CN115429890A - Conjugate and antibody drug conjugate prepared from same - Google Patents

Conjugate and antibody drug conjugate prepared from same Download PDF

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CN115429890A
CN115429890A CN202210934549.2A CN202210934549A CN115429890A CN 115429890 A CN115429890 A CN 115429890A CN 202210934549 A CN202210934549 A CN 202210934549A CN 115429890 A CN115429890 A CN 115429890A
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conjugate
group
branched alkyl
straight chain
cycloalkyl
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CN115429890B (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 which takes DBCO as a joint and contains a PEG (polyethylene glycol) -containing beta-glucuronidase cleavable linker and a cytotoxic drug, and the conjugate can be coupled with a modified azide group on an antibody at a fixed point through a simple SPAAC (SPAAC) reaction to obtain an 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 from 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 (DBCO) connector and application thereof.
Background
Antibody Drug Conjugates (ADC) are a novel targeted therapeutic drug, and are formed by coupling a monoclonal Antibody targeting a specific antigen and a small-molecule cytotoxic drug through a linker, combine the advantages of the Antibody and the cytotoxic drug, and have the characteristics of strong targeting property, high cytotoxicity, long degradation half-life, low toxic and side effects and the like compared with the traditional small-molecule antitumor drugs. With following
Figure BDA0003782964770000011
And
Figure BDA0003782964770000012
these 12 drugs are approved by the FDA for marketing, and in recent years, ADC drugs have developed rapidly, and currently about 110 ADC drugs are in clinical trials.
ADCs are generally composed of three parts, an antibody (antibody), a small molecule cytotoxin (cytoxin), and a linker (linker). The design of the linker is of great importance for ADC drugs, which themselves must be such that the drug remains stable in the blood circulation and can release the active toxin rapidly and efficiently 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 cytotoxins conjugated per molecule of antibody (DAR), the cleavable and hydrophilic properties of the linker, etc. The linker can be divided into two main classes of cleavable linker and non-cleavable linker, and the cleavable linker is divided into pH-sensitive type and enzyme-sensitive type according to the difference of 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 that relies on cathepsin B cleavage, such as Valine-citrulline (Valine-Citriline). Most ADCs currently share common structural features, such as attachment via maleimide linkers. The disclosed linker of ADC drugs has the problems of single design, poor water solubility and the like.
Regarding the conjugation mode of toxin and antibody, most of the currently marketed ADC drugs adopt lysine or cysteine random conjugation, which results in heterogeneity of the generated antibody drug conjugate due to uncertainty of conjugation number and site, and further results in heterogeneity of pharmacokinetic properties, potency and toxicity among components of the product. In order to solve the problem of ADC homogeneity, site-directed conjugation techniques have been favored recently, such as the natural glycosylation site-based GlycoConnect technique, in which the N297 glycosylation site of the Fc region of an antibody is catalyzed by an enzyme to cleave the sugar chain and attach an azide group, and then the azide group reacts with cyclooctyne on the linker via SPAAC (strained-bonded-free click) to form a conjugate with a DAR value of 2.
Patent document CN108743968A discloses a cysteine engineered antibody-toxin conjugate, the antibody is a cysteine site-specific insertion antibody, and the cysteine insertion site comprises two types of kappa/lambda light chain constant region light chain and IgG antibody heavy chain constant region heavy chain.
However, the antibody-toxin conjugate modified by genetic engineering introduces uncertainty of drugs, a reducing agent is needed to reduce the antibody, shielding on modified cysteine residues on the antibody is removed, DTT and the shielding are removed by means of cation exchange chromatography or ultrafiltration liquid exchange and the like, then an oxidizing agent is used to oxidize the antibody, so that inter-chain disulfide bonds of the antibody are reconnected, and the use of the invention is limited due to complicated and complicated processes.
Patent document CN103083680B discloses a drug conjugate with a general structural formula of polyethylene glycol-amino acid oligopeptide-eprinomectin. In the conjugate, each polyethylene glycol terminal group can be connected with a plurality of eprinomectin through amino acid oligopeptides, and the loading rate of the drug is greatly improved. The modification of the hydrophilic polymer can protect the epristokang, improve the drug absorption, prolong the action time, enhance the curative effect, reduce the administration dosage and avoid toxic and side effects.
However, the antibody drug conjugate has low uniformity, which results in non-uniform pharmacokinetic properties, potency and toxicity among the components of the product, poor repeatability of product batches and low therapeutic index.
Aiming at the defects of poor uniformity, single design of a linker, poor water solubility and the like of antibody drug conjugates in the prior art, a new linker technology is urgently needed to be developed, the aim of site-specific conjugation is fulfilled while the hydrophilicity of the linker is increased, and the uniformity of the antibody drug conjugates is improved.
Disclosure of Invention
In a first aspect, the present invention provides a conjugate having the structure of formula (i):
Q-X-L 1 -L 2 -D (I)
wherein Q is selected from:
Figure BDA0003782964770000021
Figure BDA0003782964770000022
preferably, Q is
Figure BDA0003782964770000023
R 5 And R 6 Each independently of the other having-X 1 —Q 1 Structure of (1), Q 1 Selected from-H, -F, -Cl, -Br, -I, -SO 2 、-NO 2 、C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical, X 1 Selected from the group consisting of single bonds, -O-, -S-,C 1-12 straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000031
And
Figure BDA0003782964770000032
or combinations thereof, preferably, R 5 And R 6 Is H.
X is a linking group, X is
Figure BDA0003782964770000033
Wherein m is 1 、m 2 Each independently selected from the group consisting of integers of 0 to 12 (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), xa, X b Each independently selected from: -O-, -S-, C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000034
Figure BDA0003782964770000035
Figure BDA0003782964770000036
Wherein R is 9 Selected from the group consisting of: -H, C 1-10 Straight chain/branched alkyl.
Preferably, m 1 、m 2 Each independently selected from integers of 0 to 6, more preferably, m 1 、m 2 Each independently selected from integers of 0 to 3.
In one embodiment of the present invention, m 1 Is 0.
In one embodiment of the present invention, m 2 Is 2.
Preferably, xa is selected from:
Figure BDA0003782964770000037
is more preferablyXa is
Figure BDA0003782964770000038
Preferably, X b Selected from:
Figure BDA0003782964770000039
Figure BDA00037829647700000310
more preferably, X b Is composed of
Figure BDA00037829647700000311
Preferably, R 9 is-H.
L 1 Selected from: one of a linear, Y-shaped, and multi-branched polyethylene glycol residue;
when L is 1 When Y-type and multi-branched polyethylene glycol residues are used, they may have one or more branches and
Figure BDA00037829647700000312
connecting;
L 1 including but 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 the structure represented by the general formula (III):
Figure BDA0003782964770000041
wherein n is 1 Independently selected from integers of 1 to 30, preferably 1 to 25, more preferably 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), especially 8.
L 2 Is a linking group of
Figure BDA0003782964770000042
Wherein A is glucuronide or glucosideAn acid derivative, said A being selected from:
Figure BDA0003782964770000043
wherein R is 12 Selected from: c 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl and C 6-12 One of heteroaryl, wherein, said C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl radical, C 6-12 Heteroaryl may be substituted by at least one C 1-18 Straight/branched alkyl, halogen, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C 1-10 Alkoxy and aryl substitution.
Preferably, A is
Figure BDA0003782964770000044
In one embodiment of the present invention, A is
Figure BDA0003782964770000045
The B is selected from:
Figure BDA0003782964770000046
Figure BDA0003782964770000051
wherein, Y 1 、Y 2 Each independently selected from: -H, halogen, -OC 1-10 Alkyl radical, C 1-10 Straight chain/branched alkyl, C 3-10 Cycloalkyl, -OH,
Figure BDA0003782964770000052
Preferably, Y 1 、Y 2 Are all-H.
Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000053
Figure BDA0003782964770000054
Preferably, Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight/branched alkyl; more preferably, Y 3 、Y 4 Are all-CH 3 。
In one embodiment of the present invention, Y is 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000055
X 2 Selected from the group consisting of-O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000056
Figure BDA0003782964770000057
Figure BDA0003782964770000058
And
Figure BDA0003782964770000059
or combinations thereof, wherein R 9 Selected from: -H, C 1-10 Straight chain/branched alkyl, preferably, R 9 is-H;
preferably, X 2 Selected from the group consisting of C 1-12 Straight chain/branched alkyl and
Figure BDA00037829647700000510
group (iii) of (iv).
In one embodiment of the present invention, X is 2 Is composed of
Figure BDA0003782964770000061
In one embodiment of the present invention, X is 2 Comprises the following steps:
Figure BDA0003782964770000062
R 10 selected from: -H, C 1-12 Straight chain/branched alkyl, C 1-12 Alkoxy, -NO 2 、-CN、-F、-Cl、-Br、-I、-SO 2 、C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000063
Figure BDA0003782964770000064
Figure BDA0003782964770000065
Preferably, R 10 Selected from: -H, C 1-12 Straight chain/branched alkyl, -NO 2 More preferably, R 10 Selected from: -H, -CH 3 、-NO 2 Still more preferably, R 10 is-H.
R 11 Selected from: -H, C 1-10 Straight chain/branched alkyl,
Figure BDA0003782964770000066
In one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000067
Figure BDA0003782964770000068
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000069
Figure BDA00037829647700000610
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000071
Figure BDA0003782964770000072
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000073
Figure BDA0003782964770000074
in one embodiment of the present invention, B is
Figure BDA0003782964770000075
Further, said L 2 Selected from:
Figure BDA0003782964770000076
Figure BDA0003782964770000077
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000081
Figure BDA0003782964770000082
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000083
Figure BDA0003782964770000084
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000085
Figure BDA0003782964770000086
in one embodiment of the present invention, L is 2 Is composed of
Figure BDA0003782964770000091
Further, D is a drug molecule.
Preferably, the conjugate has the structure shown in formula (II):
Figure BDA0003782964770000092
wherein, DBCO (dibenzocyclooctyne) linker in formula (II) is used for site-directed coupling with azide group of modified antibody; l is 1 Selected from: one of linear, Y-shaped and multi-branched polyethylene glycol residues, L 1 Including but 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):
Figure BDA0003782964770000093
wherein n is 1 Independently selected from integers of 1 to 30, preferably 1 to 25, more preferably 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), especially 8.
Further, the drug molecule is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, saccharides, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoid phenols, steroids, and one of their glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from: amanita, auristatin, calicheamicin, camptothecin derivatives and metabolites (such as: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, epothilones, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, irinotecan, topotecan, belotecan, irinotecan, rutotetrazone, diflutecan, geminithican, canicifukang, doxorubicin, epirubicin, morpholino doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, S.S., PBD and its derivatives, TPL, taxanes, ansamycin B and its derivatives, maytansine and its derivatives, dolastatin, mitomycin, actinomycin, aminomycin, 1- (5-acetyl-1- (1, 5-dimethylaminopyridine), vincristine, 1- (5-acetyl-adriamycin), vincristine, vindoline, doxorubicin and its derivatives, doxorubicin, and its derivatives, and their combinations.
In one embodiment of the present invention, the drug molecule is selected from the group consisting of: amastatin, auristatin, calicheamicin, camptothecin derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, 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: one of SN38 and its derivatives, dxd, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
In one embodiment of the present invention, the drug molecule is selected from the group consisting of:
Figure BDA0003782964770000101
Figure BDA0003782964770000102
Figure BDA0003782964770000111
Figure BDA0003782964770000112
to (3) is provided.
In one embodiment of the present invention, the drug molecule is selected from the group consisting of:
Figure BDA0003782964770000113
Figure BDA0003782964770000114
in one embodiment of the invention, the drug molecule is
Figure BDA0003782964770000115
The DBCO linker-drug conjugate is selected from the group consisting of:
Figure BDA0003782964770000121
Figure BDA0003782964770000131
Figure BDA0003782964770000141
in one embodiment of the invention, the DBCO linker-drug conjugate is
Figure BDA0003782964770000142
In a second aspect, the present invention provides a conjugate of formula (II) for use in a medicament for the prophylaxis and/or treatment of disease.
In particular, the disease is cancer, infection by a pathogenic organism, or an autoimmune disease.
In particular, the cancer is a hematopoietic tumor, a carcinoma, a sarcoma, a melanoma, or a glioma tumor.
In particular, the pathogenic organism is selected from the group consisting of: <xnotran> (HIV), , , , , , , , , , B , , , , , , , , , , Ⅰ , Ⅱ , , , - , , , , T , - , , , , , , , , , , , , 40, , , , , , , . </xnotran>
In particular, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, adenoidal syndrome, bullous pemphigoid, diabetes, henschel purpura, poststreptococcal nephritis, erythema nodosum, takayasu arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, ankylosing spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary affection, hashimoto thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes, giant cell arteritis/polymyalgia, anemia, acute fibrosis, and glomerulonephritis.
In a third aspect of the invention, there is provided an antibody drug conjugate of general formula (iv).
Figure BDA0003782964770000151
Wherein, ab is an antibody, and the antibody comprises a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody, and more preferably an internalization monoclonal antibody.
In the present invention, the antibody may be in the form of, for example: chimeric antibodies, humanized antibodies, human antibodies, antibody fragments that bind to an antigen (Fab, fab', F (ab) 2), subfragments (single chain constructs), or antibody Fc fusion proteins, and the like, preferably, the monoclonal antibodies are reactive against an antigen or epitope thereof associated with cancer, malignant cells, infectious organisms, or autoimmune diseases.
In one embodiment of the present invention, preferably, the monoclonal antibody is selected from the group consisting of: one of an anti-HER 2 antibody, an anti-EGFR antibody, an anti-PMSA antibody, an anti-VEGFR antibody, an anti-CD 20 antibody, an anti-CD 30 antibody, an anti-fra antibody, an anti-CD 22 antibody, an anti-CD 56 antibody, an anti-CD 29 antibody, an anti-GPNMB antibody, an anti-CD 138 antibody, an anti-CD 74 antibody, an anti-ENPP 3 antibody, an anti-Nectin-4 antibody, an anti-EGFR viii antibody, an anti-SLC 44A4 antibody, an anti-mesothelin antibody (anti-mesothelin antibody), an anti-ET 8R antibody, an anti-CD 37 antibody, an anti-CEACAM 5 antibody, an anti-CD 70 antibody, an anti-MUC 16 antibody, an anti-CD 79b antibody, an anti-MUC 16 antibody and an anti-MUC 1 antibody.
In one embodiment of the present invention, preferably, the antigen is selected from the group consisting of: <xnotran> HER-2/neu, EGFR, FR α, nectin-4, 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, (AFP), VEGF, ED-B , EGP-1, EGP-2, EGF (ErbB 1), erbB2, erbB3, H, FHL-1, flt-3, , ga733, GROB, HMGB-1, (HIF), HM1.24, (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, , HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, (PIGF), PSA, PSMA, PSMA , PAM4 , NCA-95, NCA-90, A3, A33, ep-CAM, KS-1, le (y), , </xnotran> S100, tenascin, TAC, tn antigen, thomas-Friedenreich antigen, tumor necrosis antigen, tumor angiogenesis antigen, TNF-alpha, TRAIL receptor (R1 and R2), VEGFR, RANTES, T101, cancer stem cell antigen, complement factors C3, C3a, C3b, C5a, C5, oncogene products and the like.
l is selected from an integer of 1 to 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 Is composed of
Figure BDA0003782964770000161
Wherein Z is 1 Is an integer of 1 to 10 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10), preferably, Z 1 Is an integer of 1 to 5.
In one embodiment of the present invention, Z is 1 Is 1.
In one embodiment of the present invention, Z is 1 Is 2.
L 4 Is selected from
Figure BDA0003782964770000162
n 2 Selected from integers ranging from 1 to 30 (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), preferably from 1 to 25, and more preferably from 4 to 24.
Q' is selected from
Figure BDA0003782964770000171
Figure BDA0003782964770000172
Figure BDA0003782964770000173
One of (a) and (b);
preferably, Q' is selected from
Figure BDA0003782964770000174
One of (1);
more preferably, Q' is selected from
Figure BDA0003782964770000175
R 5 And R 6 Each independently of the other having-X 1 —Q 1 Structure of (1), Q 1 Selected from-H, -F, -Cl, -Br, -I, -SO 2 、-NO 2 、C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ringsBase, X 1 Selected from the group consisting of single bonds, -O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000181
And
Figure BDA0003782964770000182
or combinations thereof, preferably, R 5 And R 6 Is H.
X is a linking group, X is
Figure BDA0003782964770000183
Wherein m is 1 、m 2 Each independently selected from the group consisting of integers of 0 to 12 (e.g., 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), xa, X b Each independently selected from: -O-, -S-, C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000184
Figure BDA0003782964770000185
Figure BDA0003782964770000186
Wherein R is 9 Selected from the group consisting of: -H, C 1-10 Straight chain/branched alkyl.
Preferably, m 1 、m 2 Each independently selected from integers of 0 to 6, more preferably, m 1 、m 2 Each independently selected from integers of 0 to 3.
In one embodiment of the present invention, m 1 Is 0.
In one embodiment of the present invention, m 2 Is 2.
Preferably, xa is selected from:
Figure BDA0003782964770000187
one of them, furtherPreferably, xa is
Figure BDA0003782964770000188
Preferably, X b Selected from the group consisting of:
Figure BDA0003782964770000189
Figure BDA00037829647700001810
more preferably, X b Is composed of
Figure BDA00037829647700001811
Preferably, R 9 is-H.
L 1 Selected from the group consisting of: one of a linear, Y-shaped, and multi-branched polyethylene glycol residue; when L is 1 When the polyethylene glycol residue is Y-shaped or multi-branched, it may have one or more branches and
Figure BDA00037829647700001812
connecting;
L 1 including but 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 the structure represented by the general formula (III):
Figure BDA0003782964770000191
wherein n is 1 Independently selected from integers of 1 to 30, preferably 1 to 25, more preferably 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), especially 8.
L 2 Is a linking group of
Figure BDA0003782964770000192
Wherein A is glucuronide or glucuronide derivative, and A is selected from：
Figure BDA0003782964770000193
Wherein R is 12 Selected from the group consisting of: c 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl and C 6-12 One of heteroaryl, wherein, said C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl radical, C 6-12 Heteroaryl may be interrupted by at least one C 1-18 Straight/branched alkyl, halogen, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C 1-10 Alkoxy and aryl substitution.
Preferably, A is
Figure BDA0003782964770000194
In one embodiment of the invention, A is
Figure BDA0003782964770000195
The B is selected from:
Figure BDA0003782964770000196
Figure BDA0003782964770000201
wherein, Y 1 、Y 2 Each independently selected from: -H, halogen, -OC 1-10 Alkyl radical, C 1-10 Straight chain/branched alkyl, C 3-10 Cycloalkyl, -OH,
Figure BDA0003782964770000202
Preferably, Y 1 、Y 2 Are all-H.
Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000203
Figure BDA0003782964770000204
Preferably, Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight/branched alkyl; more preferably, Y 3 、Y 4 Are all-CH 3 。
In one embodiment of the present invention, Y is 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000205
X 2 Selected from the group consisting of-O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000206
Figure BDA0003782964770000207
Figure BDA0003782964770000208
And
Figure BDA0003782964770000209
or combinations thereof, wherein R 9 Selected from: -H, C 1-10 Straight/branched alkyl, preferably, R 9 is-H;
preferably, X 2 Selected from the group consisting of C 1-12 Straight chain/branched alkyl and
Figure BDA00037829647700002010
group (d) of (a).
In one embodiment of the present invention, X is 2 Is composed of
Figure BDA0003782964770000211
In one embodiment of the present invention, X is 2 Comprises the following steps:
Figure BDA0003782964770000212
R 10 selected from: -H, C 1-12 Straight chain/branched alkyl, C 1-12 Alkoxy, -NO 2 、-CN、-F、-Cl、-Br、-I、-SO 2 、C 3-12 Cycloalkyl, C 6-12 Aromatic ring group,
Figure BDA0003782964770000213
Figure BDA0003782964770000214
Figure BDA0003782964770000215
Preferably, R 10 Selected from the group consisting of: -H, C 1-12 Straight chain/branched alkyl, -NO 2 More preferably, R 10 Selected from: -H, -CH 3 、-NO 2 Still more preferably, R 10 is-H.
R 11 Selected from: -H, C 1-10 A linear/branched alkyl group,
Figure BDA0003782964770000216
In one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000217
Figure BDA0003782964770000218
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000219
Figure BDA00037829647700002110
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000221
Figure BDA0003782964770000222
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000223
Figure BDA0003782964770000224
in one embodiment of the present invention, B is
Figure BDA0003782964770000225
Further, said L 2 Selected from the group consisting of:
Figure BDA0003782964770000226
Figure BDA0003782964770000227
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000231
Figure BDA0003782964770000232
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000233
Figure BDA0003782964770000234
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000235
Figure BDA0003782964770000236
in one embodiment of the present invention, L is 2 Is composed of
Figure BDA0003782964770000241
Further, D is a drug molecule.
Further, the drug molecule is selected from: amino acids, proteins, enzymes, nucleosides, saccharides, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoids, steroids, and one of their glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from: amanita, auristatin, calicheamicin, camptothecin derivatives and metabolites (such as: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, epothilones, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, irinotecan, topotecan, belotecan, irinotecan, rutotetrazone, diflutecan, geminithican, canicifukang, doxorubicin, epirubicin, morpholino doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, S.S., PBD and its derivatives, TPL, taxanes, ansamycin B and its derivatives, maytansine and its derivatives, dolastatin, mitomycin, actinomycin, aminomycin, 1- (5-acetyl-1- (1, 5-dimethylaminopyridine), vincristine, 1- (5-acetyl-adriamycin), vincristine, vindoline, doxorubicin and its derivatives, doxorubicin, and its derivatives, and their combinations.
In one embodiment of the present invention, the drug molecule is selected from the group consisting of: amastatin, auristatin, calicheamicin, camptothecin derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing.
In a particular embodiment of the invention, the drug molecule is selected from the group consisting of: one 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:
Figure BDA0003782964770000242
Figure BDA0003782964770000251
Figure BDA0003782964770000252
one kind of (1).
In one embodiment of the present invention, the drug molecule is selected from the group consisting of:
Figure BDA0003782964770000253
Figure BDA0003782964770000261
in one embodiment of the present invention, the drug molecule is
Figure BDA0003782964770000262
Preferably, the antibody drug conjugate has a structure represented by formula (V):
Figure BDA0003782964770000263
wherein i is an integer 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 present invention, i is 12.
Preferably, the antibody drug conjugate has a structure represented by formula (vi):
Figure BDA0003782964770000264
the invention also provides a pharmaceutical composition comprising the antibody drug conjugate of the invention and a pharmaceutically acceptable carrier or excipient.
In particular, the pharmaceutical compositions of the present 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 tablets, capsules, pills, granules, powder, suppositories, injections, solutions, suspensions, pastes, patches, lotions, drops, liniments, sprays and the like.
In particular, the conjugates of the invention may be administered as pure compounds or as suitable pharmaceutical compositions, using any acceptable mode of administration or agent for similar use. Thus, administration may be by oral, intranasal, parenteral, topical, transdermal or rectal means, in the form of solid, semi-solid or liquid dosage forms, for example, tablets, suppositories, pills, soft and hard gelatin capsules, powders, solutions, suspensions and injections and the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
In particular, pharmaceutical compositions which can be administered in liquid form can be prepared, for example, by dissolving, dispersing, etc., a conjugate of the invention (about 0.5 to about 20%) and, optionally, pharmaceutically acceptable adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, ethanol, etc., to form a solution or suspension.
In a fourth aspect of the present invention, there is provided a method for preparing an antibody drug conjugate of formula (V).
First, the antibody is reacted with a PEG derivative having an N3 functional group and an NHS functional group to label the antibody with the N3 functional group. Then, the N3 functional group on the antibody and the DBCO functional group of the linker are subjected to click reaction to obtain the antibody conjugated drug.
The synthetic route of the preparation method is shown as follows:
Figure BDA0003782964770000271
further, ab is an antibody;
wherein i is an integer 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 present invention, i is 12.
L 1 Selected from the group consisting of: one of linear, Y-shaped and multi-branched polyethylene glycol residues, preferably, said L 1 Is a linear polyethylene glycol residue having the structure shown below:
Figure BDA0003782964770000281
wherein n is 1 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 of
Figure BDA0003782964770000282
Wherein A is glucuronide or a glucuronide derivative, and A is selected from:
Figure BDA0003782964770000283
wherein R is 12 Selected from: c 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl and C 6-12 One of heteroaryl, wherein, said C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl radical, C 6-12 Heteroaryl may be substituted by at least one C 1-18 Straight/branched alkyl, halogen, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C 1-10 Alkoxy and aryl substitution.
Preferably, A is
Figure BDA0003782964770000284
In one embodiment of the present invention, A is
Figure BDA0003782964770000285
The B is selected from:
Figure BDA0003782964770000286
Figure BDA0003782964770000291
wherein, Y 1 、Y 2 Each independently selected from: -H, halogen, -OC 1-10 Alkyl radical, C 1-10 Straight chain/branched alkyl, C 3-10 Cycloalkyl, -OH, or,
Figure BDA0003782964770000292
Preferably, Y 1 、Y 2 Are all-H.
Y 3 、Y 4 Each is independent of othersThe land is selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000293
Figure BDA0003782964770000294
Preferably, Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight/branched alkyl; more preferably, Y 3 、Y 4 Are all-CH 3 。
In one embodiment of the present invention, Y is 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure BDA0003782964770000295
X 2 Selected from the group consisting of-O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring group,
Figure BDA0003782964770000296
Figure BDA0003782964770000297
Figure BDA0003782964770000298
And
Figure BDA0003782964770000299
or combinations thereof, wherein R 9 Selected from: -H, C 1-10 Straight/branched alkyl, preferably, R 9 is-H;
preferably, X 2 Is selected from the group consisting of C 1-12 Straight chain/branched alkyl and
Figure BDA00037829647700002910
group (d) of (a).
In one embodiment of the present invention, X is 2 Is composed of
Figure BDA0003782964770000301
In one embodiment of the present invention, X is 2 Comprises the following steps:
Figure BDA0003782964770000302
R 10 selected from: -H, C 1-12 Straight chain/branched alkyl, C 1-12 Alkoxy, -NO 2 、-CN、-F、-Cl、-Br、-I、-SO 2 、C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure BDA0003782964770000303
Figure BDA0003782964770000304
Figure BDA0003782964770000305
Preferably, R 10 Selected from: -H, C 1-12 Straight chain/branched alkyl, -NO 2 More preferably, R 10 Selected from the group consisting of: -H, -CH 3 、-NO 2 Still more preferably, R 10 is-H.
R 11 Selected from: -H, C 1-10 Straight chain/branched alkyl,
Figure BDA0003782964770000306
In one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000307
Figure BDA0003782964770000308
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000309
Figure BDA00037829647700003010
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000311
Figure BDA0003782964770000312
in one embodiment of the present invention, B is selected from:
Figure BDA0003782964770000313
Figure BDA0003782964770000314
in one embodiment of the present invention, B is
Figure BDA0003782964770000315
Further, said L 2 Selected from:
Figure BDA0003782964770000316
Figure BDA0003782964770000317
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000321
Figure BDA0003782964770000322
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000323
Figure BDA0003782964770000324
in one embodiment of the present invention, L is 2 Selected from:
Figure BDA0003782964770000325
Figure BDA0003782964770000326
in one embodiment of the present invention, L is 2 Is composed of
Figure BDA0003782964770000331
Further, D is a drug molecule.
Further, the drug molecule is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, saccharides, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanoid phenols, steroids, and one of their glycosides and alkaloids.
In one embodiment of the invention, the drug molecule is preferably selected from: amanita, auristatin, calicheamicin, camptothecin derivatives and metabolites (such as: SN-38 and its derivatives, 10-hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, epothilones, geldanamycin, maytansine and its derivatives, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, irinotecan, topotecan, belotecan, irinotecan, rutotetrazone, diflutecan, geminithican, canicifukang, doxorubicin, epirubicin, morpholino doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl doxorubicin, dxd, S.S., PBD and its derivatives, TPL, taxanes, ansamycin B and its derivatives, maytansine and its derivatives, dolastatin, mitomycin, actinomycin, aminomycin, 1- (5-acetyl-1- (1, 5-dimethylaminopyridine), vincristine, 1- (5-acetyl-adriamycin), vincristine, vindoline, doxorubicin and its derivatives, doxorubicin, and its derivatives, and their combinations.
In one embodiment of the invention, the drug molecule is selected from the group consisting of: amastatin, auristatin, calicheamicin, camptothecin derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing.
In a particular embodiment of the invention, the drug molecule is selected from the group consisting of: one 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:
Figure BDA0003782964770000332
Figure BDA0003782964770000341
Figure BDA0003782964770000342
to (3) is provided.
In one embodiment of the invention, the drug molecule is selected from the group consisting of:
Figure BDA0003782964770000343
Figure BDA0003782964770000351
in one embodiment of the present invention, the drug molecule is
Figure BDA0003782964770000352
The fifth aspect of the invention provides an antibody drug conjugate shown in the general formula (IV) or a salt thereof and an application of a pharmaceutical composition in drugs for preventing and/or treating diseases.
Further, the disease is cancer, infection by a pathogenic organism, or an autoimmune disease.
Further, the cancer is a hematopoietic tumor, a carcinoma, a sarcoma, a melanoma, or a glioma tumor.
Further, the pathogenic organism is selected from the group consisting of: human Immunodeficiency Virus (HIV), mycobacterium tuberculosis, streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, legionella pneumophila, streptococcus pyogenes, escherichia coli, netheria gonorrhoeae, neisseria meningitidis, streptococcus pneumoniae, haemophilus influenzae type B, treponema pallidum, lemer's disease spirochete, west Nile virus, pseudomonas aeruginosa, mycobacterium leprae, abortus, rabies, influenza, cytomegalovirus, herpes simplex virus type I, herpes simplex 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, verrucosis virus, bluetongue virus, sendai virus, feline leukemia virus, giardia virus, polio virus, simian virus 40, simian influenza virus, a simian equine virus, dengue virus, a rubella virus, plasmodium vivax.
Further, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, adenoidal syndrome, bullous pemphigoid, diabetes, henschel purpura, poststreptococcal nephritis, erythema nodosum, takayasu arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, ankylosing spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary affection, hashimoto thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes, giant cell arteritis/polymyalgia, anemia, acute fibrosis, and glomerulonephritis.
In a sixth aspect, the invention provides a method of treatment of a disease, said method comprising administering to a subject an antibody drug conjugate according to the invention.
Further, the antibody drug conjugate is administered in combination with one or more treatment methods selected from the group consisting of: unconjugated antibodies, radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugated antibodies, gene therapy, chemotherapy, therapeutic peptides, oligonucleotides, local radiotherapy, surgery, and interfering RNA therapy.
Further, the disease is cancer, infection by a pathogenic organism, or an autoimmune disease.
Further, the cancer is a hematopoietic tumor, a carcinoma, a sarcoma, a melanoma, or a glioma tumor.
Further, the pathogenic organism is selected from the group consisting of: human Immunodeficiency Virus (HIV), mycobacterium tuberculosis, streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, legionella pneumophila, streptococcus pyogenes, escherichia coli, netheria gonorrhoeae, neisseria meningitidis, streptococcus pneumoniae, haemophilus influenzae type B, treponema pallidum, lemer's disease spirochete, west Nile virus, pseudomonas aeruginosa, mycobacterium leprae, abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus type I, herpes simplex 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, verruca virus, bluetongue virus, sendai virus, feline leukemia virus, reovirus, polio virus, simian virus 40, murine mammary tumor virus, dengue virus, rubella virus, plasmodium falciparum, plasmodium vivax, toxoplasma gondii, trypanosoma donii
Further, the autoimmune disease is selected from the group consisting of: immune-mediated thrombocytopenia, dermatomyositis, sjogren's syndrome, multiple sclerosis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, adenoidal syndrome, bullous pemphigoid, diabetes, henschel purpura, poststreptococcal nephritis, erythema nodosum, takayasu arteritis, addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, igA nephropathy, polyarteritis nodosa, ankylosing spondylitis, goodpasture's syndrome, thromboangiitis obliterans, primary biliary affection, hashimoto thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes, giant cell arteritis/polymyalgia, anemia, acute fibrosis, and glomerulonephritis.
The conjugate of the PEG-containing beta-glucuronidase cleavable linker and the cytotoxic drug, which takes DBCO as a linker, can realize site-directed coupling with a modified azide group on an antibody through a simple SPAAC reaction to obtain the antibody coupling drug with higher uniformity; meanwhile, PEG groups are introduced into the linker, so that the purpose of improving the hydrophilicity of the linker can be achieved, the water solubility of the linker-drug conjugate is increased, and the problem of poor solubility of the linker-drug conjugate is solved.
Term C as used herein 1-8 Straight/branched alkyl radicals including 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.
Term C in the present invention 1-10 Straight/branched alkyl radicals including 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.
Term C as used herein 1-5 Straight/branched alkyl radicals including methyl, ethyl, C 3 Straight chain/branched alkyl, C 4 Straight chain/branched alkyl, C 5 Straight chain/branched alkyl. Term C in the present invention 3-12 Cycloalkyl radicals including C 3 Cycloalkyl radical, C 4 Cycloalkyl radical, C 5 Cycloalkyl, C 6 Cycloalkyl, C 7 Cycloalkyl, C 8 Cycloalkyl radical, C 9 Cycloalkyl radical, C 10 Cycloalkyl radical, C 11 Cycloalkyl radical, C 12 A cycloalkyl group.
Term C in the present invention 1-12 Straight/branched alkyl radicals including 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.
Term C in the present invention 3-12 Cycloalkyl radicals including, C 3 Cycloalkyl radical, C 4 Cycloalkyl, C 5 Cycloalkyl radical, C 6 Cycloalkyl radical, C 7 Cycloalkyl radical, C 8 Cycloalkyl radical, C 9 Cycloalkyl radical, C 10 Cycloalkyl radical, C 11 Cycloalkyl radical, C 12 A cycloalkyl group.
Term C in the present invention 3-10 Cycloalkyl radicals including, C 3 Cycloalkyl radical, C 4 Cycloalkyl, C 5 Cycloalkyl, C 6 Cycloalkyl radical, C 7 Cycloalkyl radical, C 8 Cycloalkyl radical, C 9 Cycloalkyl radical, C 10 A cycloalkyl group.
Term C in the present invention 6-12 Aromatic ring radicals including C 6 Aromatic ring radical (phenyl), C 7 Aromatic ring radical, C 8 Aromatic ring radical, C 9 Aromatic ring radical, C 10 Aromatic ring radical, C 11 Aromatic ring radical, C 12 An aromatic ring group.
Term C in the present invention 3-12 Heterocycloalkyl radicals including C 3 Heterocycloalkyl, C 4 Heterocycloalkyl radical, C 5 Heterocycloalkyl radical, C 6 Heterocycloalkyl radical, C 7 Heterocycloalkyl radical, C 8 Heterocycloalkyl radical, C 9 Heterocycloalkyl, C 10 Heterocycloalkyl radical, C 11 Heterocycloalkyl, C 12 A heterocycloalkyl group.
Term C as used herein 6-12 Heteroaryl of, including C 6 Heteroaryl group, C 7 Heteroaryl group, C 8 Heteroaryl group, C 9 Heteroaryl, C 10 Heteroaryl, C 11 Heteroaryl group, C 12 A heteroaryl group.
Term C in the present invention 3-8 Cycloalkyl radicals including, C 3 Cycloalkyl, C 4 Cycloalkyl, C 5 Cycloalkyl radical, C 6 Cycloalkyl, C 7 Cycloalkyl, C 8 A cycloalkyl group.
Term C in the present invention 3-8 Cycloalkoxy radicals, including C 3 Cycloalkoxy, C 4 Cycloalkoxy, C 5 Cycloalkoxy, C 6 Cycloalkoxy, C 7 Cycloalkoxy, C 8 A cycloalkoxy group.
Term C as used herein 1-12 Alkoxy radicals including methoxy, ethoxy, C 3 Alkoxy radical, C 4 Alkoxy radical, C 5 Alkoxy radical, C 6 Alkoxy radical, C 7 Alkoxy radical, C 8 Alkoxy radical, C 9 Alkoxy radical, C 10 Alkoxy radical, C 11 Alkoxy radical, C 12 An alkoxy group.
Term C in the present invention 1-10 Alkoxy radicals including methoxy, ethoxy, C 3 Alkoxy radical, C 4 Alkoxy radical, C 5 Alkoxy radical, C 6 Alkoxy radical, C 7 Alkoxy radical, C 8 Alkoxy radical, C 9 Alkoxy radical, C 10 An alkoxy group.
The term MMAE as used herein 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 used herein refers to monomethyl auristatin D.
The term PBD as used herein refers to pyrrolobenzodiazepines.
The term TPL as used herein refers to triptolide.
The term Dxd as used herein refers to Delutikang.
Drawings
FIG. 1 is a MS map of Compound 1.
Detailed Description
In order to clearly understand the technical content of the present invention, the following embodiments are specifically illustrated in detail to clearly and completely describe the technical scheme of the present invention, which is only for better understanding of the content of the present invention and is not intended to limit all other embodiments obtained by a person of ordinary skill in the art without creative efforts to the protection scope of the present invention, and all other embodiments are within the protection scope of the present invention.
EXAMPLE 1 Synthesis of Compound 1
Figure BDA0003782964770000391
1.1 Synthesis of Compound 1a
The raw material Glucose-NO is mixed 2 OH (48.5g, 0.10mol) was added to 480mL of DCM and 100mL of DMF, and DIEA (25.8g, 0.20mol) and TBSCl (16.5g, 0.111mol) were sequentially added to the solution, and the reaction was stirred at room temperature for 6H, and TLC showed completion of the reaction. The reaction was washed three times with 400ml × 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, the solvent was dried to give a 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 (45.1g, 0.08mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added, hydrogen was replaced, the reaction was stirred under hydrogen atmosphere for 4H, TLC showed completion of the reaction, the filter cake was removed by filtration, and the resulting filtrate was dried by spinning to give a 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 (6.32g, 11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83g, 12.7 mmol), TEA (2.1g, 21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC showed the starting material was reacted completely and was directly purified by C-18 medium pressure flash (H2O/CH 3OH 40% -90%) 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 (5.5g, 4.3mmol) was dissolved in THF (55 mL), 2N HCl aqueous solution (4.0 mL) was added, the reaction was stirred at room temperature 2H, TLC and HPLC showed complete reaction of the starting material, naHCO was added 3 The reaction was quenched with 50mL of saturated aqueous solution, then extracted twice with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate, and spin dried to give the crude product. The crude product was dissolved in 10mL DCM and the resulting solution was not clear, the solid was removed by filtration through a filter and the filtrate was rotary dried to give compound 1d (3.1 g, product as a yellow oil) in 62% yield.
MS m/z(ESI):1166.5[M+1]
1.5 Synthesis of Compound 1e
Dissolving compound 1d (2.9g, 2.5 mmol) in 40mL DCM, cooling to 0-5 ℃ in an ice-water bath, adding p-nitrochloroformic acid phenyl ester (0.75g, 3.7mmol) and DMAP (0.61g, 5.0mmol) in sequence into the reaction system, slowly heating to room temperature and stirring for 4h, after TLC shows that the reaction is complete, adding 30mL of 5% NaHSO 4 Washed once with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, dried, wet loaded and purified on 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-neck flask, compound 1e (2.3g, 1.7mmol), MMAE (1.5g, 2.1mmol), HOBt (116mg, 0.9mmol) pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48H, after completion of the reaction by HPLC, the solvent was removed by rotary evaporation, and flash (H) at medium pressure C-18 (H-18) 2 O/CH 3 OH50% -90%) 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 (144mg, 12eq) 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. Flash (H) with C-18 medium pressure 2 O/CH 3 OH50% -90%) to give 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
Figure BDA0003782964770000411
2.1 Synthesis of Compound 2a
The raw material Glucose-NO is mixed 2 -OH(48.5g，0.10 mol) was added to 480mL of DCM and 100mL of DMF, and DIEA (25.8g, 0.20mol) and TBSCl (16.5g, 0.11mol) were added to the solution in this order, and the reaction was stirred at room temperature for 6h, and TLC showed completion of the reaction. The reaction was washed three times with 400ml × 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, the solvent was dried to give a 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 (45.1g, 0.08mol) was dissolved in 300mL methanol and 150mL ethyl acetate, 10% Pd/C (4.5 g) was added, hydrogen was replaced, the reaction was stirred under hydrogen atmosphere for 4h, TLC showed completion of the reaction, the cake was removed by filtration, and the resulting filtrate was spun dry to give a 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), 88% yield.
MS m/z(ESI):570.2[M+1]
2.3 Synthesis of Compound 2c
Compound 2b (6.32g, 11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83g, 12.7 mmol) and TEA (2.1 g,21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC shows that after the raw material reaction is completed, the direct C-18 medium-pressure flash (H) 2 O/CH 3 OH 40% -90%) to give compound 2c (8.1 g, product as a yellow oil) in 60% yield.
MS m/z(ESI):1280.6[M+1]
2.4 Synthesis of Compound 2d
Compound 2c (5.5g, 4.3mmol) was dissolved in THF (55 mL), 2N HCl aqueous solution (4.0 mL) was added, the reaction solution was stirred at room temperature 2H, TLC and HPLC showed complete reaction of the starting materials, naHCO was added 3 The reaction was quenched with 50mL of saturated aqueous solution, then extracted twice with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate, and spin dried to give the crude product. The crude product was dissolved in 10mL of DCM and the resulting solution was not clear, the solid was removed by filtration through a filter, and the filtrate was dried by spinning 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 (2.9g, 2.5mmol) is dissolved in 40mL DCM, the temperature is reduced to 0-5 ℃ in an ice water bath, phenyl p-nitrochloroformate (0.75g, 3.7mmol) and DMAP (0.61g, 5.0mmol) are sequentially added into the reaction system, the reaction is slowly heated to room temperature and stirred for 4h, after TLC shows that the reaction is complete, 30mL of 5% NaHSO is added 4 Washed once with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, dried, wet loaded and purified on 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 neck flask, compound 2e (2.3g, 1.7 mmol), TBS-SN38 (1.1g, 2.1mmol), 4-dimethylaminopyridine (0.4g, 3.4mmol), DIEA (2 mL) and THF (50 mL) were added, the reaction stirred at room temperature for 48h, after completion of the reaction as shown by HPLC, the solvent was removed by rotary evaporation, and flash (H) at medium pressure C-18 was used 2 O/CH 3 OH50% -90%) to give compound 2f (1.4 g, off-white solid) in 50% yield.
MS m/z(ESI):1699.0[M+1]
2.7 Synthesis of 2g Compound
Compound 2f (1.0g, 0.6mmol) was dissolved in 50mL of tetrahydrofuran, tetrabutylammonium fluoride (0.52g, 2.0mmol) was added, stirring was carried out at room temperature for 30min, the progress of the reaction was monitored by TLC, the solvent was dried after completion of the reaction, and the purification preparation was carried out with C-18flash (MeOH: H) 2 O =50% -90%) to obtain 2g (590 mg, yellow solid) of the compound in 62% yield.
MS m/z(ESI):792.8[M/2+1]
2.8 Synthesis of Compound 2
Compound 2g (500mg, 0.3mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (72mg, 12eq) 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. Flash (H) with C-18 medium pressure 2 O/CH 3 OH50% -90%) to give 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
Figure BDA0003782964770000441
3.1 Synthesis of Compound 3a
The raw material Glucose-NO is mixed 2 OH (48.5g, 0.10mol) was added to 480mL of DCM and 100mL of DMF, and DIEA (25.8g, 0.20mol) and TBSCl (16.5g, 0.111mol) were sequentially added to the solution, and the reaction was stirred at room temperature for 6H, and TLC showed completion of the reaction. The reaction was washed three times with 400ml × 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, the solvent was dried to give a 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 (45.1g, 0.08mol) was dissolved in 300mL methanol and 150mL ethyl acetate, 10% Pd/C (4.5 g) was added, hydrogen was replaced, the reaction was stirred under hydrogen atmosphere for 4h, TLC showed completion of the reaction, the cake was removed by filtration, and the resulting filtrate was spun dry to give a 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), 88% yield.
MS m/z(ESI):570.2[M+1]
3.3 Synthesis of Compound 3c
Compound 3b (6.32g, 11.1 mmol), DBCO-PEG8-PA (7.7 g,10.6 mmol), HATU (4.83g, 12.7 mmol), TEA (2.1g, 21.2 mmol) were dissolved in DMF (80 mL) and the reaction stirred at room temperature for 4h. TLC shows that after the raw material reaction is completed, the direct C-18 medium-pressure flash (H) 2 O/CH 3 OH 40% -90%) to give compound 3c (8.1 g, product as a yellow oil) in 60% yield.
MS m/z(ESI):1280.6[M+1]
3.4 Synthesis of Compound 3d
Compound 3c (5.5g, 4.3mmol) was dissolved in THF (55 mL), and 2N aqueous HCl (4.0 mL) was added to the solutionStirring at room temperature for 2h, TLC and HPLC showed complete reaction of the starting material, naHCO was added 3 The reaction was quenched with 50mL of saturated aqueous solution, then extracted twice with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate, and spin dried to give the crude product. The crude product was dissolved in 10mL of DCM and the resulting solution was not clear, the solid was removed by filtration through a filter, and the filtrate was dried by spinning 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
Dissolving compound 3d (2.9g, 2.5 mmol) in 40mL DCM, cooling to 0-5 ℃ in an ice-water bath, adding p-nitrochloroformic acid phenyl ester (0.75g, 3.7mmol) and DMAP (0.61g, 5.0mmol) in sequence into the reaction system, slowly heating to room temperature and stirring for 4h, after TLC shows that the reaction is complete, adding 30mL of 5% NaHSO 4 The mixture was washed once with water, once with 30mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, spun dry, wet loaded, and purified on 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 (1.96g, 5.0mmol) and 4-dimethylaminopyridine (915mg, 7.5mmol) were dissolved in 15mL of dichloromethane, triphosgene (670 mg) was added under nitrogen protection, the reaction was stirred at room temperature for 5 minutes, a solution of Boc-DMEA (1.14g, 6.3mmol) in dichloromethane (4.0 mL) was added to the reaction solution, the reaction was stirred at room temperature for 5 minutes, and LCMS showed that the starting materials reacted completely. The reaction was washed 2 times with water to give 3f (2.3 g, yellow solid) in 76% yield.
MS m/z(ESI):607.2[M+1]
3.7 Synthesis of 3g of Compound
Compound 3f (2.3g, 3.7mmol) was dissolved in a mixed solvent of dichloromethane (4 mL) and trifluoroacetic acid (1 mL) and the reaction stirred at room temperature under nitrogen for 2h and LCMS indicated essentially complete reaction of starting material. The reaction was diluted with acetonitrile, concentrated at low temperature and purified in the medium pressure preparative liquid phase to give the product 3g (1.6 g, yellow solid) in 82% yield.
MS m/z(ESI):507.2[M+1]
3.8 Synthesis of Compound 3h
In a 100mL single-neck flask, compound 3e (2.3g, 1.7 mmol), 3g (1.1g, 2.1mmol), HOBt (116mg, 0.9mmol) pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48h, after completion of the reaction by HPLC, the solvent was removed by rotary evaporation, and flash (H) was added under C-18 medium pressure 2 O/CH 3 OH50% -90%) 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 (1.9g, 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 (288mg, 12eq) 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
Figure BDA0003782964770000471
4.1 Synthesis of Compound 4a
The raw material Glucose-NO is mixed 2 OH (49.9g, 0.10mol) was added to 480mL of DCM and 100mL of DMF, and then DIEA (25.8g, 0.20mol) and TBSCl (16.5g, 0.111mol) were sequentially added to the solution, and the reaction was stirred at room temperature for 6H, and TLC indicated that the reaction was complete. The reaction was washed three times with 400ml × 3 saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, the solvent was dried to give a 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, 0.08mol) was dissolved in 300mL of methanol and 150mL of ethyl acetate, 10% Pd/C (4.5 g) was added, hydrogen was replaced, the reaction was stirred under a hydrogen atmosphere for 4h, TLC showed completion of the reaction, the filter cake was removed by filtration, and the resulting filtrate was dried by spinning to give a 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 (6.42g, 11.0mmol), DBCO-PEG8-PA (7.7g, 10.6 mmol), HATU (4.83g, 12.7mmol) and TEA (2.1g, 21.2mmol) were dissolved in DMF (80 mL) and the reaction was stirred at room temperature for 4h. TLC shows that after the raw material reaction is completed, the direct C-18 medium-pressure flash (H) 2 O/CH 3 OH 40% -90%) to give compound 4c (8.8 g, product as a yellow oil) 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 was stirred at room temperature for 2H, TLC and HPLC showed complete reaction of starting material, naHCO was added 3 The reaction was quenched with 50mL of saturated aqueous solution, then extracted twice with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate, and spin dried to give the crude product. The crude product was dissolved in 10mL DCM and the resulting solution was not clear, the solid was removed by filtration through a filter and the filtrate was rotary dried to give compound 4d (3.3 g, product as a yellow oil) in 66% yield.
MS m/z(ESI):1180.5[M+1]
4.5 Synthesis of Compound 4e
Dissolving compound 4d (3.0g, 2.5 mmol) in 40mL DCM, cooling to 0-5 ℃ in an ice-water bath, adding p-nitrophenyl chloroformate (0.75g, 3.7mmol) and DMAP (0.61g, 5.0mmol) into the reaction system in sequence, slowly heating to room temperature and stirring for 4 hours, after TLC shows that the reaction is complete, adding 30mL of 5% NaHSO 4 Washed once with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, dried, wet loaded and purified on 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-neck flask, compound 4e (2.3g, 1.7mmol), MMAE (1.5g, 2.1mmol), HOBt (116mg, 0.9mmol) pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48h, after completion of the reaction by HPLC, the solvent was removed by rotary evaporation, and flash (H) at medium pressure C-18 (H-18) 2 O/CH 3 OH50% -90%) to give compound 4f (2.0 g, off-white solid) in 61% yield.
MS m/z(ESI):962.5[M/2+1]
4.7 Synthesis of Compound 4
Compound 4f (961mg, 0.5mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (144mg, 12eq) 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. Flash (H) with C-18 medium pressure 2 O/CH 3 OH50% -90%) to give 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
Figure BDA0003782964770000491
5.1 Synthesis of Compound 5b
Compound 5a (6.76g, 11.0mmol), DBCO-PEG8-PA (7.7g, 10.6 mmol), HATU (4.83g, 12.7mmol), TEA (2.1g, 21.2mmol) were dissolved in DMF (80 mL) and the reaction was stirred at room temperature for 4h. TLC showed the starting material was reacted completely, directly to C-18 medium pressure flash (H) 2 O/CH 3 OH 40% -90%) 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 (5.7g, 4.3mmol) was dissolved in THF (55 mL), 2N HCl aqueous solution (4.0 mL) was added, the reaction was stirred at room temperature 2H, TLC and HPLC showed complete reaction of starting material, naHCO was added 3 Quenching reaction by saturated aqueous solution 50mL, extracting twice by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, and spin-drying to obtainTo a crude product. The crude product was dissolved in 10mL DCM and the resulting solution was not clear, the solid was removed by filtration through a filter and the filtrate was rotary 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
Dissolving compound 5c (3.0g, 2.5 mmol) in 40mL DCM, cooling to 0-5 ℃ in an ice-water bath, adding p-nitrophenyl chloroformate (0.75g, 3.7mmol) and DMAP (0.61g, 5.0mmol) into the reaction system in sequence, slowly heating to room temperature and stirring for 4 hours, after TLC shows that the reaction is complete, adding 30mL of 5% NaHSO 4 Washed once with 30mL of saturated brine, and the organic phase dried over anhydrous sodium sulfate, dried, wet loaded and purified on 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-neck flask, compound 5d (2.3g, 1.7mmol), MMAE (1.5g, 2.1mmol), HOBt (116mg, 0.9mmol) pyridine (20 mL), DIEA (2 mL) and THF (50 mL) were added, the reaction was stirred at room temperature for 48h, after completion of the reaction by HPLC, the solvent was removed by rotary evaporation, and flash (H) at medium pressure C-18 (H-18) 2 O/CH 3 OH50% -90%) to give compound 5e (2.1 g, off-white solid) in 65% yield.
MS m/z(ESI):978.5[M/2+1]
5.5 Synthesis of Compound 5
Compound 5e (979mg, 0.5mmol) was dissolved in a mixed solvent of THF and MeOH, cooled in an ice bath, and an aqueous solution of lithium hydroxide (144mg, 12eq) 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. Flash (H) medium pressure with C-18 2 O/CH 3 OH50% -90%) to give 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
According to the molar ratio of 1mAb to PEG12-SPA20 in the ratio of the raw materials. First, 208. Mu.L of mAb solution (concentration: 9.6 mg/mL) was measured and placed in a 1.5mL centrifuge tube, and the amount of mAb measured was 2mg. 10mg/mL of N was prepared using a 5mM pH3.0 PBS solution 3 Measuring 20 mu L of PEG12-SPA solution, adding into a centrifuge tube, fully mixing, and reacting for 2h with shaking. Removing unreacted N by ultrafiltration after the reaction 3 -PEG12-SPA。
6.2mAb-PEG12 reaction procedure with linker-drug
Taking a prepared mAb-PEG12 sample, measuring the protein concentration to be 2.05mg/mL, and measuring 490 mu L of sample solution to be placed in a 1.5mL centrifuge tube. 10mg of linker-drug was weighed, dissolved in DMSO, and prepared into a solution of 1 mg/mL. According to the mAb and linker-drug feeding ratio of 1. After the reaction, unreacted small molecules were 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 value
And detecting the DAR value of the ADC by using a liquid phase method.
The liquid chromatography setup was as follows:
Figure BDA0003782964770000511
under the liquid chromatography condition, the separation degree of SN38 from ADC1 and ADC2 is more than 1.5. Preparing standard curves of different concentrations, establishing a linear standard curve between the SN38 peak area and the concentration, and quantifying the SN38 on the ADC1 and the ADC2 respectively. And calculating the SN38 coupling number on the ADC according to the antibody concentration of the ADC and the SN38 concentration. ADC1 and ADC2 prepared in example 6 were measured, and the DAR values were both 4;
example 8 determination of the affinity of ADC with EGFR
The affinity of ADC drug was tested by SPR, which is a product biacore without GE. The simple operation steps are as follows: EGFR antigen was coupled to CM chip. The affinity of the antibody or free antibody of the ADC drug to the antigen was tested with different concentrations of ADC drug or mab drug. The results show that the affinity of the antibodies of ADC1 and ADC2 prepared in example 6 is not significantly decreased, and that the affinity of ADC1 is higher than that of ADC2.
Example 9 in vitro cytotoxicity assay
BXPC-3 (human pancreatic cancer cells) cells were used for in vitro cytotoxicity assays. The test procedure was as follows:
1. cells were added at 100 μ L per well in 96-well plates (same volume of medium was added, leaving 2 blanks without cells). 5% of cells at 37% 2 Culturing in a cell culture box for 24h. Cytotoxicity assay approximately 10000 cells per well were added at 100. Mu.L.
2. Each well was dosed with 10 μ L of different concentrations of ADC or SN38 drug.
3. The 96 well plates were incubated at 37 ℃ with 5% CO 2 Incubate in a cell incubator with air and 100% humidity for 24 hours.
4. Add 10. Mu.L of CCK-8 solution to each well. 37 ℃ C., 5% CO 2 Incubate in incubator for 3h.
5. The absorbance at 450nm was measured with a microplate reader.
6. And (4) analyzing results:
A. cell survival rate: background OD values (blank) were subtracted from the OD values of each test well and the OD values of each replicate well were averaged. + -. SD.
Cell viability% = (medicated cell OD/control cell OD) × 100%.
B. The drug concentration (IC 50) at T/C =50% and the drug concentration (IC 90) at T/C =10% were obtained.
The results show that the IC50 values of ADC1 prepared in example 6 are smaller than the IC50 values of ADC2, comparable to the IC50 values of SN 38.
The result shows that the ADC1 obtained by the invention not only can effectively exert partial biological functions of an EGFR antibody, but also has the high-efficiency killing activity of SN38 on tumor cells. Compared with ADC2 prepared by the traditional dipeptide linker, ADC1 obtained by the method has better activity.

Claims (20)

1. A conjugate having the structure of formula (i):
Q-X-L 1 -L 2 -D (I)
wherein Q is selected from:
Figure FDA0003782964760000011
Figure FDA0003782964760000012
one of (a) and (b);
R 5 and R 6 Each independently of the other has-X 1 —Q 1 Structure of (1), Q 1 Selected from-H, -F, -Cl, -Br, -I, -SO 2 、-NO 2 、C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical, X 1 Selected from the group consisting of single bonds, -O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring group,
Figure FDA0003782964760000013
And
Figure FDA0003782964760000014
or a combination thereof;
x is a linking group, X is
Figure FDA0003782964760000015
Wherein m is 1 、m 2 Each independently selected from the integers of 0 to 12, xa and X b Each independently selected from: -O-, -S-, C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000016
Figure FDA0003782964760000017
Figure FDA0003782964760000018
One of (1), R 9 Selected from: -H, C 1-10 Straight/branched alkyl;
L 1 selected from: one of a linear, Y-shaped, and multi-branched polyethylene glycol residue;
L 2 is a linking group of
Figure FDA0003782964760000019
Wherein A is glucuronide or a glucuronide derivative;
the B is selected from:
Figure FDA00037829647600000110
Figure FDA0003782964760000021
wherein Y is 1 、Y 2 Each independently selected from: -H, halogen, -OC 1-10 Alkyl radical, C 1-10 Straight chain/branched alkyl, C 3-10 Cycloalkyl, -OH, or,
Figure FDA0003782964760000022
Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure FDA0003782964760000023
Figure FDA0003782964760000024
X 2 Selected from the group consisting of-O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000025
Figure FDA0003782964760000026
Figure FDA0003782964760000027
And
Figure FDA0003782964760000028
or a combination thereof;
R 10 selected from the group consisting of: -H, C 1-12 Straight chain/branched alkyl, C 1-12 Alkoxy, -NO 2 、-CN、-F、-Cl、-Br、-I、-SO 2 、C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring group,
Figure FDA0003782964760000029
Figure FDA00037829647600000210
Figure FDA0003782964760000031
R 11 Selected from: -H, C 1-10 A linear/branched alkyl group,
Figure FDA0003782964760000032
D is a drug molecule.
2. The conjugate of claim 1, wherein Q is
Figure FDA0003782964760000033
Preferably, R is 5 And R 6 Are all H;
preferably, R is 9 is-H;
preferably, said Y is 1 、Y 2 Are all-H.
3. The conjugate of claim 1, wherein R is 10 Selected from: -H, C 1-12 Straight chain/branched alkyl, -NO 2 Preferably, R 10 Selected from: -H, -CH 3 、-NO 2 More preferably, R 10 is-H.
4. The conjugate of claim 1, wherein Y is 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, preferably, Y 3 、Y 4 Are all-CH 3 。
5. The conjugate of claim 1, wherein m is 1 、m 2 Each independently selected from integers of 0 to 3;
preferably, m is 1 Is 0;
preferably, m is 2 Is 2.
6. The conjugate of claim 1, wherein Xa is selected from the group consisting of:
Figure FDA0003782964760000034
Figure FDA0003782964760000035
preferably, xa is
Figure FDA0003782964760000036
7. The conjugate of claim 1, wherein X is b Selected from:
Figure FDA0003782964760000037
Figure FDA0003782964760000038
preferably, X b Is composed of
Figure FDA0003782964760000039
8. The conjugate of claim 1, having the structure of formula (ii):
Figure FDA0003782964760000041
9. the conjugate of any one of claims 1 to 8, wherein L is 1 Is a linear polyethylene glycol residue having the structure represented by the general formula (III):
Figure FDA0003782964760000042
wherein n is 1 Independently selected from integers from 1 to 30, preferably from 1 to 25, more preferably from 4 to 24, and especially 8.
10. The conjugate of any one of claims 1 to 8, wherein a is selected from the group consisting of:
Figure FDA0003782964760000043
wherein R is 12 Selected from the group consisting of: c 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical, C 3-12 Heterocycloalkyl and C 6-12 One of the heteroaryl groups.
11. The conjugate of claim 1, wherein a is
Figure FDA0003782964760000044
Preferably, A is
Figure FDA0003782964760000045
12. The conjugate of claim 1, wherein X is 2 Is selected from the group consisting of C 1-12 Straight chain/branched alkyl and
Figure FDA0003782964760000051
preferably, said X 2 Is composed of
Figure FDA0003782964760000052
More preferably, X is 2 Comprises the following steps:
Figure FDA0003782964760000053
13. the conjugate of claim 1, wherein B is selected from the group consisting of:
Figure FDA0003782964760000054
preferably, said B is selected from:
Figure FDA0003782964760000055
Figure FDA0003782964760000056
preferably, said B is selected from:
Figure FDA0003782964760000057
Figure FDA0003782964760000058
preferably, B is
Figure FDA0003782964760000059
14. The conjugate of claim 1, wherein L is 2 Selected from:
Figure FDA0003782964760000061
Figure FDA0003782964760000062
preferably, said L 2 Selected from:
Figure FDA0003782964760000063
Figure FDA0003782964760000064
preferably, said L 2 Selected from:
Figure FDA0003782964760000065
Figure FDA0003782964760000071
preferably, said L 2 Is composed of
Figure FDA0003782964760000072
15. The conjugate of claim 1, wherein the drug molecule is selected from the group consisting of: amastatin, auristatin, calicheamicin, camptothecin derivatives and metabolites (SN-38), cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, geldanamycin, maytansine, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoids, or derivatives of any of the foregoing;
preferably, the drug molecule is selected from: one of SN38 and its derivatives, dxd, MMAE, MMAF, MMAD, PBD and its derivatives, calicheamicin and TPL.
16. The conjugate of claim 1, wherein the drug molecule is selected from the group consisting of:
Figure FDA0003782964760000073
preferably, the drug molecule is
Figure FDA0003782964760000074
17. The conjugate of claim 1, wherein the conjugate is selected from the group consisting of:
Figure FDA0003782964760000081
Figure FDA0003782964760000091
Figure FDA0003782964760000101
preferably, the conjugate is
Figure FDA0003782964760000102
18. An antibody drug conjugate having the structure of formula (iv):
Figure FDA0003782964760000103
wherein Ab is an antibody, and the antibody comprises a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody, and more preferably an internalized monoclonal antibody;
l is an integer from 1 to 50;
L 3 is composed of
Figure FDA0003782964760000111
Wherein, Z 1 Is an integer of 1 to 10;
L 4 is selected from
Figure FDA0003782964760000112
Wherein n is 2 An integer selected from 1 to 30;
q' is selected from
Figure FDA0003782964760000113
Figure FDA0003782964760000114
Figure FDA0003782964760000115
One of (a) and (b);
R 5 and R 6 Each independently of the other having-X 1 —Q 1 Structure of (1), Q 1 Selected from-H, -F, -Cl, -Br, -I, -SO 2 、-NO 2 、C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical, X 1 Selected from the group consisting of single bonds, -O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000116
And
Figure FDA0003782964760000117
or a combination thereof;
x is a linking group, X is
Figure FDA0003782964760000118
Wherein m is 1 、m 2 Each independently selected from integers of 0 to 12, xa, X b Each independently selected from: -O-, -S-, C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000121
Figure FDA0003782964760000122
Figure FDA0003782964760000123
One of (1), R 9 Selected from the group consisting of: -H, C 1-10 Straight/branched alkyl;
L 1 selected from: one of a linear, Y-shaped, and multi-branched polyethylene glycol residue;
L 2 is a linking group of
Figure FDA0003782964760000124
Wherein A is a glucuronide or a glucuronide derivative;
the B is selected from:
Figure FDA0003782964760000125
Figure FDA0003782964760000126
wherein, Y 1 、Y 2 Each independently selected from: -H, halogen, -OC 1-10 Alkyl radical, C 1-10 Straight chain/branched alkyl, C 3-10 Cycloalkyl, -OH,
Figure FDA0003782964760000127
Y 3 、Y 4 Each independently selected from: -H, C 1-10 Straight chain/branched alkyl, C 3-10 A cycloalkyl group, a,
Figure FDA0003782964760000128
Figure FDA0003782964760000131
X 2 Selected from the group consisting of-O-, -S-, and C 1-12 Straight chain/branched alkyl, C 3-12 Cycloalkyl radical, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000132
Figure FDA0003782964760000133
Figure FDA0003782964760000134
And
Figure FDA0003782964760000135
or a combination thereof;
R 10 selected from: -H, C 1-12 Straight chain/branched alkyl, C 1-12 Alkoxy, -NO 2 、-CN、-F、-Cl、-Br、-I、-SO 2 、C 3-12 Cycloalkyl, C 6-12 Aromatic ring radical,
Figure FDA0003782964760000136
Figure FDA0003782964760000137
R 11 Selected from: -H, C 1-10 A linear/branched alkyl group,
Figure FDA0003782964760000138
D is a drug molecule;
preferably, the antibody drug conjugate has a structure represented by formula (V):
Figure FDA0003782964760000139
wherein i is selected from an integer of 1 to 20;
preferably, the antibody drug conjugate has a structure represented by formula (vi):
Figure FDA0003782964760000141
19. a method of preparing an antibody drug conjugate of claim 18, the method comprising the following synthetic route:
Figure FDA0003782964760000142
wherein i is an integer of 1 to 20.
20. Use of the conjugate of claim 1 or the antibody drug conjugate of claim 18 for the prevention and/or treatment of a disease, which is cancer, infection with a pathogenic organism, or an autoimmune disease.
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