepine.
The term TPL as used herein refers to triptolide.
The term Dxd as described in the present invention refers to delutinacon.
Drawings
FIG. 1 is an MS plot of Compound 1.
Detailed Description
In order to make the technical content of the present invention more clearly understood, the following detailed description of embodiments is given for clarity and completeness of description, and the purpose of the present invention is to better understand the content of the present invention and not to limit all other embodiments obtained by those skilled in the art without making any creative effort, which fall within the protection scope of the present invention.
EXAMPLE 1 Synthesis of Compound 1
1.1 Synthesis of Compound 1a
Raw material Glucose-NO 2 -OH (48.5 g,0.10 mol) was added to 480mL DCM and 100mL DMF, then DIEA (25.8 g,0.20 mol) and TBSCl (16.5 g,0.11 mol) were added sequentially to the solution, the reaction stirred at room temperature for 6H, and TLC showed completion. The reaction was washed three times with 400ml of 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 20/1-8/1) to give compound 1a (50 g, white solid) in 83% yield.
MS m/z(ESI):600.2[M+1]
1.2 Synthesis of Compound 1b
Compound 1a (45.1 g,0.08 mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added to displace hydrogen, the reaction was stirred under hydrogen atmosphere for 4H, TLC showed complete reaction, filter cake was removed, and the obtained filtrate was dried by spin-drying to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 10/1-3/1) to give compound 1b (white solid, 38 g) in 88% yield.
MS m/z(ESI):570.2[M+1]
1.3 Synthesis of Compound 1c
Compound 1b (6.32 g,11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83 g,12.7 mmol), TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed that after the starting material had reacted completely, direct C-18 medium pressure flash (H2O/CH 3OH 40% -90%) had been purified to give compound 1C (8.1 g, yellow oily product) in 60% yield.
MS m/z(ESI):1280.6[M+1]
1.4 Synthesis of Compound 1d
Compound 1c (5.5 g,4.3 mmol) was dissolved in THF (55 mL), 2N aqueous HCl (4.0 mL) was added, the reaction stirred at room temperature for 2H, TLC and HPLC showed complete reaction, 50mL of saturated aqueous NaHCO 3 was added to quench the reaction, then extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and spun-dried to give the crude product. The crude product was dissolved in 10mL DCM, the resulting solution was not clear, the solids were removed by filtration through a filter membrane, and the filtrate was spin-dried to give compound 1d (3.1 g, yellow oily product) in 62% yield.
MS m/z(ESI):1166.5[M+1]
1.5 Synthesis of Compound 1e
Compound 1d (2.9 g,2.5 mmol) was dissolved in 40mL DCM, cooled to 0-5℃in an ice-water bath, phenyl p-nitrochloroformate (0.75 g,3.7 mmol) and DMAP (0.61 g,5.0 mmol) were added sequentially to the reaction system, the reaction was slowly warmed to room temperature and stirred for 4h, TLC showed complete reaction, 30mL of 5% NaHSO 4 was added and washed once with 30mL of saturated saline, the organic phase was dried over anhydrous sodium sulfate, dried over wet, and purified on a silica gel column (DCM/THF 10/1-2/1) to give compound 1e (2.4 g, yellow oil) in 73% yield.
MS m/z(ESI):1331.5[M+1]
1.6 Synthesis of Compound 1f
In a 100mL single-necked flask, compound 1e (2.3 g,1.7 mmol), MMAE (1.5 g,2.1 mmol), HOBt (116 mg,0.9 mmol), pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature 48H, after which the HPLC showed complete reaction, the solvent was removed by rotary evaporation and flash (H 2O/CH3 OH50% -90%) was purified to give Compound 1f (2.0 g, off-white solid) in 61% yield.
MS m/z(ESI):1909.0[M+1]
1.7 Synthesis of Compound 1
Compound 1f (1.0 g,0.5 mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (144 mg,12 eq) was slowly added, and the temperature was gradually increased to room temperature. After the reaction was complete, the pH was adjusted to 7 with acetic acid. Purification by C-18 medium pressure flash (H 2O/CH3 OH 50% -90%) afforded compound 1 (500 mg, off-white solid) in 55% yield.
MS m/z(ESI):885.9[M/2+1]
EXAMPLE 2 Synthesis of Compound 2
2.1 Synthesis of Compound 2a
The starting material Glucose-NO 2 -OH (48.5 g,0.10 mol) was added to 480mL DCM and 100mL DMF, then DIEA (25.8 g,0.20 mol) and TBSCl (16.5 g,0.11 mol) were added sequentially to the solution and the reaction stirred at room temperature for 6h, and TLC showed completion. The reaction was washed three times with 400ml of 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 20/1-8/1) to give compound 2a (50 g, white solid) in 83% yield.
MS m/z(ESI):600.2[M+1]
2.2 Synthesis of Compound 2b
Compound 2a (45.1 g,0.08 mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added to replace hydrogen, the reaction was stirred under hydrogen for 4h, TLC showed complete reaction, filter cake was removed, and the resulting filtrate was dried by spin-on to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 10/1-3/1) to give compound 2b (white solid, 38 g) in 88% yield.
MS m/z(ESI):570.2[M+1]
2.3 Synthesis of Compound 2c
Compound 2b (6.32 g,11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83 g,12.7 mmol), TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed that after the starting material had reacted completely, direct C-18 medium pressure flash (H 2O/CH3 OH 40% -90%) had been purified to give compound 2C (8.1 g, yellow oily product) in 60% yield.
MS m/z(ESI):1280.6[M+1]
2.4 Synthesis of Compound 2d
Compound 2c (5.5 g,4.3 mmol) was dissolved in THF (55 mL), 2N aqueous HCl (4.0 mL) was added, the reaction stirred at room temperature for 2H, TLC and HPLC showed complete reaction, 50mL of saturated aqueous NaHCO 3 was added to quench the reaction, then extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and spun-dried to give the crude product. The crude product was dissolved in 10mL DCM, the resulting solution was not clear, the solids were removed by filtration through a filter membrane, and the filtrate was spin-dried to give compound 2d (3.1 g, yellow oily product) in 62% yield.
MS m/z(ESI):1166.5[M+1]
2.5 Synthesis of Compound 2e
Compound 2d (2.9 g,2.5 mmol) was dissolved in 40mL DCM, cooled to 0-5℃in an ice-water bath, phenyl p-nitrochloroformate (0.75 g,3.7 mmol) and DMAP (0.61 g,5.0 mmol) were added sequentially to the reaction system, the reaction was slowly warmed to room temperature and stirred for 4h, TLC showed complete reaction, 30mL of 5% NaHSO 4 was added and washed once with 30mL of saturated saline, the organic phase was dried over anhydrous sodium sulfate, dried over wet, and purified on a silica gel column (DCM/THF 10/1-2/1) to give compound 2e (2.4 g, yellow oil) in 73% yield.
MS m/z(ESI):1331.5[M+1]
2.6 Synthesis of Compound 2f
In a 100mL single-necked flask, compound 2e (2.3 g,1.7 mmol), TBS-SN38 (1.1 g,2.1 mmol), 4-dimethylaminopyridine (0.4 g,3.4 mmol), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48H, after HPLC showed complete reaction, the solvent was removed by rotary evaporation and purification with C-18 medium pressure flash (H 2O/CH3 OH50% -90%) gave Compound 2f (1.4 g, off-white solid) in 50% yield.
MS m/z(ESI):1699.0[M+1]
2.7 Synthesis of Compound 2g
Compound 2f (1.0 g,0.6 mmol) was dissolved in 50mL tetrahydrofuran, tetrabutylammonium fluoride (0.52 g,2.0 mmol) was added, stirring was performed at room temperature for 30min, TLC monitored the progress of the reaction, and the solvent was spun-dried after completion of the reaction and purified by C-18flash (MeOH: H 2 O=50% -90%), yielding compound 2g (590 mg, yellow solid) in 62% yield.
MS m/z(ESI):792.8[M/2+1]
2.8 Synthesis of Compound 2
2G (500 mg,0.3 mmol) of the compound was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (72 mg,12 eq) was slowly added thereto, and the temperature was gradually increased to room temperature. After the reaction was complete, the pH was adjusted to 7 with acetic acid. Purification by C-18 medium pressure flash (H 2O/CH3 OH 50% -90%) afforded compound 2 (198 mg, off-white solid) in 58% yield.
MS m/z(ESI):568.7[M/2+1]
EXAMPLE 3 Synthesis of Compound 3
3.1 Synthesis of Compound 3a
Raw material Glucose-NO 2 -OH (48.5 g,0.10 mol) was added to 480mL DCM and 100mL DMF, then DIEA (25.8 g,0.20 mol) and TBSCl (16.5 g,0.11 mol) were added sequentially to the solution, the reaction stirred at room temperature for 6H, and TLC showed completion. The reaction was washed three times with 400ml of 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 20/1-8/1) to give compound 3a (50 g, white solid) in 83% yield.
MS m/z(ESI):600.2[M+1]
3.2 Synthesis of Compound 3b
Compound 3a (45.1 g,0.08 mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added to replace hydrogen, the reaction was stirred under hydrogen for 4h, TLC showed complete reaction, filter cake was removed, and the resulting filtrate was dried by spin-on to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 10/1-3/1) to give compound 3b (white solid, 38 g) in 88% yield.
MS m/z(ESI):570.2[M+1]
3.3 Synthesis of Compound 3c
Compound 3b (6.32 g,11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83 g,12.7 mmol), TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed that after the starting material had reacted completely, direct C-18 medium pressure flash (H 2O/CH3 OH 40% -90%) had been purified to give compound 3C (8.1 g, yellow oily product) in 60% yield.
MS m/z(ESI):1280.6[M+1]
3.4 Synthesis of Compound 3d
Compound 3c (5.5 g,4.3 mmol) was dissolved in THF (55 mL), 2N aqueous HCl (4.0 mL) was added, the reaction stirred at room temperature for 2h, TLC and HPLC showed complete reaction, 50mL of saturated aqueous NaHCO 3 was added to quench the reaction, then extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and spun-dried to give the crude product. The crude product was dissolved in 10mL DCM, the resulting solution was not clear, the solids were removed by filtration through a filter membrane, and the filtrate was spin-dried to give compound 3d (3.1 g, yellow oily product) in 62% yield.
MS m/z(ESI):1166.5[M+1]
3.5 Synthesis of Compound 3e
Compound 3d (2.9 g,2.5 mmol) was dissolved in 40mL DCM, cooled to 0-5℃in an ice-water bath, phenyl p-nitrochloroformate (0.75 g,3.7 mmol) and DMAP (0.61 g,5.0 mmol) were added sequentially to the reaction system, the reaction was slowly warmed to room temperature and stirred for 4h, TLC showed complete reaction, 30mL of 5% NaHSO 4 was added and washed once with 30mL of saturated saline, the organic phase was dried over anhydrous sodium sulfate, dried over wet, and purified on a silica gel column (DCM/THF 10/1-2/1) to give compound 3e (2.4 g, yellow oil) in 73% yield.
MS m/z(ESI):1331.5[M+1]
3.6 Synthesis of Compound 3f
SN38 (1.96 g,5.0 mmol) and 4-dimethylaminopyridine (910 mg,7.5 mmol) were dissolved in 15mL of dichloromethane, triphosgene (670 mg) was added under nitrogen protection, the reaction was stirred at room temperature for 5 minutes, boc-DMEA (1.14 g,6.3 mmol) in dichloromethane (4.0 mL) was added to the reaction solution, and the reaction was stirred at room temperature for 5 minutes, LCMS indicated complete reaction of the starting materials. The reaction solution was washed with water 2 times to give 3f (2.3 g, yellow solid) in 76% yield.
MS m/z(ESI):607.2[M+1]
3.7 Synthesis of Compound 3g
Compound 3f (2.3 g,3.7 mmol) was dissolved in a mixed solvent of dichloromethane (4 mL) and trifluoroacetic acid (1 mL) and the reaction was stirred at room temperature under nitrogen for 2 hours, LCMS showed the starting material to be essentially complete. The reaction solution was diluted with acetonitrile, concentrated at low temperature, and purified in medium pressure to give 3g (1.6 g, yellow solid) of the product in 82% yield.
MS m/z(ESI):507.2[M+1]
3.8 Synthesis of Compounds for 3h
In a 100mL single-necked flask, 3e (2.3 g,1.7 mmol), 3g (1.1 g,2.1 mmol), HOBt (116 mg,0.9 mmol) pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48H, after HPLC showed complete reaction, the solvent was removed by rotary evaporation, and flash (H 2O/CH3 OH 50% -90%) was purified to give compound 3H (1.9 g, off-white solid) in 65% yield.
MS m/z(ESI):850.0[M/2+1]
3.9 Synthesis of Compound 3
Compound 3h (1.9 g,1.0 mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (288 mg,12 eq) was slowly added, and the temperature was gradually increased to room temperature. After the reaction was complete, the pH was adjusted to 7 with acetic acid. Purification by C-18 medium pressure flash (H2O/CH 3OH 50% -90%) afforded compound 3 (810 mg, off-white solid) in 52% yield.
MS m/z(ESI):780.5[M/2+1]
EXAMPLE 4 Synthesis of Compound 4
4.1 Synthesis of Compound 4a
Raw material Glucose-NO 2 -OH (49.9 g,0.10 mol) was added to 480mL DCM and 100mL DMF, then DIEA (25.8 g,0.20 mol) and TBSCl (16.5 g,0.11 mol) were added sequentially to the solution, the reaction stirred at room temperature for 6H, and TLC showed completion. The reaction was washed three times with 400ml of 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 20/1-8/1) to give compound 4a (49 g, white solid) in 80% yield.
MS m/z(ESI):614.2[M+1]
4.2 Synthesis of Compound 4b
Compound 4a (49.0 g,0.08 mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added to replace hydrogen, the reaction was stirred under hydrogen for 4h, TLC showed complete reaction, filter cake was removed, and the obtained filtrate was dried by spin-drying to give crude product, which was purified by silica gel column chromatography (n-hexane/EA 10/1-3/1) to give compound 4b (40 g, white solid) in 87% yield.
MS m/z(ESI):584.2[M+1]
4.3 Synthesis of Compound 4c
Compound 4b (6.42 g,11.0 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83 g,12.7 mmol), TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed that after the starting material had reacted completely, direct C-18 medium pressure flash (H 2O/CH3 OH 40% -90%) had been purified to give compound 4C (8.8 g, yellow oily product) in 62% yield.
MS m/z(ESI):1294.6[M+1]
4.4 Synthesis of Compound 4d
Compound 4c (5.6 g,4.3 mmol) was dissolved in THF (55 mL), 2N aqueous HCl (4.0 mL) was added, the reaction stirred at room temperature for 2H, TLC and HPLC showed complete reaction, 50mL of saturated aqueous NaHCO 3 was added to quench the reaction, then extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and spun-dried to give the crude product. The crude product was dissolved in 10mL DCM and the resulting solution was not clear, the solids removed by filtration through a filter membrane and the filtrate was spin-dried to give compound 4d (3.3 g, yellow oily product) in 66% yield.
MS m/z(ESI):1180.5[M+1]
4.5 Synthesis of Compound 4e
Compound 4d (3.0 g,2.5 mmol) was dissolved in 40mL DCM, cooled to 0-5℃in an ice-water bath, phenyl p-nitrochloroformate (0.75 g,3.7 mmol) and DMAP (0.61 g,5.0 mmol) were added sequentially to the reaction system, the reaction was slowly warmed to room temperature and stirred for 4h, TLC showed complete reaction, 30mL of 5% NaHSO 4 was added and washed once with 30mL of saturated saline, the organic phase was dried over anhydrous sodium sulfate, dried over wet, and purified on a silica gel column (DCM/THF 10/1-2/1) to give compound 4e (2.3 g, yellow oil) in 70% yield.
MS m/z(ESI):1345.5[M+1]
4.6 Synthesis of Compound 4f
In a 100mL single-necked flask, compound 4e (2.3 g,1.7 mmol), MMAE (1.5 g,2.1 mmol), HOBt (116 mg,0.9 mmol), pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48H, after HPLC showed complete reaction, the solvent was removed by rotary evaporation, and Compound 4f (2.0 g, off-white solid) was purified by flash (H 2O/CH3 OH50% -90%) in C-18 in 61% yield.
MS m/z(ESI):962.5[M/2+1]
4.7 Synthesis of Compound 4
Compound 4f (961 mg,0.5 mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (144 mg,12 eq) was slowly added, and the temperature was gradually increased to room temperature. After the reaction was complete, the pH was adjusted to 7 with acetic acid. Purification by C-18 medium pressure flash (H 2O/CH3 OH 50% -90%) afforded compound 4 (472 mg, off-white solid) in 53% yield.
MS m/z(ESI):892.5[M/2+1]
EXAMPLE 5 Synthesis of Compound 5
5.1 Synthesis of Compound 5b
Compound 5a (6.76 g,11.0 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83 g,12.7 mmol), TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed that after the starting material had reacted completely, direct C-18 medium pressure flash (H 2O/CH3 OH 40% -90%) had been purified to give compound 5b (8.4 g, yellow oily product) in 60% yield.
MS m/z(ESI):1325.6[M+1]
5.2 Synthesis of Compound 5c
Compound 5b (5.7 g,4.3 mmol) was dissolved in THF (55 mL), 2N aqueous HCl (4.0 mL) was added, the reaction stirred at room temperature for 2H, TLC and HPLC showed complete reaction, 50mL of saturated aqueous NaHCO 3 was added to quench the reaction, then extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate and spun-dried to give the crude product. The crude product was dissolved in 10mL DCM and the resulting solution was not clear, the solids removed by filtration through a filter membrane and the filtrate was spin-dried to give compound 5c (3.2 g, yellow oily product) in 61% yield.
MS m/z(ESI):1211.5[M+1]
5.3 Synthesis of Compound 5d
Compound 5c (3.0 g,2.5 mmol) was dissolved in 40mL DCM, cooled to 0-5℃in an ice-water bath, phenyl p-nitrochloroformate (0.75 g,3.7 mmol) and DMAP (0.61 g,5.0 mmol) were added sequentially to the reaction system, the reaction was slowly warmed to room temperature and stirred for 4h, TLC showed complete reaction, 30mL of 5% NaHSO 4 was added and washed once with 30mL of saturated saline, the organic phase was dried over anhydrous sodium sulfate, dried over wet, and purified on a silica gel column (DCM/THF 10/1-2/1) to give compound 5d (2.1 g, yellow oil) in 61% yield.
MS m/z(ESI):1376.5[M+1]
5.4 Synthesis of Compound 5e
In a 100mL single-necked flask, compound 5d (2.3 g,1.7 mmol), MMAE (1.5 g,2.1 mmol), HOBt (116 mg,0.9 mmol), pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48H, after HPLC showed complete reaction, the solvent was removed by rotary evaporation, and Compound 5e (2.1 g, off-white solid) was purified by flash (H 2O/CH3 OH50% -90%) in C-18 in 65% yield.
MS m/z(ESI):978.5[M/2+1]
5.5 Synthesis of Compound 5
Compound 5e (979 mg,0.5 mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (144 mg,12 eq) was slowly added, and the temperature was gradually increased to room temperature. After the reaction was complete, the pH was adjusted to 7 with acetic acid. Purification by C-18 medium pressure flash (H 2O/CH3 OH 50% -90%) afforded compound 5 (435 mg, off-white solid) in 48% yield.
MS m/z(ESI):908.0[M/2+1]
Example 6 preparation of ADC
6.1MAb reaction procedure with PEG12
The reaction was carried out at a molar ratio of mAb to PEG12-SPA of 1:20. mu.L of mAb solution (concentration: 9.6 mg/mL) was first weighed and placed in a 1.5mL centrifuge tube, and then the mAb was weighed to be 2mg. 10mg/mL of N 3 -PEG12-SPA solution was prepared by using 5mM PBS solution with pH3.0, 20. Mu.L was measured and added into a centrifuge tube, and the mixture was thoroughly mixed and reacted for 2 hours under shaking. After the reaction is finished, unreacted N 3 -PEG12-SPA is removed by ultrafiltration.
6.2 Reaction Process of mAb-PEG12 with linker-drug
The prepared mAb-PEG12 sample was taken, the protein concentration was determined to be 2.05mg/mL, 490. Mu.L of the sample solution was measured and placed in a 1.5mL centrifuge tube. 10mglinker-drug was weighed and dissolved in DMSO to prepare a 1mg/mL solution. According to the charging ratio of mAb to linker-drug being 1:4, 41 mu Llinker-drug solution is measured and added into a centrifuge tube, 510 mu L of PBS and 59 mu L of DMSO are added into the centrifuge tube, the protein concentration is kept at 1mg/mL, the ratio of water to DMSO is 10:1, and the mixture is fully mixed and subjected to shaking reaction for 2 hours. After the reaction, unreacted small molecules are removed by ultrafiltration to obtain ADC molecules, as shown in the following table.
Linker-drug ADC molecules
Compound 2 ADC1
DBCO-VC-PAB-SN38 ADC2
Example 7 measurement of DAR values
The DAR value of the ADC is detected by a liquid phase method.
The liquid chromatography was set as follows:
Under the condition of liquid chromatography, the separation degree of SN38 from ADC1 and ADC2 is more than 1.5. Different concentration standard curves are prepared, linear standard curves between the peak area and the concentration of the SN38 are established, and the quantitative analysis of the SN38 on the ADC1 and the SN38 on the ADC2 are respectively carried out. The number of conjugates of SN38 on the ADC was calculated based on the antibody concentration and the concentration of SN38 on the ADC. Measuring ADC1 and ADC2 prepared in example 6, wherein DAR values are 4;
Example 8 affinity assay of ADC with EGFR
The affinity of ADC drugs was measured by SPR, the product biacore of the experimental instrument not shown GE. The simple operation steps are as follows: EGFR antigen was coupled to CM chip. Antibodies to ADC drugs or free antibodies to antigens were tested for affinity with different concentrations of ADC drugs or monoclonal antibodies. The results show that the decrease in affinity of the antibodies was not significantly pronounced for ADC1 and ADC2 prepared in example 6, ADC1 having a higher affinity than ADC2.
Example 9 in vitro cytotoxicity assay
In vitro cytotoxicity assays were performed with BXPC-3 (human pancreatic cancer cells) cells. The test procedure was as follows:
1. 100. Mu.L of cells were added to each well of the 96-well plate (2 blank groups were left without cells and the same volume of medium was added). Cells were incubated in a 5% CO 2 cell incubator at 37℃for 24h. Cytotoxicity experiments about 10000 cells were added per well in 100 μl.
2. 10 Μl of different concentrations of ADC or SN38 drug was added per well.
3. The 96-well plates were incubated in a cell incubator at 37℃with 5% CO 2 air and 100% humidity for 24 hours.
4. Mu.L of CCK-8 solution was added to each well. Incubate at 37℃in a 5% CO 2 incubator for 3h.
5. The absorbance at 450nm was measured by a microplate reader.
6. Analysis of results:
A. Cell viability: the OD value of each test well was subtracted by the background OD value (blank), and the OD value of each duplicate well was averaged ± SD.
Cell viability% = (dosed cell OD/control cell OD) ×100%.
B. the drug concentration at T/c=50% (IC 50) and the drug concentration at T/c=10% (IC 90) were obtained.
The results show that the IC50 value of ADC 1 prepared in example 6 is less than that of ADC 2, and is comparable to that of SN 38.
The result shows that the ADC1 obtained by the invention not only can effectively exert the biological function of an EGFR antibody part, but also has the high-efficiency killing activity of SN38 on tumor cells. Compared with the ADC2 prepared by the traditional dipeptide linker, the ADC1 prepared by the invention has better activity.

Claims (3)

1. An antibody drug conjugate having a structure represented by formula (V):
（V），
Wherein i is an integer selected from 1 to 20,
Wherein Ab is an antibody, and the antibody comprises a monoclonal antibody and a polyclonal antibody;
l is selected from integers from 1 to 50;
L 1 is a linear polyethylene glycol residue having the structure shown below:
Wherein n 1 is an integer from 4 to 24;
L 2 is a linking group, which is Wherein A is/>；
The B is；
D is；
The L 2 is。
2. A method of preparing the antibody drug conjugate of claim 1, which is synthesized by the following route:
；
Wherein i is an integer of1 to 20.
3. Use of the antibody drug conjugate of claim 1 in the preparation of a medicament for the treatment of a disease, said disease being pancreatic cancer.
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