CN115505028A - Branched polyethylene glycol linker and application thereof - Google Patents

Abstract

The invention relates to a branched polyethylene glycol linker, which has a structure shown in a formula (I):

the linker can be connected with two identical or different cytotoxic drugs, so that the drug loading of the antibody conjugate drug is improved, and the construction of the high-DAR antibody conjugate drug can be realized; meanwhile, the introduction of the PEG structure increases the hydrophilicity of the compound, and adjusts the physicochemical property of the antibody coupled drug, thereby affecting the drug effect and pharmacokinetics of the antibody coupled drug; the connecting chain takes DBCO as a joint, and can be coupled with modified azide groups on an antibody through a simple SPAAC reaction to obtain the antibody coupling drug with high uniformity.

Description

A branched polyethylene glycol linker and its use

technical field

The invention belongs to the field of medicine, and in particular relates to a branched polyethylene glycol linker and its application.

Background technique

和

等14个药物由FDA批准上市，近年来ADC药物发展迅速，目前大约有110个ADC药物处于临床试验中。Antibody drug conjugates (Antibodydrugconjugate, ADC) is a new type of targeted therapy drug, which is composed of a monoclonal antibody targeting a specific antigen and a small molecule cytotoxic drug coupled through a linker, which combines antibodies and cells Compared with traditional small molecule antineoplastic drugs, it has the characteristics of strong targeting, high cytotoxicity, long degradation half-life, and low toxic and side effects. along with

and

14 other drugs have been approved by the FDA. In recent years, ADC drugs have developed rapidly. Currently, about 110 ADC drugs are in clinical trials.

ADC generally consists of three parts: antibody, small molecule cytotoxin and linker. Among them, the design of the linker is of great significance to the ADC drug, which must keep the drug stable in the blood circulation system and release the active toxin quickly and effectively after reaching the target tissue. In the development of ADC linkers, there are many important considerations, including the conjugation site of the antibody, the average number of cytotoxins per molecule of antibody (drug to antibody ratio, DAR), the cleavability and hydrophilicity of the linker, etc. Linkers can be divided into two categories: cleavable linkers and non-cleavable linkers, and cleavable linkers are divided into pH-sensitive and enzyme-sensitive types according to the different cleavage mechanisms. Currently, enzyme-sensitive linkers have become the mainstream choice for ADCs. The more mature enzyme-sensitive linker is a dipeptide linker dependent on cathepsin B cleavage, such as Valine-Citriline. Most current ADCs share common structural features, such as linkage via maleimide linkers. The published linkers of ADC drugs have problems such as relatively simple design and poor water solubility.

Regarding the coupling method of toxins and antibodies, currently marketed ADC drugs are mostly randomly coupled with lysine or cysteine. Due to the uncertainty of the number and sites of coupling, the generated antibody-drug conjugates are inconsistent. Uniformity, which in turn leads to inhomogeneity in pharmacokinetic properties, potency and toxicity among the components of the product. In order to solve the problem of ADC uniformity, site-specific coupling technology has gained more favor in recent years, such as GlycoConnect technology based on natural glycosylation sites. After cleavage and connection of the azide group, the azide group reacts with the cyclooctyne on the linker by SPAAC (strain-promotedcopper-freeclick) to form a uniform conjugate with a DAR value of 2.

Patent document CN108743968A discloses a cysteine modified antibody-toxin conjugate, the antibody is a cysteine site-directed insertion antibody, and the cysteine insertion site contains kappa/λ light chain constant region light chain and IgG anti- There are two types of heavy chains in the constant region of the heavy chain.

However, the antibody-toxin conjugates introduced by genetic engineering introduce uncertainty in the drug, and it is necessary to use a reducing agent to reduce the antibody, release the shield on the modified cysteine residue on the antibody, and pass through cation exchange chromatography or ultrasonography. Removing DTT and shielding materials by means of filtration and replacement, and then using an oxidizing agent to oxidize the antibody to reconnect the interchain disulfide bonds of the antibody, the cumbersome and complicated process limits the application of this invention.

Patent document CN103083680B discloses a drug conjugate having a general structural formula of polyethylene glycol-amino acid oligopeptide-irnotecan. In the conjugate, each end group of polyethylene glycol can be connected with multiple irinotecans through amino acid oligopeptides, and the drug loading rate is greatly improved. The modification of the hydrophilic polymer can protect inotecan, improve drug absorption, prolong action time, enhance curative effect, reduce dosage and avoid toxic and side effects.

However, the homogeneity of the antibody-drug conjugate is not high, which leads to inhomogeneity in pharmacokinetic properties, potency and toxicity among the components of the product, poor reproducibility of product batches, and low therapeutic index.

In view of the defects in the above-mentioned existing technologies such as poor uniformity of antibody-drug conjugates, single linker design, and poor water solubility, it is urgent to develop new linker technologies. The polyethylene glycol group is introduced to increase the hydrophilicity of the linker, and the branched linker can also achieve the purpose of increasing the drug loading. At the same time, the DBCO linker can be used to achieve fixed-point coupling and improve the uniformity of the antibody-drug conjugate.

Contents of the invention

In order to solve the problems mentioned in the prior art, the present invention provides a branched polyethylene glycol linker, which has the structure shown in formula (I):

中的一种；Wherein, the Q is selected from:

one of

R ₅ and R ₆ each independently have the structure of -X ₄ -Q ₁ , Q ₁ is selected from -H, -F, -Cl, -Br, -I, -SO ₂ , -NO ₂ , C _1-12 chains Alkyl, C _3-12 cycloalkyl, C _6-12 aralkyl;

及

中的一种或多种组成的组；X _is selected from single bond, -O-, -S-, C _1-12 chain alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of them;

及

中的一种或多种组成的组，其中R₉选自：-H、C_1-10直链/支链烷基；X is a linking group selected from single bond, -O-, -S-, C _1-12 straight chain/branched alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of, wherein R ₉ is selected from: -H, C _1-10 straight chain/branched alkyl;

The W ₁ and W ₂ are independently selected from single bond, linear or branched C _1-6 alkyl groups;

L _1a , L _1b , L _1c are independently selected from single bonds,

The n is an integer selected from 1 to 24;

For example, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

及

中的一种或多种组成的组；X ₁ and X ₂ are linking groups, independently selected from single bond, -O-, -S-, C _1-12 linear/branched alkylene, C _3-12 cycloalkylene, C _{6- 12} aralkylene,

and

A group consisting of one or more of them;

其中A为肽类连接子，B为自降解基团或不存在，L ₂ and L ₃ are linking groups, which are

Wherein A is a peptide linker, B is a self-degrading group or does not exist,

When B is a self-degrading group, B is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH,

Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

及

或其组合组成的组；X ₃ is selected from -O-, -S-, C _1-12 straight chain/branched chain alkyl, C _3-12 cycloalkyl, C _6-12 aromatic ring group,

and

or a combination thereof;

R ₂₀ is selected from: -H, C _1-12 straight chain/branched chain alkyl, C _1-12 alkoxy, -NO ₂ , -CN, -F, -Cl, -Br, -I, -SO ₂ , C _3-12 cycloalkyl, C _6-12 aromatic ring group,

A is a peptide linker, including one or more identical or different polypeptide residues, and said polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives.

优选的，所述Q为

Further, the Q is

Preferably, the Q is

The R ₅ and R ₆ are independently selected from H and C _1-3 alkyl groups, preferably, both R ₅ and R ₆ are H;

C_1-12直链/支链亚烷基、

组成的组，其中R₉为H、C_1-3直链/支链烷基；Said X is selected from

C _1-12 straight chain/branched chain alkylene,

The group consisting of, wherein R ₉ is H, C _1-3 straight chain/branched alkyl;

所述R₇为C_1-12的亚烷基；更优选的，所述R₇为C_1-4的亚烷基；Preferably, the X is

The R ₇ is a C _1-12 alkylene group; more preferably, the R ₇ is a C _1-4 alkylene group;

The L _1a , L _1b , L _1c are independently selected from single bonds,

The W ₄ is selected from a single bond, C _1-6 linear or branched alkylene;

Preferably, said n is an integer selected from 1 to 12;

The amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine , lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine and cystine One or a combination of two or more;

Preferably, the amino acid is selected from one or more of valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine and arginine A combination of two or more;

More preferably, the polypeptide residues include one of valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine.

Further, when B is a self-degrading group, said A is selected from

Wherein, A ₁ , A ₂ , A ₃ ... A _y are the same or different, and the structures of A ₁ , A ₂ , A ₃ ... A _y are independently selected from: single bond,

The y is an integer selected from 1 to 5;

The j is an integer selected from 1 to 10;

For example, j can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, the j is an integer of 2-4;

中的一种；The Y _a is selected from -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH, -(C _1-6 alkylene base)-OH, -(C _1-6 alkylene)-SH, -(CH ₂ ) _1-6 -cycloalkyl, -(C _1-6 alkylene)-aromatic ring group, -(C _{1 -6} alkylene)-heterocycloalkyl, -(C _1-6 alkylene)-heterocyclic aryl, -(C _1-6 alkylene)-COOH, -(C _1-6 alkylene )-CONH ₂ , -(C _1-6 alkylene)-NH ₂ and

one of

结构；Said A ₁ , A ₂ , A ₃ ... A _y has at least one

structure;

-(C_1-6亚烷基)-芳环基；Preferably, said Y _a is selected from: -H, -C _1-6 straight chain/branched chain alkyl,

-(C _1-6 alkylene)-aromatic ring group;

More preferably, the Y _a is selected from: -H, methyl, ethyl, propyl, isopropyl,

Further, said B does not exist or is selected from:

Preferably, said B is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH;

Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

Further, the Q is

The X is

Said L _1a , L _1b , L _1c are independently selected from a single bond or

Said A is selected from

或不存在。Said B is:

or does not exist.

Further, said L ₂ and L ₃ are independently selected from:

Further, the linker is selected from:

Another aspect of the present invention also provides a branched polyethylene glycol linker-drug conjugate, which has the structure shown in formula (II):

中的一种；Wherein, the Q is selected from:

one of

R ₅ and R ₆ each independently have the structure of -X ₄ -Q ₁ , Q ₁ is selected from -H, -F, -Cl, -Br, -I, -SO ₂ , -NO ₂ , C _1-12 chains Alkyl, C _3-12 cycloalkyl, C _6-12 aralkyl;

及

中的一种或多种组成的组；X _is selected from single bond, -O-, -S-, C _1-12 chain alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of them;

及

中的一种或多种组成的组，其中R₉选自：-H、C_1-10直链/支链烷基；X is a linking group selected from single bond, -O-, -S-, C _1-12 straight chain/branched alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of, wherein R ₉ is selected from: -H, C _1-10 straight chain/branched alkyl;

The W ₁ and W ₂ are independently selected from single bond, linear or branched C _1-6 alkyl groups;

L _1a , L _1b , L _1c are independently selected from single bonds,

The n is an integer selected from 1 to 24, and may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

及

中的一种或多种组成的组，其中R₁₀选自：-H、C_1-10直链/支链烷基；X ₁ and X ₂ are linking groups, independently selected from single bond, -O-, -S-, C _1-12 linear/branched alkylene, C _3-12 cycloalkylene, C _{6- 12} aralkylene,

and

A group consisting of one or more of, wherein R ₁₀ is selected from: -H, C _1-10 straight chain/branched alkyl;

其中A’为肽类连接子，B’为自降解基团，B’选自：

其中，Y₁、Y₂各自独立地选自：-H、卤素、-OC_1-10烷基、C_1-10直链/支链烷基、C_3-10环烷基、-OH、

L ₂ ', L ₃ ' are linking groups, which are

Wherein A' is a peptide linker, B' is a self-degrading group, and B' is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH,

Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

及

或其组合组成的组；X ₃ is selected from -O-, -S-, C _1-12 straight chain/branched chain alkyl, C _3-12 cycloalkyl, C _6-12 aromatic ring group,

and

or a combination thereof;

R ₂₀ is selected from: -H, C _1-12 straight chain/branched chain alkyl, C _1-12 alkoxy, -NO ₂ , -CN, -F, -Cl, -Br, -I, -SO ₂ , C _3-12 cycloalkyl, C _6-12 aromatic ring group,

W ₃ is C _1-6 straight chain/branched chain alkylene;

The peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives;

Preferably, the amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Citrulline, Ornithine, and Cystine One or more combinations of acids, preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, One or more combinations of leucine and arginine; more preferably, the polypeptide residues include valine-citrulline, valine-alanine and glycine-glycine-benzene One of alanine-glycine;

The D is a drug molecule.

优选的，所述Q为

Further, the Q is

Preferably, the Q is

The R ₅ and R ₆ are independently selected from H and C _1-3 alkyl groups, preferably, both R ₅ and R ₆ are H;

C_1-12直链/支链亚烷基、

组成的组，其中R₉为H、C_1-3直链/支链烷基；Said X is selected from

C _1-12 straight chain/branched chain alkylene,

The group consisting of, wherein R ₉ is H, C _1-3 straight chain/branched alkyl;

所述R₇为C_1-12的亚烷基；更优选的，所述R₇为C_1-4的亚烷基；Preferably, the X is

The R ₇ is a C _1-12 alkylene group; more preferably, the R ₇ is a C _1-4 alkylene group;

The L _1a , L _1b , L _1c are independently selected from single bonds,

The W ₄ is selected from a single bond, C _1-6 straight chain or branched alkyl;

Preferably, the n is an integer selected from 1-12, for example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

For example, said A' is selected from

Wherein, A ₁ , A ₂ , A ₃ ... A _y are the same or different, and the structures of A ₁ , A ₂ , A ₃ ... A _y are independently selected from: single bond,

The y is an integer selected from 1 to 10;

For example, the y can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, the y is an integer of 2 to 4;

The j is an integer selected from 1 to 10;

For example, the j may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, the j is an integer of 2-4;

中的一种；The Y _a is selected from -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH, -(C _1-6 alkylene base)-OH, -(C _1-6 alkylene)-SH, -(CH ₂ ) _1-6 -cycloalkyl, -(C _1-6 alkylene)-aromatic ring group, -(C _{1 -6} alkylene)-heterocycloalkyl, -(C _1-6 alkylene)-heterocyclic aryl, -(C _1-6 alkylene)-COOH, -(C _1-6 alkylene )-CONH ₂ , -(C _1-6 alkylene)-NH ₂ and

one of

结构；Said A ₁ , A ₂ , A ₃ ... A _y has at least one

structure;

-(C_1-6亚烷基)-芳环基；Preferably, said Y _a is selected from: -H, -C _1-6 straight chain/branched chain alkyl,

-(C _1-6 alkylene)-aromatic ring group;

More preferably, the Y _a is selected from: -H, methyl, ethyl, propyl, isopropyl,

The B' is selected from:

其中，Y₁、Y₂各自独立地选自：-H、-OC_1-10烷基、C_1-10直链/支链烷基、-OH；Y₃、Y₄各自独立地选自：-H、C_1-10直链/支链烷基；

Wherein, Y ₁ , Y ₂ are each independently selected from: -H, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, -OH; Y ₃ , Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl;

W ₃ is selected from C _1-6 straight chain/branched chain alkylene;

Preferably, Y ₁ , Y ₂ , Y ₃ , and Y ₄ are each independently selected from H, C _1-5 straight chain/branched chain alkyl.

Further, the Q is

The X is

Said L _1a , L _1b , L _1c are independently selected from a single bond or

The A' is selected from

The B' is selected from:

Further, said L ₂ ' and L ₃ ' are independently selected from:

Further, the D is a chemotherapeutic agent, selected from: alkylating agents, nitrogen mustards: chlorpheniramine, chlorpronazine, cyclophosphamide, dacarbazine, estramustine, Ifosfamide, nitrogen mustard, dimethoxamine hydrochloride, dinitrogen mustard, amlodipine hydrochloride, mycophenolic acid, dulcitol, pipbromide, new nitrogen mustard, benzene mustard cholesterol, turpentine mustard, thiotegua, trifosamide, uracil; CC-1065 and adolaisine, cardzelesine, bizelesine or their synthetic analogues; duocamycin and its synthetic analogues, KW- 2189 or CBI-TMI, benzodiazepine dimer or pyrrolobenzodiazepine (PBD) dimer, tomemycin dimer, indolobenzodiazepine dimer, imidazobenzene Dimers of thiadiazepines, or dimers of oxazolidinebenzodiazepines; nitrosoureas: including carmustine, lomustine, clomustine, formustine , nimustine, lamustine; alkyl sulfonates: including basiphen, shusuphen, sulfaprosulfone, and pisuphen; triazenes or dacarbazine; platinum-containing compounds: including carboplatin , cisplatin, oxaliplatin; aziridines, chromanone, caloxone, methutepa, and uredopa; ethyleneimine and methylmelamine, including hexamethylmelamine, Sanya Ethyltriamine, triethylphosphoramide, triethylenethiophosphoramide and trimethylolmethylamine;

The D is a plant alkaloid: selected from vinca alkaloids, including vincristine, vinblastine, vindesine, vinorelbine, norvinblastine; paclitaxel: including paclitaxel, docetaxel and analogs thereof; Maytansinoids include DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine, ansamycin and their analogs); cryptophycin (including cryptophycin1 and cryptophycin8); Molide, bryostatin, dolastatin, auristin, tubulysins, cephalostatin; pancratistatin; erbulins; sarcodictyn; spongostatin;

The D is a DNA topoisomerase inhibitor: selected from etoposide tinib, including 9-aminocamptothecin, camptothecin, clinator, doramycin, etoposide, etoposide phosphate, Irinotecan, exinotecan, Dxd, SN38, mitoxantrone, nodiclofen, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin or RFS2000, Mitomycin and its analogues;

Said D is selected from antifolates (DHFR inhibitors, including methotrexate, trimact, dimethyl folic acid, pteroxin, aminopterin (4-aminobenzoic acid) or other folic acid analogues); IMP Dehydrogenase inhibitors (including mycophenolic acid, thiazofurin, ribavirin, EICAR); ribonucleotide reductase inhibitors (including hydroxyurea, deferoxamine); pyrimidine analogs: uracil analogs (including ancitabine, azacitidine, 6-azuracil, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enoxitabine , 5-fluorouracil, floxuridine, ratitrexed (Tomudex)) and cytosine analogs (including cytarabine, cytosine arabinoside, fludarabine); purine analogs (including azathioprine, fludarabine , mercaptopurine, thiamine, thioguanine); folic acid supplements, florinic acid; and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT);

The D is a hormone therapy agent: selected from receptor antagonists: antiestrogens (including megestrol, raloxifene, tamoxifen), LHRH stimulants (including gostaline, leuprolide acetate Lin); anti-androgen drugs (including bicalutamide, flutamide, caluspristone, betandrosterone propionate, epiandrosterol, goserelin, leuprolide, metilidine, nitric acid Luthimide, testolide, trilosteine and other androgen inhibitors); retinoids: vitamin D3 analogs (including CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (including verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocretin A); cytokines (interferon-α, interferon-γ, tumor necrosis factor (TNF), TNF-containing human protein);

The D is a kinase inhibitor selected from BIBW2992 (anti-EGFR/Erb2), imatinib, gefitinib, guagatanib, sorafenib, dasatinib, sunitinib, erb2 Lotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406 ), bosutinib (SKI-606), cabozantinib, vimodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab Anti-, trastuzumab, ranibizumab, panitumumab, Espins;

Described D is poly (ADP-ribose) polymerase (PARP) inhibitor, is selected from Olapari, Nirapari, Inipari, Tarazopari, Velipari, Velipari , CEP9722 (Cephalon), E7016 (Eisai), BGB-290 (Beigene), 3-aminobenzamide;

Said D is an antibiotic: selected from enediyne antibiotics (selected from calicheamicin, calicheamicin γ1, δ1, α1 and β1, dynemycin, including dynemycin A and deoxyoryzae esperamycin, catamycin, C-1027, maduropeptin, or neocarbazine Austin and related chromopin enediyne antibiotics), aclacinomysins, actinomycin, antramycin, azaserine, Bleomycin, kanomycin, karamycin, carmine, carcinophilin, chromomycin, daunorubicin, daunorubicin, dedaunorubicin, 6-diazo-5-oxo Di-L-norleucine, doxorubicin, morpholine-doxorubicin, cyanomorpholine-doxorubicin, 2-pyrroline doxorubicin and deoxydaunorubicin, epirubicin, a Ruubicin, idarubicin, marcomycin, nitomycin, mycophenolic acid, nogamycin, olivine, peplomycin, potfiromycin, puromycin, quilamycin, rodaumycin, streptomycin streptozotocin, tuberculin, ubenimex, netastatin, zorubicin;

The D is a polyketide compound (Annonaline), bullatacin and bullatacinone; gemcitabine, cyclooxygenase and carfilomib, bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allowctin-7, Xegeva, Provenge, Yervoy, prenylation inhibitors and lovastatin, dopaminergic neurotoxin (selected from staurosporine), actinomycin (contains Actinomycin D, dactinomycin), amanitin, bleomycin (including bleomycin A2, bleomycin B2, pelomycin), anthracyclines, including daunorubicin, Adriamycin (adriamycin), idarubicin, epirubicin, zorubicin, mitoxantrone, MDR inhibitors (or verapamil), Ca2+ATPase inhibitors (or thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, entinostat, SB939, Resminostat, Givinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); celecoxib, glitazones, epigallocatechin gallate, disulfiram, Salinosporamide A); anti Adrenal drug selected from the group consisting of: aminoglutethimide, mitotane, trolosteine, aceglucuronide, aldophosphamide, aminolevulinic acid, amsacrine, arabinoside, bestrabucil, bisantrene, edatraxate, defofamine, methacin, decquinone, eflornithine (DFMO), elfomithine, etricetium, etoglu, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, clonida Ming, Mitoguanidine Hydrazone, Mitoxantrone, Mopraditol, Diamine Niacridine, Pentostatin, Methionine, Pirarubicin, Podophyllic Acid, 2-Ethylhydrazine, Procarbazine ; piperazine diketopane; rhizoctidine; sizole; spirogermanium; A, bacitracin A and anguidine), polyurethane, siRNA, antisense drugs;

The D is an autoimmune disease drug selected from cyclosporin, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (including Amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flurcoron (danazol), dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA , etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus;

The D is an anti-infective disease drug, including aminoglycosides: selected from amikacin, asemicin, gentamicin (netilmicin, sisomicin, isopamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribomycin ), netilmicin, spectinomycin, streptomycin, tobramycin, methylzithramycin;

The D is an anti-infective disease drug, including amide alcohols: selected from azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol;

The D is an anti-infective disease drug, including ansamycin: selected from geldanamycin or herbimycin;

The D is an anti-infective disease drug, including carbapenems: selected from biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem ;

The D is an anti-infective disease drug, including cephems: selected from carbacephem (loracarb), cefuroxime, ampicillin, cephradine, cefadroxidine, cefuroxime, cefotaxime, cephalothin or Cephalosporin, cephalexin, cephalosporin, cefamandole, cefapirin, cephalosporin oxamazole, cephalosporin fluxazone, fenidone, cephalosporin oxazoline, cefbuperazone, cefcapine, cephalosporin Daxime, cefepime, cefixime, cefoxitin, cefprozil, cefmethoxam, ceftezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, ceftazidime The United States, cefmenoxime, cefodizime, cefanixime, cefoperazone, cefreide, cefotaxime, cefazolin, cefazolan, cephalexin, cefamizole, cefpiramide, cefpirin Luo, cefpodoxime, cefprozil, cefquinol, cefsulodin, ceftazidime, cefditoren, ceftibuten, cefotaxime, ceftizoxime, ceftazidime, ceftriaxone, cefuroxime, ceftizole South, cephamycin (cefoxitin, cefotetan, ceftriaxone), oxygen (carbon) cephem (fluoxyceph, latamoxef);

The D is an anti-infective disease drug, including glycopeptide: selected from bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), remora rather;

The D is an anti-infective disease drug, including glycylcycline: such as tigecycline;

The D is an anti-infective disease drug, including a β-lactamase inhibitor: selected from penicillane (sulbactam, tazobactam), oxypenicillane (clavulanic acid);

The D is an anti-infective disease drug, including lincosamide: selected from clindamycin or lincomycin;

The D is an anti-infective disease drug, including lipopeptide: selected from daptomycin, A54145, calcium-dependent antibiotics (CDA);

The D is an anti-infective disease drug, including macrolides: selected from azithromycin, clindamycin, clarithromycin, dirithromycin, erythromycin, fluremycin, josamycin, ketone Esters (telithromycin, erythromycin), midecamycin, mikamycin, oleandomycin, rifamycins (isoniazid, rifampicin, rifabutin, rifapentine), Piramycin, Roxithromycin, Spectinomycin, Spiramycin, Tacrolimus (FK506), Troleandomycin, Telithromycin;

The D is an anti-infective disease drug, including monocyclic amine: selected from aztreonam or tigemonan;

The D is an anti-infective disease drug, including oxazolidinones: such as linezolid;

The D is an anti-infective disease drug, including penicillins: selected from amoxicillin, ampicillin (piamicillin, heroicillin, bamicillin, ampicillin, doxorubicin), amoxicillin, azlocillin , benzathine penicillin, benzathine penicillin benzathine penicillin, benzathine penicillin phenoxymethyl penicillin, clotillin, procaine penicillin (methenicillin), mezlocillin, methicillin, nafcillin, oxacillin, Acemethicillin, penicillin, phenecillin, phenoxymethylpenicillin, gualacillin, ampicillin, sulfbenzillin, temoxicillin, ticarcillin;

The D is an anti-infectious disease drug, including a polypeptide: selected from bacitracin, colistin, and polymyxin B;

The D is an anti-infective disease drug, including quinolones: selected from alatrafloxacin, baloxacin, ciprofloxacin, Kelinsha, dafloxacin, difloxacin, enoxacin, Enrofloxacin, Garafloxacin, Gatifloxacin, Gemifloxacin, Gepafloxacin, Canotrovafloxacin, Levofloxacin, Lomefloxacin, Mabofloxacin, Moxifloxacin, Nafloxacin , Norfloxacin, Orbifloxacin, Ofloxacin, Pefloxacin, Trovafloxacin, Gepafloxacin, Sitafloxacin, Sparfloxacin, Temafloxacin, Tofloxacin, Qu Valfloxacin;

The D is an anti-infective disease drug, including streptavidin: selected from prunamycin, quinupristin/dalfopristin;

The D is an anti-infective disease drug, including sulfonamides: selected from ambenzylsulfonamide, azosulfonamide, sulfadiazine, sulfamethoxazole, sulfimide, sulfapyridine, sulfisoxazole, trimethoprim, sulfamethoxazole Methoxazole (compound sulfamethoxazole);

The D is an anti-infective disease drug, including a steroid antibacterial drug: such as fusidic acid;

The D is an anti-infective disease drug, including tetracyclines: selected from doxycycline, aureomycin, clomixicycline, demecycline, ramoxiline, mexicycline, metacycline, Minocycline, oxytetracycline, pamecycline, pyrrolidinylmethyltetracycline, tetracycline, glycylcycline (such as tigecycline);

Said D is an anti-infective disease drug, including other types of antibiotics: anemone, arsenadine, bacterial terpene alcohol inhibitor (bacitracin), DANAL/AR inhibitor (cycloserine), dictyostatin, and disc sponge Lactone, soft coralline, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthesis inhibitor (e.g. fosfomycin), nitrofurantoin, paclitaxel, prancimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin, tazobactantinidazole, sclerosin;

The D is an antiviral drug, including an invasion/fusion inhibitor: selected from apavero, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO140 or CD4 (ibalizumab);

The D is an antiviral drug, including an integrase inhibitor: selected from Raltegravir, elvite-gravir or globoidnanA;

The D is that the D is an antiviral drug, including a maturation inhibitor: selected from bevirimat or vivecon;

The D is an antiviral drug, including a neuraminidase inhibitor: selected from oseltamivir, zanamivir or peramivir;

The D is an antiviral drug, including nucleosides and nucleotides: selected from abacavir, aciximavir, adefovir, amoxivir, abciximab, brovudine, cidofo vir, clavudine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluracillin (5-FU), 3'-fluoro substituted 2', 3'-Deoxynucleoside analogs, including 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), Fumi Weisheng, 9-guanine, iodine, lamivudine (3TC), 1-nucleosides (including β-1-thymidine and β-1-2'-deoxycytidine), penciclovir, racivir , ribavirin, detidine, stavudine (d4T), talibavirin (viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclo Wei, zalcitabine (ddC) or zidovudine (AZT);

The D is an antiviral drug, including non-nucleosides: selected from amantadine, atipyridine, capravirine, diarylpyrimidine (etravirine, rilpivirine), delavudine, docosanol , emivirine, efavirenz, foscarnet (phosphoroformic acid), imiquimod, peginterferon, loviramide, lord adenosine, inthizone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848) or amantadine;

The D is an antiviral drug, including protease inhibitors: selected from amprenavir, atazanavir, boceprevir, darunavir, fosanavir, indinavir, lopinavir, nelfinavir, Leconali, ritonavir, saquinavir, telaprevir (VX-950) or tipranavir;

The D is an antiviral drug, including other types of antiviral drugs: such as abzyme, arbidol, calanolidea, ceragenin, cyanverine-n, diarylpyrimidine, epigallocatechin gallate (EGCG ), foscarnet, griffithine, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, mixed inhibitors, ribavirin or seliciclib;

The D is a radioisotope, which can be selected from (radionuclide) 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Ga, 86Y, 99Tc, 111In, 123I, 124I, 125I, 131I, 133Xe, 177Lu, 211At , or 213Bi.

The D is a chromophore molecule capable of absorbing ultraviolet rays, fluorescence, infrared light, near-infrared light, and visible light; yellow pigments, red blood cells, iridescent pigments, white blood cells, melanin, and a class or a subclass of blue-green pigments, fluorescent molecules absorb Fluorescent chemicals that glow after light. Visual phototransduction molecules, fluorophore molecules, luminescent molecules, luciferin compounds. Non-proteinaceous organic fluorophores selected from the group consisting of: xanthene derivatives (including fluorescein, rhodamine, Oregon green, eosin and Texas red); cyanine derivatives (including cyanine, indocarbocyanine, oxacyanines, thiocyanines, and merocyanines); squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; naphthalene derivatives (including dansyl and sodium fluorosilicate derivatives); Coumarin derivatives; oxadiazole derivatives (including pyridyloxazole, nitrobenzoxazole, and benzoxadiazole); anthracene derivatives (including anthraquinones, including DRAQ5, DRAQ7, and CyTRAK orange); Pyrene derivatives (including cascade blue, etc.); oxazine derivatives (including Nile red, Nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (including flavin, acridine orange, acridine yellow, etc.); arylmethylamine derivatives (including auramine, crystal violet, malachite green) and tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin).

Said D is a chromogenic molecule selected from any analog and derivative of the following fluorescent compounds: CF dyes, DRAQ and CyTRAK probes, BODIPY, AlexaFluor, DyLightFluor, Atto and Tracy, FluoProbes, AbberiorDyes, DY and MegaStokesDyes, SulfoCy dyes , HiLyteFluor, Seta, SeTau and SquareDyes, Quasar and CalFluordyes, SureLightDyes (APC, RPEPerCP, Phycobilisomes), APC, APCXL, RPE, BPE, Allophycocyanin (APC), Aminonopain, APC-Cy7 Conjugate, BODIPY -FL, CascadeBlue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, F Fluorescein, FluorX, Hydroxycoumarin, Lissamine Rhodamine B, Fluorescent Yellow, Me-Methoxy Coumarin, NBD, PacificBlue, PacificOrange, PE-Cy5 Conjugate, PE-Cy7 Conjugate, PerCP, R-Phycoerythrin (PE), Red613, Seta-555-Azide, Seta-555-DBCO , Seta-555-NHS, Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-670, Seta-380-NHS , SeTau-405-maleimide, SeTau-405-NHS, SeTau-425-NHS, SeTau-647-NHS, TexasRed, TRITC, TruRed, X-Rhodamine, 7-AAD (7-aminoactinomycin D, CG-selective), acridine orange, chromomycin A3, CyTRAK orange (reexcitationdark), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst33258, Hoechst33342, LDS751, mithromycin, propidium iodide ( PI), SYTOX blue, SYTOX green, SYTOX orange, thiazole orange, TO-PRO: cyanine dye monomer, TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, YOSeta-1, YOYO- 1. Fluorescent compounds: including DCFH (2', 7'-dichlorodihydrofluorescein, oxidized form), DHR (dihydrorhodamine 123, oxidized form, photocatalytic oxidation), Fluo-3 (AM ester, pH> 6), Fluo-4 (AM ester, pH7.2), Indo-1 ( AM Ester, Low/High Calcium (Ca2+), SNARF (pH6/9), Allophycocyanin (APC), AmCyan1 (Tetramer, Clontech), AsRed2 (Tetramer, Clontech), AzamiGreen (Monomer) , Azurite, B-phycoerythrin (BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP"), EBFP, EBFP2, ECFP, EGFP (weak dimer), Emerald (weak dimer ), EYFP (weak dimer), GFP (S65A mutation), GFP (S65C mutation), GFP (S65L mutation), GFP (S65T mutation), GFP (Y66F mutation), GFP (Y66H mutation), GFP (Y66W mutation ), GFPuv, HcRed1, J-Red, Katusha, KusabiraOrange (monomer, MBL), mCFP, mCherry, mCitrine, MidoriishiCyan (dimer, MBL), mKate (TagFP635, monomer), mKeima-Red (monomer) , mKO, mOrange, mPlum, mRaspberry, mRFP1 (monomer), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome complex), peridinoxanthin-chlorophyll-protein complex (PerCP), R-phycoerythrin (RPE ), T-Sapphire, TagCFP (dimer), TagGFP (dimer), TagRFP (dimer), TagYFP (dimer), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer) , TurboFP635 (dimer), TurboGFP (dimer), TurboRFP (dimer), TurboYFP (dimer), Venus, WildTypeGFP, YPet, ZsGreen1 (tetramer), ZsYellow1 (tetramer) and derivative.

The D is a cell-binding ligand or receptor agonist, which can be selected from: folic acid derivatives, glutamic acid urea derivatives, somatostatin and analogs thereof (selected from octreotide (Sandostatin) and lanreotide (Somatuline) ), aromatic sulfonamides, Aromaticsulfonamides, pituitary adenylate cyclase activating peptide (PACAP) (PAC1), vasoactive intestinal peptide (VIP/PACAP) (VPAC1, VPAC2), α-melanocyte stimulating hormone (α-MSH ), cholecystokinin (CCK)/gastrin receptor agonist, bombesin (selected from Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met -NH2)/gastrin-releasing peptide (GRP), neurotensin receptor and its receptor subtypes (NTR1, NTR2, NTR3), substance P (NK1 receptor) ligand, neuropeptide Y (Y1–Y6 ), homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and polycyclic RGD peptides (selected from cRGDfV), TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor body) and F3 peptides, cell-penetrating peptides (CPPs), peptide hormones selected from luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, and gonadotropin-releasing hormone (GnRH) agonists, by targeting follicle stimulation Hormone (FSH) and Luteinizing Hormone (LH) act, and testosterone product, selected from Buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt) , Gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-gly-NH2), Goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu) -Leu-Arg-Pro-Azgly-NH2), Histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-Phenyl)-Leu-Arg-Pro-NHEt), Leuprolide Acetate Lin (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), Nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly- NH2), triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), deserelin, abarelix (Ac-D-2Nal-D -4-Chloro-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-Isopropyl-Lys-Pro-D-Ala-NH2) , Cetrorelix (Ac-D-2Nal-D-4-chloro-Phe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2 ), Degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe( Carbamoyl)-Leu-IsopropylLys-Pro-D-Ala-NH2) and Ganyrelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala- Ser-Tyr-D-(N9,N10-diethyl)-homoArg-Leu-(N9,N10-diethyl)-homoArg-Pro-D-Ala-NH2), pattern recognition receptors (PRRs), selected From Toll receptors (TLRs), C-type lectins and Nodlike receptors (NLRs), calcitonin receptor agonists, integrin receptors and their receptor subtypes (selected from αVβ1, αVβ3, αVβ5, αVβ6, α6β4 , α7β1, αLβ2, αIIbβ3) agonist (selected from GRGDSPK, cyclic (RGDfV) (L1) and its derivatives [cyclic (-N(Me)R-GDfV), cyclic (R-Sar-DfV), cyclic (RG -N(Me)D-fV), Cyclo(RGD-N(Me)f-V), Cyclo(RGDf-N(Me)V-)(Cilengitide)], Nanobodies (Derivatives of VHH (Camel Ig)), Domain antibody (dAb, derivative of VH or VL domain), bispecific T cell Engager (BiTE, bispecific diabody), double affinity retargeting (DART, bispecific diabody), tetravalent tandem antibody (TandAb , dimeric bispecific diabodies), Anticalins (derivatives of Lipocalins), Adnectins (10thFN3 (Fibronectin)), Designer Ankyrin Repeat Proteins (DARPins), Avimers, EGF receptor and VEGF receptor agonists.

Said D is a pharmaceutically acceptable salt of the above drug, acid or its derivative, hydrate or hydrated salt, or crystal structure or any optical isomer, racemate, diastereoisomer or Enantiomers.

Preferably, said D is selected from the group consisting of: amino acids, proteins, enzymes, nucleosides, sugars, organic acids, glycosides, flavonoids, quinones, terpenes, phenylpropanol phenols, steroids and their glycosides and One of the alkaloids.

More preferably, the D is a drug for killing tumor cells;

And/or, said D is preferably selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites (such as: SN-38 and derivatives thereof, 10 -Hydroxycamptothecin, 9-aminocamptothecin, 9-nitrocamptothecin), exitecan, Dxd, cryptomycin, daunomycin, dolastatin, doxorubicin, Duocamycin, Epothilone, Espamycin, Geldanamycin, Maytansine and its derivatives, Methotrexate, Monomethyl Auristatin E (“MMAE”), Monomethyl Auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizobin, SG2285, tubulysin, vindesine, toxoid, irinotecan, topotecan, belotecan, exatecan , rutotecan, diflutecan, genitecan, canitecan, doxorubicin, epirubicin, morpholino doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrole Phenyl doxorubicin, MMAD, PBD and its derivatives, TPL, taxanes, cyclomycin, bleomycin B and its derivatives, maytansine and its derivatives, dolastatin, mitomyces Aminopterin, actinomycin, bleomycin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonyl hydrazide, talithromycin, a Cytoside, etoposide, paclitaxel, laramycin, podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy One or a combination of two or more of doxorubicin and its derivatives.

In some embodiments of the present invention, the D is selected from

Further, the conjugate is selected from:

The conjugates of the present invention belong to branched monodisperse polyethylene glycol linker-drug conjugates.

Another aspect of the present invention also provides the application of the aforementioned conjugates in the preparation of drugs for the prevention and/or treatment of diseases, characterized in that the diseases are cancer, inflammation and autoimmune diseases.

Another aspect of the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned conjugate and a pharmaceutically acceptable salt, a carrier, a diluent or an excipient or a combination thereof.

The pharmaceutical composition is in the form of tablets, suppositories, pills, soft and hard gelatin capsules, powders, solutions, suspensions or aerosols.

Another aspect of the present invention also provides an application of the aforementioned conjugate in the preparation of a drug for the prevention and/or treatment of diseases, the drug has anticancer activity, specifically, the anticancer activity includes anticancer activity against intestinal cancer, ovarian cancer and Lung cancer has inhibitory activity.

The term "alkyl" appearing in the present invention can be a linear or branched alkyl group, and a typical alkyl group contains 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12) carbon atoms, namely C1-12 alkyl, such as C1-8 alkyl, C1-6 alkyl, C1-3 alkyl, examples of alkyl such as methyl, ethyl, propyl, iso Propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 2-ethylbutyl, n-pentyl, isopentyl, 1-methylpentyl, 1,3-dimethylbutyl , n-hexyl, 1-methylhexyl, n-heptyl, isoheptyl, 1,1,3,3-tetramethylbutyl, 1-methylheptyl, 3-methylheptyl, n-octyl, 2-ethylhexyl, 1,1,3-trimethylhexyl, 1,1,3,3-tetramethylpentyl, nonyl, decyl, undecyl, 1-methylundecyl , dodecyl, 1,1,3,3,5,5-hexamethylhexyl.

The term "alkylene" appearing in the present invention can be a linear or branched alkyl group, and a typical alkylene group contains 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12) carbon atoms, that is, C1-12 alkylene, such as C1-18 alkylene, C1-12 alkylene, C1-8 alkylene, C1-6 alkylene, C1- 3 Alkylene, examples of alkylene are ethylene, propylene, isopropylene, butylene, sec-butylene, isobutylene, 2-ethylbutylene, pentylene, isobutylene Pentyl, 1-methylpentylene, 1,3-dimethylbutylene, hexylene, 1-methylhexylene, heptylene, 1,1,3,3-tetramethylbutylene, 1 -Methylheptylene, 3-methylheptylene, octylene, 2-ethylhexylene, 1,1,3-trimethylhexylene, 1,1,3,3-tetramethylhexylene Branched and straight-chain isomers of pentyl, nonylene, decylene, undecylene and dodecylene.

The term "cycloalkyl" appearing in the present invention includes substituted cycloalkyl and unsaturated cycloalkyl, typical cycloalkyl contains 1 to 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10) carbon atoms, that is, C _1-10 cycloalkyl, such as C3-6 cycloalkyl, examples of cycloalkyl such as cyclopropyl, cyclobutyl, methylcyclobutyl, cyclopentyl, methyl Cyclopentyl, dimethylcyclopentyl, trimethylcyclopentyl, ethylcyclopentyl, propylcyclopentyl, isopropylcyclopentyl, cyclohexyl, methylcyclohexyl, dimethylcyclopentyl Hexyl, ethylcyclohexyl, cycloheptyl, methylcycloheptyl, cyclooctyl.

In the present invention, examples of the heterocycloalkyl group are cycloalkyl groups containing elements such as N, O, S, and Si.

The term aryl in the present invention includes full carbon aryl and heteroaryl, which can be replaced by one or more C ₁ -C ₁₂ alkyl, halogen, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkane Oxygen, C ₁ -C ₁₈ alkoxy, aryl substitution.

Among the present invention, term "DMAP" is a kind of super-strong nucleophilic acylation catalyst, and Chinese name is 4-dimethylaminopyridine, and it is widely used in the novel high-efficiency catalyst of chemical synthesis, has higher catalytic ability, to Increasing the yield has an extremely obvious effect.

In the present invention, the term "TFA" means trifluoroacetic acid.

In the present invention, the term "ADC" refers to Antibody-Drug Conjugate, antibody-drug conjugate. Antibody-drug conjugate ADC is a combination of a monoclonal antibody with a targeting effect and a compound with specific pharmacological properties (such as cytotoxicity), and the two parts are coupled into a whole through a linker.

In the present invention, the term "DAR" (Drug-to-Antibody Ratio, drug-to-antibody ratio) is a key attribute of antibody-drug conjugates and an important quality control link in the ADC drug development process.

In the present invention, the term "mAb" is an antibody.

In the present invention, the term "DCM" refers to a pure solution of dichloromethane.

In the present invention, the term "ACN" refers to a pure solution of acetonitrile.

In the present invention, the term "MPA" is mobile phase A;

In the present invention, the term "MPB" refers to mobile phase B.

In the present invention, the term "SPR" (SPR, Surface Plasmon Resonance) refers to optical surface plasmon resonance, which can obtain specific signals of binding and interaction between biomolecules by monitoring the dynamic change of the SPR angle during the biological reaction process.

In the present invention, the term "h" stands for hours.

In the present invention, the term "room temperature" means 25±5°C.

In the present invention, the term "DIEA" refers to N,N-diisopropylethylamine, which is widely used as a solvent for the synthesis of amino acid polypeptides and the like.

In the present invention, the term "DMF" means that dimethylformamide (DMF) is a transparent liquid, commonly used as a solvent, and can be miscible with water and most organic solvents.

In the present invention, the term "HATU" is a commonly used polypeptide condensation reagent, which is used in the reaction of carboxyl and amino groups to synthesize peptide bonds. Its system name is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, English name 2-(7-Azabenzotriazol-1-yl) -N,N,N',N'-tetramethyluronium hexafluorophosphate.

In the present invention, the term "MMAE" refers to monomethyl auristatin E, referred to as MMAE, which is an anti-mitotic agent that can inhibit cell division by blocking the polymerization of tubulin, and has attracted much attention in antibody-drug conjugates (ADC) .

Among the present invention, term " KD " refers to the expression of affinity constant KD (unit is liter/mass molar concentration), and its calculation formula is

KD＝[Ab·H]/([Ab][H])

Wherein [ ] represents the molar concentration, Ab represents the antibody, H represents the hapten, and Ab—H represents the conjugate of the antibody and the hapten

The higher the affinity constant KD, the stronger the ability of the antibody to bind the hapten. The strength of antibody affinity depends on the degree of coordination between the paratope of the antibody and the epitope used, including the size of the contact area, the closeness of the matching, and the distribution of the dotted group and the hydrophobic group, etc. .

Beneficial effects of the present invention:

(1) The branched polyethylene glycol linker-drug conjugate provided by the present invention can simultaneously couple two cytotoxic drugs to construct a double payload antibody-drug conjugate. When connecting two identical toxins, the drug loading capacity can be increased to realize the purpose of constructing antibody-drug conjugates with high DAR value; when connecting different toxins with the same mechanism of action, synergistic effect can be achieved and the curative effect can be improved; connecting two different mechanisms of action Toxins can reduce the incidence of drug resistance.

(2) The branched polyethylene glycol linker-drug conjugate provided by the present invention, its specific structure endows it with high hydrophilicity, improves its retention time in blood circulation; at the same time, balances cytotoxicity The hydrophobicity of the payload alleviates the problem of easy aggregation of ADCs with hydrophobic toxins.

(3) Compared with traditional antibody-drug conjugates, the branched polyethylene glycol linker-drug conjugate provided by the present invention is simpler to prepare, has a narrower distribution of DAR values, and has higher product uniformity.

(4) For the branched polyethylene glycol linker-drug conjugate provided by the present invention, the ratio of naked antibody and ADC with low cross-linking degree is almost zero (mass spectrometry cannot detect components with DAR of 0 and 1).

(5) The branched polyethylene glycol linker-drug conjugate provided by the present invention, after coupling, the hydrophilicity endowed by ethylene glycol can be used to adjust the characteristics of biomolecules, and has certain safety and safety in treating tumors. effectiveness.

(6) The PEG chain is introduced into the linker of the branched polyethylene glycol linker-drug conjugate of the present invention, which increases the solubility of cytotoxic drugs, makes the ADC coupling reaction more complete and the coupling efficiency is higher.

(7) The coupling method provided by the present invention is applicable to most antibodies, so it has a wide application prospect and has a relatively fast cross-linking speed, and the cross-linking reaction time can usually be completed within 2-4 hours.

Description of drawings

Figure 1 shows the mass spectrum of compound 1.

detailed description

In order to understand the technical content of the present invention more clearly, the following examples are given in detail to describe the technical solution of the present invention clearly and completely, the purpose of which is only to better understand the content of the present invention rather than limit the protection of the present invention Scope All other embodiments obtained by persons of ordinary skill in the field without creative efforts fall within the protection scope of the present invention.

The reagents used in the examples of the present invention are commercially available conventional reagents.

The synthesis of embodiment 1 compound 1

1. Synthesis of Compound 1a

The raw material DBCO-NHS (8.0g, 19mmol) was added to 80mL DMF, followed by the addition of L-glutanicacid (3.89g, 23mmol) and TEA (4.0g, 39mmol), and the reaction was stirred at room temperature for 15h. HPLC showed that the reaction was complete. The reaction solution was washed three times with 200 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 6.5 g of light yellow solid (Compound 1a), with a yield of 75.5%.

MSm/z(ESI):435.2[M+1]

2. Synthesis of compound 1b

Compound 1a (6.2g, 14.3mmol) was added to 60mL DCM, and then NH2-PEG8-PAOMe (14.3g, 31.3mmol), DIEA (5.5g, 43mmol), HATU (16.3g, 43mmol) were added sequentially. The reaction system was stirred at room temperature for 6 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated ammonium chloride, the organic phase was washed once with saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 15 g of crude product. The crude product was purified by silica gel column chromatography (DCM/MeOH 50/1-20/1) to obtain 10.5 g of light yellow oil (compound 1b), with a yield of 58%.

MSm/z(ESI):1309.7[M+1]

3. Synthesis of Compound 1c

Compound 1b (10g, 7mmol) was dissolved in 50mL THF, and then 50mL LiOH (1.7g, 74mmol) aqueous solution dissolved in advance was added. The reaction system was stirred at room temperature for 1 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 8.0 g of light yellow oil (compound 1c), with a yield of 81%.

MSm/z(ESI):1281.6[M+1]

4. Synthesis of Compound 1d

Compound 1c (8.0g, 6mmol) was added into 80mL DMF, followed by VC-PAB (4.97g, 13mmol), DIEA (2.4g, 18mmol), HATU (7.1g, 18mmol). The reaction system was stirred at room temperature for 2 h, and HPLC showed that the reaction was complete. The reaction solution was directly prepared and purified. Finally, 8.5 g of yellow solid (compound 1d) was obtained with a yield of 68%.

MSm/z(ESI):2004.1[M+1]

5. Synthesis of compound 1e

The raw material SN38 (30g, 76mmol) was added to 300mL DCM, and then DIEA (14.8g, 0.1mol) and TBSCl (13.8g, 91mmol) were added in sequence, and the reaction was stirred at room temperature for 5h. TLC showed that the reaction was complete. The reaction solution was washed three times with 400 mL of saturated sodium chloride solution, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain a crude product, which was then purified by silica gel column chromatography (n-hexane/EA20/1-8/1) to obtain 42 g of a white solid (Compound 1e), the yield was 82.3%.

MSm/z(ESI):507.2[M+1]

6. Synthesis of Compound 1f

Compound 1e (10g, 14.8mmol) was dissolved in 70mL DCM, cooled to 0-5°C in an ice-water bath, and p-nitrophenylchloroformate (3.3g, 16mmol) and DMAP (2.89g, 24mmol) were added successively, and allowed to cool to room temperature After stirring for 2H, TLC showed that the reaction was complete. Wash once with 50 mL of saturated sodium bisulfate solution and 50 mL of NaCl, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain 15 g of crude product. Silica gel column purification (DCM/EA20/1-1/1) gave 8 g of yellow solid (compound 1f), yield 61.5%.

MSm/z(ESI):672.2[M+1]

7. Synthesis of Compound 1g

Compound 1f (5.6g, 8mmol) was dissolved in 50mL DCM and 10mL DMSO, then compound 1d (8.0g, 4mmol) and DMAP (1.07g, 8.7mmol) were added, stirred at room temperature for 1h, HPLC showed that the reaction was complete. 100mL DCM and 50mL saturated aqueous sodium bisulfate solution were added to the reaction system. After the layers were separated, the organic phase was washed once with 50mL saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain 13g of crude product (compound 1g).

MSm/z(ESI):1534.7[M/2+1]

7. Synthesis of compound 1

The crude product from the previous step (compound 1g) was dissolved in THF (80 mL), and then 1 mol/L HCl aqueous solution (20 mL) was added, and stirred at room temperature for 3 h. HPLC showed that the reaction of the raw material was complete. The reaction system was adjusted to pH=7-8 with 100 mL of NaHCO3 saturated aqueous solution, then extracted twice with dichloromethane (100 mL), the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified with a high-pressure preparation system to finally obtain 5.1 g of a yellow solid (compound 1), with a yield of 46%, which was compound 1.

MSm/z(ESI):1421.6[M/2+1]

The synthesis of embodiment 2 compound 2

1. Synthesis of Compound 2a

The raw material DBCO-NHS (4.0g, 10mmol) was added to 40mL DMF, NH ₂ -PEG ₈ -PA (4.9g, 11mmol) and TEA (2.0g, 20mmol) were added in sequence, and the reaction was stirred at room temperature for 15h. HPLC showed that the reaction was complete. The reaction solution was washed three times with 200 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 5.1 g of a light yellow solid (compound 2a), with a yield of 71%.

MSm/z(ESI):729.4[M+1]

2. Synthesis of compound 2b

Compound 2a (5.0g, 7.0mmol) and N-hydroxysuccinimide (1.0g, 8.40mmol) were dissolved in 60mL DCM, and 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine (1.6g, 8.40mmol) was stirred at room temperature for 4h to obtain the reaction solution of compound 2b, which was directly used in the next reaction.

MSm/z(ESI):826.4[M+1]

3. Synthesis of compound 2c

Add L-glutanic acid (1.2g, 8mmol) to the reaction solution of compound 2b obtained above, add TEA (0.8g, 8mmol), stir at room temperature for 15h, HPLC shows that the reaction is complete. The reaction solution was washed three times with 100 mL of saturated sodium bisulfate solution and 60 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 4.2 g of light yellow oil (compound 2c), with a yield of 70.2%.

MSm/z(ESI):858.4[M+1]

4. Synthesis of compound 2d

Compound 2c (4.0g, 4.6mmol) was added to 40mL DMF, followed by VC-PAB (3.8g, 10mmol), DIEA (1.9g, 14mmol), HATU (5.5g, 14mmol). The reaction system was stirred at room temperature for 2 h, and HPLC showed that the reaction was complete. The reaction solution was directly prepared and purified. Finally, 3.9 g of yellow solid (compound 2d) was obtained with a yield of 54%.

MSm/z(ESI):1580.8[M+1]

5. Synthesis of Compound 2e

Compound 1f (2.8g, 4mmol) was dissolved in 30mL DCM and 5mL DMSO, then compound 2d (3.2g, 2mmol) and DMAP (0.5g, 4.4mmol) were added, stirred at room temperature for 2h, HPLC showed that the reaction was complete. Add 50 mL of DCM and 50 mL of saturated aqueous sodium bisulfate to the reaction system, wash the organic phase once with 50 mL of saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate to obtain 2.6 g of crude product.

MSm/z(ESI):1323.1[M/2+1]

6. Synthesis of compound 2

The crude product from the previous step was dissolved in THF (20 mL), and then 1 mol/L HCl aqueous solution (5 mL) was added, and stirred at room temperature for 3 h. HPLC showed that the reaction of the raw material was complete. The reaction system was adjusted to pH=7-8 with saturated NaHCO ₃ aqueous solution, then extracted twice with dichloromethane (100 mL), the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by a high-pressure preparation system to finally obtain 1.2 g of a yellow solid (compound 2), with a yield of 48%.

MSm/z(ESI):1209.0[M/2+1]

The synthesis of embodiment 3 compound 3

1. Synthesis of compound 3a

The raw material DBCO-NHS (8.0g, 19mmol) was added to 80mL DMF, followed by the addition of L-glutanicacid (3.89g, 23mmol) and TEA (4.0g, 39mmol), and the reaction was stirred at room temperature for 15h. HPLC showed that the reaction was complete. The reaction solution was washed three times with 200 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 6.5 g of a light yellow solid (compound 3a), with a yield of 75.5%.

MSm/z(ESI):435.2[M+1]

2. Synthesis of compound 3b

Compound 3a (6.2g, 14.3mmol) was added to 60mL DCM, and then NH ₂ -PEG ₈ -PAOMe (14.3g, 31.3mmol), DIEA (5.5g, 43mmol), HATU (16.3g, 43mmol) were added in sequence. The reaction system was stirred at room temperature for 6H, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated ammonium chloride, the organic phase was washed once with saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 15 g of crude product. The crude product was purified by silica gel column chromatography (DCM/MeOH 50/1-20/1) to obtain 10.5 g of light yellow oil (compound 3b), with a yield of 58%.

MSm/z(ESI):1309.7[M+1]

3. Synthesis of compound 3c

Compound 3b (10g, 7mmol) was dissolved in 50mL THF, and then 50mL LiOH (1.7g, 74mmol) aqueous solution dissolved in advance was added. The reaction system was stirred at room temperature for 1 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 8.0 g of light yellow oil (compound 3c), with a yield of 81%.

MSm/z(ESI):1281.6[M+1]

4. Synthesis of Compound 3d

Compound 3c (8.0g, 6mmol) was added into 80mL DMF, and then VC-PAB (4.97g, 13mmol), DIEA (2.4g, 18mmol), HATU (7.1g, 18mmol) were added sequentially. The reaction system was stirred at room temperature for 2 h, and HPLC showed that the reaction was complete. The reaction solution was directly prepared and purified. Finally, 8.5 g of yellow solid (compound 3d) was obtained with a yield of 68%.

MSm/z(ESI):2004.1[M+1]

6. Synthesis of compound 3

Compound 3d (8g, 4mmol) was dissolved in 50mL DCM, the temperature was cooled to 0-5°C in an ice-water bath, p-nitrophenylchloroformate (2.1g, 10mmol) and DMAP (1.9g, 16mmol) were added sequentially, and stirred at room temperature for 2h, After TLC showed the reaction was complete, MMAE (6.5 g, 9 mmol) and DMAP (1.1 g, 9 mmol) were added. Wash once with 50 mL of saturated sodium bisulfate solution and 50 mL of NaCl, dry the organic phase with anhydrous sodium sulfate, and concentrate the organic phase to obtain a crude product. 4.7 g of yellow solid (compound 3) was obtained by silica gel column chromatography and high-pressure preparation, and the yield was 34%.

MSm/z(ESI):1746[M/2+1]

The synthesis of embodiment 4 compound 4

1. Synthesis of compound 4a

The raw material DBCO-NHS (8.0g, 19mmol) was added to 80mL DMF, followed by the addition of L-glutanicacid (3.89g, 23mmol) and TEA (4.0g, 39mmol), and the reaction was stirred at room temperature for 15h. HPLC showed that the reaction was complete. The reaction solution was washed three times with 200 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 6.5 g of a light yellow solid (compound 4a), with a yield of 75.5%.

MSm/z(ESI):435.2[M+1]

2. Synthesis of Compound 4b

Compound 4a (6.2g, 14.3mmol) was added to 60mL DCM, and NH ₂ -PEG ₈ -PAOMe (14.3g, 31.3mmol), DIEA (5.5g, 43mmol), HATU (16.3g, 43mmol) were added in sequence. The reaction system was stirred at room temperature for 6 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated ammonium chloride, the organic phase was washed once with saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 15 g of crude product. The crude product was purified by silica gel column chromatography (DCM/MeOH 50/1-20/1) to obtain 10.5 g of light yellow oil (compound 4b), with a yield of 58%.

MSm/z(ESI):1309.7[M+1]

3. Synthesis of Compound 4c

Compound 4b (10g, 7mmol) was dissolved in 50mL THF, and then 50mL LiOH (1.7g, 74mmol) aqueous solution dissolved in advance was added. The reaction system was stirred at room temperature for 1 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 8.0 g of light yellow oil (compound 4c), with a yield of 81%.

MSm/z(ESI):1281.6[M+1]

4. Synthesis of compound 4d

Compound 4c (8.0g, 6mmol) was added into 80mL DMF, and then VC-PAB (4.97g, 13mmol), DIEA (2.4g, 18mmol), HATU (7.1g, 18mmol) were added sequentially. The reaction system was stirred at room temperature for 2 h, HPLC showed that the reaction was complete, and a reaction solution was obtained. The aforementioned reaction solution was directly prepared and purified. Finally, 8.5 g of yellow solid (compound 4d) was obtained with a yield of 68%.

MSm/z(ESI):2004.1[M+1]

5. Synthesis of Compound 4e

Compound 4d (8g, 4mmol) was dissolved in 50mL DCM, the temperature was cooled to 0-5°C in an ice-water bath, p-nitrochloroformate (2.1g, 10mmol) and DMAP (1.9g, 16mmol) were added successively, and stirred at room temperature for 2h, After TLC showed that the reaction was complete, the reaction system was washed once with 50 mL of saturated sodium bisulfate solution and 50 mL of NaCl, and the reaction system was separated. The organic phase was dried with anhydrous sodium sulfate and concentrated to obtain 6.0 g of crude product (compound 4e), with a yield of 65%.

MSm/z(ESI):1167.5[M/2+1]

6. Synthesis of compound 4f

Compound 4e (4.6g, 2mmol) was dissolved in 20mL DCM and 5mLDMSO, then compound SN38-OTBS (0.5g, 1mmol) and DMAP (0.2g, 1.5mmol) were added, stirred at room temperature for 2h, HPLC showed that the reaction was complete. Add 50mL DCM and 50mL saturated sodium bisulfate aqueous solution to the reaction system to separate the reaction system, wash the organic phase once with 50mL saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate to obtain the crude product, which is separated by high-pressure preparation to obtain a yellow solid 1.6 g (compound 4f), yield 61%.

MSm/z(ESI):1351.2[M/2+1]

7. Synthesis of compound 4g

Compound 4f (1.3g, 0.5mmol) was dissolved in 10mL DCM, compound MMAE (0.4g, 0.6mmol) and DMAP (0.1g, 1.0mmol) were added, stirred at room temperature for 2H, HPLC showed that the reaction was complete. Add 20mL DCM and 20mL saturated sodium bisulfate aqueous solution to the reaction system to separate the reaction system, wash the organic phase once with 20mL saturated sodium chloride solution, dry over anhydrous sodium sulfate, and concentrate to obtain the crude product, which is separated by high-pressure preparation to obtain a yellow solid 1.6 g (compound 4g), yield 70%.

MSm/z(ESI):1640.4[M/2+1]

8. Synthesis of Compound 4

Compound 4g (1.5g, 0.46mmol) was dissolved in THF (10mL), then 1mol/L HCl aqueous solution (3mL) was added, stirred at room temperature for 3h, HPLC showed that the reaction of the raw materials was complete. The reaction system was adjusted to pH=7-8 with saturated NaHCO ₃ aqueous solution, then extracted twice with dichloromethane (100 mL), the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by high-pressure preparation, and finally 700 mg of yellow solid (compound 4) was obtained with a yield of 48%.

MSm/z(ESI):1583.4[M/2+1]

The synthesis of embodiment 5 compound 5

1. Synthesis of Compound 5a

The raw material DBCO-NHS (8.0g, 19mmol) was added to 80mL DMF, followed by the addition of L-glutanicacid (3.89g, 23mmol) and TEA (4.0g, 39mmol), and the reaction was stirred at room temperature for 15h. HPLC showed that the reaction was complete. The reaction solution was washed three times with 200 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 6.5 g of a light yellow solid (compound 5a), with a yield of 75.5%.

MSm/z(ESI):435.2[M+1]

2. Synthesis of Compound 5b

Compound 5a (6.2g, 14.3mmol) was added to 60mL DCM, and then NH2-PEG8-PAOMe (14.3g, 31.3mmol), DIEA (5.5g, 43mmol), HATU (16.3g, 43mmol) were added sequentially. The reaction system was stirred at room temperature for 6H, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated ammonium chloride, the organic phase was washed once with saturated sodium chloride solution, then dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 15 g of crude product. The crude product was purified by silica gel column chromatography (DCM/MeOH 50/1-20/1) to obtain 10.5 g of light yellow oil (compound 5b), with a yield of 58%.

MSm/z(ESI):1309.7[M+1]

3. Synthesis of compound 5c

Compound 5b (10g, 7mmol) was dissolved in 50mL THF, and then 50mL LiOH (1.7g, 74mmol) aqueous solution dissolved in advance was added. The reaction system was stirred at room temperature for 1 h, and HPLC showed that the reaction was complete. The reaction solution was washed three times with 100 mL of saturated sodium bisulfate solution and 100 mL of DCM, the organic phase was dried with anhydrous sodium sulfate, and the solvent was spin-dried to obtain 8.0 g of light yellow oil (compound 5c), with a yield of 81%.

MSm/z(ESI):1281.6[M+1]

4. Synthesis of compound 5d

Compound 5c (8.0g, 6mmol) was added into 80mL DMF, and then VC-PAB (4.97g, 13mmol), DIEA (2.4g, 18mmol), HATU (7.1g, 18mmol) were added sequentially. The reaction system was stirred at room temperature for 2 h, and HPLC showed that the reaction was complete. The reaction solution is directly purified by a high-pressure preparation system. Finally, 8.5 g of yellow solid (.compound 5d) was obtained with a yield of 68%.

MSm/z(ESI):2004.1[M+1]

5. Synthesis of compound 5e

Compound 5d (8g, 4mmol) was dissolved in 50mL DCM, the temperature was cooled to 0-5°C in an ice-water bath, p-nitrophenylchloroformate (2.1g, 10mmol) and DMAP (1.9g, 16mmol) were added sequentially, and stirred at room temperature for 2h, After TLC showed that the reaction was complete, it was washed once with 50 mL of saturated sodium bisulfate solution and 50 mL of saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain 6.0 g of crude product (compound 5e), with a yield of 65%.

MSm/z(ESI):1167.5[M/2+1]

6. Synthesis of compound 5f

Compound 5e (4.6g, 2mmol) was dissolved in 20mL DCM and 5mL DMSO, then compound MMAE (0.7g, 1mmol) and DMAP (0.2g, 1.5mmol) were added, stirred at room temperature for 2h, HPLC showed that the reaction was complete. The reaction system was added 50mL DCM and 50mL saturated aqueous sodium bisulfate solution, the organic phase was washed once with 50mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated by high-pressure preparation to obtain 1.9 g of a yellow solid (compound 5f). Yield 65%.

MSm/z(ESI):1456.8[M/2+1]

7. Synthesis of Compound 5g

SN38 (5.5g, 14mmol) (purchased from Hubei Xinkang, ≥95%) was dissolved in 50mL DCM, cooled to 0-5°C in an ice-water bath, and p-nitrochloroformate phenyl (3.3g, 16mmol) and DMAP ( 2.89g, 24mmol), naturally to room temperature and stirred for 2h, TLC showed that the reaction was complete. Wash once with 50 mL of saturated sodium bisulfate solution and 50 mL of NaCl, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain 15 g of crude product. Silica gel column purification (DCM/EA20/1-1/1) gave 4.6 g of yellow solid (compound 5 g), yield 60%.

MSm/z(ESI):558.1[M+1]

8. Synthesis of compound 5h

Compound 5g (4.5g, 8mmol) was dissolved in 30mLDCM and 5mLDMSO, then N,N'-dimethylethylenediamine (0.8g, 9.0mmol) and DMAP (1.07g, 8.7mmol) were added, and stirred at room temperature for 1h , HPLC showed that the reaction was complete. The reaction system was added 100mL DCM and 50mL saturated aqueous sodium bisulfate solution, the organic phase was washed once with 50mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain 2.3g of a yellow solid (compound 5h), yield 58 %.

MSm/z(ESI):507.2[M+1]

9. Synthesis of Compound 5

Compound 5f (1.4g, 0.5mmol) was dissolved in 10mL DCM and 2mL DMSO, then compound 5h (0.3g, 0.6mmol) and DMAP (0.1g, 1.0mmol) were added, stirred at room temperature for 2h, HPLC showed that the reaction was complete. The reaction system was added 20mL DCM and 20mL saturated aqueous sodium bisulfate solution, the organic phase was washed once with 20mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated by high-pressure preparation to obtain 700 mg of a yellow solid (compound 5). rate 47%.

MSm/z(ESI):1554.3[M/2+1]

The preparation of embodiment 6 ADC

6.1 Reaction process of mAb and PEG12

The reaction was carried out according to the molar ratio of mAb and N ₃ -PEG12-SPA being 1:20. First measure 208 μL of mAb solution (concentration: 9.6 mg/mL) and place it in a 1.5 mL centrifuge tube, then the measured mAb is 2 mg. A 10 mg/mL N ₃ -PEG12-SPA solution was prepared using 5 mM PBS solution at pH 3.0, and 20 μL was added to a centrifuge tube, mixed thoroughly, and oscillated for 2 hours. After the reaction, the unreacted N ₃ -PEG12-SPA was removed by ultrafiltration.

mAb is EGFR monoclonal antibody-Necitumumab (Necitumumab), purchased from Eli Lilly (EliLilly)

N ₃ -PEG12-SPA is self-made, with a purity of 95%, MSm/z (ESI): 741.4[M+1].

6.2 Reaction process of mAb-PEG12 and linker-drug

Take the prepared mAb-PEG12 sample, determine the protein concentration to be 2.05 mg/mL, measure 490 μL of the sample solution and place it in a 1.5 mL centrifuge tube. Weigh 10 mg linker-drug, dissolve in DMSO, and prepare a 1 mg/mL solution. According to the calculation that the ratio of mAb to linker-drug is 1:4, measure 41 μL of linker-drug solution and add it to the centrifuge tube, then add 510 μL of PBS and 59 μL of DMSO to the centrifuge tube to keep the protein concentration at 1 mg/mL, water The ratio with DMSO is 10:1, mix well, and shake for 2 hours. After the reaction was completed, unreacted small molecules were removed by ultrafiltration to obtain ADC molecules, as shown in the table below.

Table 1 Types of Linker-drugs and names of corresponding ADC molecules

Linker-drugs ADC molecule Compound 1 ADC1 Compound 4 ADC2 DBCO-VC-PAB-SN38 ADC3

Among them, compound 1 was prepared in Example 1, compound 4 was prepared in Example 4, DBCO-VC-PAB-SN38, self-made, purity 90%, MSm/z (ESI): 1087.4 [M+1]

The structural formula of DBCO-VC-PAB-SN38 is shown in the figure below:

The measurement of embodiment 7 DAR value

The DAR value of the ADC in Example 6 was detected by liquid chromatography.

The LC settings were as follows:

Table 2 Liquid Chromatography Conditions

Dissolve the ADC1, ADC2, and ADC3 prepared in Example 6, and detect ADC1, ADC2, and ADC3 according to the liquid chromatography conditions in Table 2. Under the liquid chromatography conditions, the separation between SN38 and ADC1, ADC2, and ADC3 is greater than 1.5. Prepare standard curves of different concentrations, establish a linear standard curve between the peak area of SN38 and the concentration, and quantify SN38 on ADC1, ADC2 and ADC3, respectively. According to the antibody concentration of ADC and the concentration of SN38, the conjugation number of SN38 on ADC was calculated. The DAR value of ADC1 prepared in Example 6 is 8, the DAR value of ADC2 is 8, the DAR value of ADC3 is 4, and the DAR value of ADC1 and ADC2 is higher than that of ADC3. The experimental results show that compared with the traditional ADC1 obtained by this invention and ADC2 have higher DAR values.

Example 8 Affinity Determination of ADC and EGFR

The affinity of the ADC drug is detected by the method of SPR, and the test instrument is biacore, a product of GE Company.

The simple operation steps are: coupling the EGFR antigen to the CM chip. Use different concentrations of ADC drugs or monoclonal antibody drugs to test the affinity of ADC drug antibodies or free antibodies to antigens.

The results showed that ADC1, ADC2 and ADC3 prepared in Example 6, the affinity decrease of the antibodies was not obvious, and the affinity of ADC1 and ADC2 was higher than that of ADC3.

Table 3 Experimental results:

Analyze KD mAb 0.287nM ADC1 0.435nM ADC2 0.397nM ADC3 0.542nM

Example 9 In Vitro Cytotoxicity Experiment

In vitro cytotoxicity tests were performed with BXPC-3 (human pancreatic cancer cell) cells. The test operation is as follows:

1. Add 100 μL of cells to each well of the 96-well plate (keep 2 blank groups without adding cells, and add the same volume of medium). The cells were cultured in a 5% CO ₂ incubator at 37°C for 24 h. For cytotoxicity experiments, add 100 μL to each well to contain about 10,000 cells.

2. Add 10 μL of ADC or SN38 drugs with different concentrations to each well.

3. Incubate the 96-well plate at 37°C for 24 hours in a cell culture incubator containing 5% CO ₂ air and 100% humidity.

4. Add 10 μL of CCK-8 solution to each well. Incubate for 3 h at 37°C in a 5% CO ₂ incubator.

5. Measure the absorbance at 450nm with a microplate reader.

6. Result analysis:

A. Cell survival rate: subtract the background OD value (blank group) from the OD value of each test well, and take the mean ± SD of the OD value of each repeated well.

Cell viability%=(OD of drug-added cells/OD of control cells)×100%.

B. Determine the drug concentration (IC50) when T/C=50% and the drug concentration (IC90) when T/C=10%.

The results showed that the IC50 value of ADC1 prepared in Example 6 was less than that of ADC2, and both were less than the IC50 value of SN38.

Table 4 Cytotoxicity Results:

drug molecule IC50 ADC1 0.097nM ADC2 0.083nM ADC3 0.267nM FreeSN38 0.072μM

The results show that the ADC1 and ADC2 obtained by the invention can not only effectively play the biological function of the EGFR antibody part, but also have the high-efficiency killing activity of SN38 on tumor cells. Compared with ADC3 prepared by traditional linear linker, ADC1 and ADC2 with branched linker obtained in the present invention have better activity.

It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.

Claims (14)

1. A branched polyethylene glycol linker, characterized in that it has a structure shown in formula (I):

Wherein, the Q is selected from:

one of R ₅ and R ₆ each independently have the structure of -X ₄ -Q ₁ , Q ₁ is selected from -H, -F, -Cl, -Br, -I, -SO ₂ , -NO ₂ , C _1-12 chains Alkyl, C _3-12 cycloalkyl, C _6-12 aralkyl; X _is selected from single bond, -O-, -S-, C _1-12 chain alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of them; X is a linking group selected from single bond, -O-, -S-, C _1-12 straight chain/branched alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of, wherein R ₉ is selected from: -H, C _1-10 straight chain/branched alkyl; The W ₁ and W ₂ are independently selected from single bond, linear or branched C _1-6 alkyl groups; L _1a , L _1b , L _1c are independently selected from single bonds,

The n is an integer selected from 1 to 24; X ₁ and X ₂ are linking groups, independently selected from single bond, -O-, -S-, C _1-12 linear/branched alkylene, C _3-12 cycloalkylene, C _{6- 12} aralkylene,

and

A group consisting of one or more of them; L ₂ and L ₃ are linking groups

Wherein A is a peptide linker, B is a self-degrading group or does not exist, When B is a self-degrading group, B is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH,

Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

X ₃ is selected from -O-, -S-, C _1-12 straight chain/branched chain alkyl, C _3-12 cycloalkyl, C _6-12 aromatic ring group,

and

or a combination thereof; R ₂₀ is selected from: -H, C _1-12 straight chain/branched chain alkyl, C _1-12 alkoxy, -NO ₂ , -CN, -F, -Cl, -Br, -I, -SO ₂ , C _3-12 cycloalkyl, C _6-12 aromatic ring group,

A is a peptide linker, including one or more identical or different polypeptide residues, and said polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives. 2. The linker according to claim 1, wherein said Q is

Preferably, the Q is

The R ₅ and R ₆ are independently selected from H and C _1-3 alkyl groups, preferably, both R ₅ and R ₆ are H; Said X is selected from

C _1-12 straight chain/branched chain alkylene,

The group consisting of, wherein R ₉ is H, C _1-3 straight chain/branched alkyl; Preferably, the X is

The R ₇ is a C _1-12 alkylene group; more preferably, the R ₇ is a C _1-4 alkylene group; The L _1a , L _1b , L _1c are independently selected from single bonds,

The W ₄ is selected from a single bond, C _1-6 linear or branched alkylene; Preferably, said n is an integer selected from 1 to 12; The amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine , lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, citrulline, ornithine and cystine One or a combination of two or more; Preferably, the amino acid is selected from one or more of valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, leucine and arginine A combination of two or more; More preferably, the polypeptide residues include one of valine-citrulline, valine-alanine and glycine-glycine-phenylalanine-glycine. 3. The linker of claim 1, wherein said B does not exist or is selected from:

Preferably, said B is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH; Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

4. The linker according to claim 2, wherein said Q is

The X is

Said L _1a , L _1b , L _1c are independently selected from a single bond or

Said A is selected from

The B is

or does not exist. 5. The linker according to claim 1, wherein said L ₂ and L ₃ are independently selected from:

6. The linker according to any one of claims 1 to 5, wherein the linker is selected from:

7. A branched polyethylene glycol linker-drug conjugate, characterized in that it has a structure of formula (II):

Wherein, the Q is selected from:

one of R ₅ and R ₆ each independently have the structure of -X ₄ -Q ₁ , Q ₁ is selected from -H, -F, -Cl, -Br, -I, -SO ₂ , -NO ₂ , C _1-12 chains Alkyl, C _3-12 cycloalkyl, C _6-12 aralkyl; X _is selected from single bond, -O-, -S-, C _1-12 chain alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of them; X is a linking group selected from single bond, -O-, -S-, C _1-12 straight chain/branched alkylene, C _3-12 cycloalkylene, C _{6-12 aralkylene} ,

and

A group consisting of one or more of, wherein R ₉ is selected from: -H, C _1-10 straight chain/branched alkyl; The W ₁ and W ₂ are independently selected from single bond, linear or branched C _1-6 alkyl groups; L _1a , L _1b , L _1c are independently selected from single bonds,

The n is an integer selected from 1 to 24; X ₁ and X ₂ are linking groups, independently selected from single bond, -O-, -S-, C _1-12 linear/branched alkylene, C _3-12 cycloalkylene, C _{6- 12} aralkylene,

and

A group consisting of one or more of, wherein R ₁₀ is selected from: -H, C _1-10 straight chain/branched alkyl; L ₂ ', L ₃ ' are linking groups, which are

Wherein A' is a peptide linker, B' is a self-degrading group, and B' is selected from:

Wherein, Y ₁ and Y ₂ are each independently selected from: -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH,

Y ₃ and Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl,

X ₃ is selected from -O-, -S-, C _1-12 straight chain/branched chain alkyl, C _3-12 cycloalkyl, C _6-12 aromatic ring group,

and

or a combination thereof; R ₂₀ is selected from: -H, C _1-12 straight chain/branched chain alkyl, C _1-12 alkoxy, -NO ₂ , -CN, -F, -Cl, -Br, -I, -SO ₂ , C _3-12 cycloalkyl, C _6-12 aromatic ring group,

W ₃ is C _1-6 straight chain/branched chain alkylene; The peptide linker includes one or more identical or different polypeptide residues, and the polypeptide residues include two or more identical or different amino acid residues or amino acid residue derivatives; Preferably, the amino acid is selected from: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Citrulline, Ornithine, and Cystine One or more combinations of acids, preferably, the amino acid is selected from: valine, citrulline, glycine, alanine, phenylalanine, tyrosine, isoleucine, One or more combinations of leucine and arginine; more preferably, the polypeptide residues include valine-citrulline, valine-alanine and glycine-glycine-benzene One of alanine-glycine; The D is a drug molecule. 8. The conjugate according to claim 7, wherein said Q is

Preferably, the Q is

The R ₅ and R ₆ are independently selected from H and C _1-3 alkyl groups, preferably, both R ₅ and R ₆ are H; Said X is selected from

C _1-12 straight chain/branched chain alkylene,

The group consisting of, wherein R ₉ is H, C _1-3 straight chain/branched alkyl; Preferably, the X is

The R ₇ is a C _1-12 alkylene group; more preferably, the R ₇ is a C _1-4 alkylene group; The L _1a , L _1b , L _1c are independently selected from single bonds,

The W ₄ is selected from a single bond, C _1-6 linear or branched alkyl; Preferably, the n is an integer selected from 1-12. 9. The conjugate as claimed in claim 7, wherein said A' is selected from

Wherein, A ₁ , A ₂ , A ₃ ... A _y are the same or different, and the structures of A ₁ , A ₂ , A ₃ ... A _y are independently selected from: single bond,

The y is an integer selected from 1 to 10; The j is an integer selected from 1 to 10; Preferably, the j is an integer of 2-4; The Y _a is selected from -H, halogen, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, C _3-10 cycloalkyl, -OH, -(C _1-6 alkylene base)-OH, -(C _1-6 alkylene)-SH, -(CH ₂ ) _1-6 -cycloalkyl, -(C _1-6 alkylene)-aromatic ring group, -(C _{1 -6} alkylene)-heterocycloalkyl, -(C _1-6 alkylene)-heterocyclic aryl, -(C _1-6 alkylene)-COOH, -(C _1-6 alkylene )-CONH ₂ , -(C _1-6 alkylene)-NH ₂ and

one of Said A ₁ , A ₂ , A ₃ ... A _y has at least one

structure; Preferably, said Y _a is selected from: -H, -C _1-6 straight chain/branched chain alkyl,

-(C _1-6 alkylene)-aromatic ring group; More preferably, the Y _a is selected from: -H, methyl, ethyl, propyl, isopropyl,

The B' is selected from:

Wherein, Y ₁ , Y ₂ are each independently selected from: -H, -OC _1-10 alkyl, C _1-10 straight chain/branched chain alkyl, -OH; Y ₃ , Y ₄ are each independently selected from: -H, C _1-10 straight chain/branched chain alkyl; W ₃ is selected from C _1-6 straight chain/branched chain alkylene; Preferably, Y ₁ , Y ₂ , Y ₃ , and Y ₄ are each independently selected from H, C _1-5 straight chain/branched chain alkyl. 10. The conjugate according to claim 8, wherein said Q is

The X is

Said L _1a , L _1b , L _1c are independently selected from a single bond or

The A' is selected from

The B' is selected from:

11. The conjugate according to claim 7, wherein said L ₂ ', L ₃ ' are independently selected from:

12. The conjugate according to claim 11, wherein the D is a drug for killing tumor cells; And/or, the D is selected from: amanitin, auristatin, calicheamicin, camptothecin, camptothecin derivatives and metabolites, exinotecan, Dxd, cryptomycin, Daunomycin, dolastatin, doxorubicin, duocamycin, epothilone, espamycin, geldanamycin, maytansine and its derivatives, methotrexate , monomethyl auristatin E, monomethyl auristatin F, pyrrolobenzodiazepines, rhizobin, SG2285, tubulysin, vindesine, toxoid, irinotecan, topotecan, belotide Kang, exatecan, rutotecan, diflutecan, geninotecan, canitecan, doxorubicin, epirubicin, morpholino doxorubicin, cyanomorpholino-A Mycin, 2-pyrrolidinyl doxorubicin, Dxd, MMAD, PBD and its derivatives, TPL, taxanes, cyclomycin, bleomycin B and its derivatives, maytansine and its derivatives, multi Lalastatin, mitomycin, aminopterin, actinomycin, bleomycin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide , talithromycin, cytarabine, etoposide, paclitaxel, laramycin, podophyllotoxin, serpentine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5 - one or a combination of two or more of diacetoxy-pentyl) doxorubicin and its derivatives; Preferably, D is selected from,

13. The conjugate according to any one of claims 7-12, wherein the conjugate is selected from:

14. A use of the conjugate according to any one of claims 7 to 13 in the preparation of drugs for the prevention and/or treatment of diseases, characterized in that the diseases include: cancer, inflammation, autoimmune diseases .

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