Amatoxin conjugates containing branched linkers

Abstract

The invention relates to the coupling of an amatoxin homologue and a cell binding molecule by a branched chain (side chain) linker, and the generated conjugate has a better targeted killing effect on abnormal cells. The invention also relates to methods of coupling amatoxin homologues to cell binding agents, and methods of using the conjugates for targeted therapy of cancer, infection and immune diseases.

Description

[0001] This case is a divisional application of a case with an application date of January 31, 2019, an invention name of Amanita toxin conjugate containing branched linkers, and an application number of CN201980088973.6. Technical Field

[0002] The present invention relates to the coupling of an amatoxin homologue and a cell binding molecule with a branched (side chain) linker, and the resulting conjugate has better pharmacokinetic properties, thereby being able to more accurately target and kill abnormal cells. The present invention also relates to a method for coupling an amatoxin homologue with a cell binding agent, and a method for using the conjugate to target and treat cancer, infection and immune diseases. Technical Background

[0003] Antibody-drug conjugates (ADCs) are synergistic combinations of small molecule chemotherapeutic drugs and monoclonal antibodies coupled via stable linkers. Over the past 20 years, they have become a promising new type of biological drug, and their clinical pipeline is growing rapidly and on a large scale. The combination of the three components of ADC produces a powerful anti-tumor agent that can deliver normally intolerable cytotoxins directly to cancer cells, which then internalize and release drugs that destroy the cells (L. Ducry and B Stump, Bioconjugate Chem., 2010, 21, 5–13; GS Hamilton / Biologicals 2015, 43, 318-32).

[0004] Compared with standard chemotherapy drugs, the therapeutic window of early ADC therapy is smaller. However, the development of linker technology and the use of highly active cytotoxic agents that cannot be directly administered have greatly improved the effectiveness of ADC (Bander, NH et al., Adv. Hematol. Oncol., 2012, 10, 1-16; Behrens, CR and Liu, B., mAbs, 2014.6, 46-53). However, off-target toxicity is currently one of the main challenges faced in ADC drug development (Roberts, SA et al., Regul. Toxicol. Pharmacol. 2013, 67, 382-91). For example, in clinical practice, Ado-trastuzumabemtansine (T-DM1, ) used a stable (non-cleavable) MCC linker, which has great benefit for patients with HER2-positive metastatic breast cancer (mBC), who have received mBC treatment, or whose tumor relapsed within six months of adjuvant therapy (Peddi, P. and Hurvitz, S., Ther. Adv. Med. Oncol. 2014, 6(5), 202–209; Piwko C. et al., Clin Drug Investig. 2015, 35(8), 487-93; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57, 6949-64). However, clinical trials of T-DM1 as a first-line treatment for patients with HER2-positive unresectable locally advanced or metastatic breast cancer, or as a second-line treatment for HER2-positive advanced gastric cancer, have failed, with the benefits to these patients being modest compared with the toxic side effects (Ellis, PA, et al., J. Clin. Oncol. 2015, 33 (2015 ASCO Abstract 507); Shen, K. et al., Sci Rep. 2016, 6, 23262; de Goeij, BE and Lambert, JM Curr Opin Immunol 2016, 40, 14-23; Barrios, CH et al., J Clin Oncol 2016, 34, (2016 ASCO Abstract 593)).

[0005] In order to solve the problem of off-target toxicity, the scope of linker-payload and conjugation chemistry is being expanded from the chemical aspect in the design of ADC, including expanding the types of payloads to improve the activity of linker-payloads against targets / targeted diseases (Lambert, JM Ther Deliv 2016, 7, 279-82; Zhao, RY et al., 2011, J. Med. Chem. 54, 3606-23). There are also many drug developers and academic institutions that are highly focused on developing novel, reliable and specific conjugation linkers suitable for ADC site-specific conjugation. These conjugates appear to have longer circulation half-life, better efficacy, potentially less off-target toxicity, and better in vivo pharmacokinetic (PK) properties of ADCs as well as good batch-to-batch consistency during production (Hamblett, KJ et al., Clin. Cancer Res. 2004, 10, 7063-70; Adem, YT et al., Bioconjugate Chem. 2014, 25, 656-664; Boylan, NJ Bioconjugate Chem. 2013, 24, 1008-1016; Strop, P., et al 2013 Chem. Biol. 20, 161-67; Wakankar, A. mAbs, 2011, 3, 161-172). So far, the specific coupling methods reported include the introduction of engineered cysteine ​​(Junutula, JR et al., Nat. Biotechnol. 2008, 26, 925-32; Junutula, JR et al., 2010 Clin. Cancer Res. 16, 4769; U.S. Patents 8,309,300; 7,855,275; 7,521,541; 7,723,485, WO2008 / 141044), selenocysteine ​​(Hofer, T., et al., Biochemistry 2009, 48, 12047-57; Li, X. et al., Methods 2014, 65, 133-8; U.S. Patents 8916159 US National Cancer Institute). Institute), cysteine ​​containing aromatic fluorine label (Zhang, C. et al. Nat. Chem. 2015, 8, 1–9), thiofucose (Okeley, NM et al., 2013 Bioconjugate Chem. 24, 1650), unnatural amino acids (Axup, JY et al., Proc. Nat. Acad. Sci. USA. 2012, 109, 16101-6; Zimmerman, ES et al., 2014, Bioconjug. Chem. 25, 351–361; Wu, P. et al., 2009 Proc. Natl.Acad. Sci. 106, 3000-5; Rabuka, D. et al., Nat. Protoc. 2012, 7, 1052-67; U.S. Patent 8778631 and Patent Application 20100184135, WO2010 / 081110 Sutro Biopharma; WO2006 / 069246, 2007 / 059312, U.S. Patents 7,332,571, 7,696,312, and 7,638,299 Ambrx; WO2007 / 130453, U.S. Patents 7,632,492 and 7,829,659 Allozyne), re-bridging reduced intramolecular disulfide bonds by the following functional groups: dibromomaleimide (Jones, MW et al., J. Am. Chem. Soc. 2012, 134, 1847–52), bissulfonyl reagents (Badescu, G. et al., Bioconjug. Chem. 2014, 25, 1124–36; WO2013 / 190272, WO2014 / 064424 PolyTherics Ltd), dibromopyridopyridazinedione (Maruani, A. et al., Nat. Commun. 2015, 6, 6645), by galactosyl and sialyltransferase (Zhou, Q. et al., Bioconjug. Chem. 2014, 25, 510–520; U.S. Patent Application 20140294867 Sanofi-Genzyme), formylglycine generating enzyme (FGE) (Drake, PM et al. Bioconj. Chem. 2014, 25, 1331–41; Carrico, IS et al. U.S. Patents 7,985,783; 8,097,701; 8,349,910, Patent Application 20140141025, 20100210543 Redwood Bioscience), phosphopantetheinyltransferases (PPTases) (Grünewald, J. et al. Bioconjug. Chem. 2015, 26, 2554–62), sortase A (Beerli, RR, et al. PLoS One 2015, 10, e0131177), introduction of glutamine tags using Streptoverticillium mobaraense transglutaminase (mTG) (Strop, P., Bioconj. Chem., 2014, 25, 855–62; Strop, P., et al., Chem. Biol. 2013, 20, 161–7; U.S. Patent 8,871,908 Rinat-Pfizer) or microbial transglutaminase (MTGase) (Dennler, P.et al., 2014, Bioconjug. Chem. 25, 569–78; Siegmund, V. et al., Angew. Chemie-Int. Ed. 2015, 54, 13420–4; U.S. Patent Application 20130189287 Innate Pharma; U.S. Patent 7,893,019 Bio-Ker Srl (IT)), through an enzyme / bacteria that forms isopeptide-peptide bonds outside the protein backbone (Kang, HJ et al., Science 2007, 318, 1625–8; Zakeri, B. et al., Proc. Natl. Acad. Sci. USA 2012, 109, E690–7; Zakeri, B. & Howarth, MJ Am. Chem. Soc. 2010, 132, 4526–7). .

[0006] We have disclosed several conjugation methods that can rebridge the pair of thiols generated by the reduction of the interchain disulfide of the native antibody, such as using bromomaleimide and dibromomaleimide linkers (WO2014 / 009774), 2,3-disubstituted succinic acid / 2-monosubstituted / 2,3-disubstituted fumaric acid or maleic acid linkers (WO2015 / 155753, WO20160596228), acetylene dicarboxyl linkers (WO2015 / 151080, WO20160596228) or hydrazine linkers (WO2015 / 151081). Compared with the traditional non-selective methods of conjugation to cysteine ​​or lysine residues on antibodies, the therapeutic window of ADCs prepared using these linkers and conjugation methods is larger. Here we disclose an amatoxin conjugate containing a long branched linker. Long branched-chain linkers can prevent the antibody-drug conjugate from being hydrolyzed by hydrolases such as proteases or esterases, making the conjugate more stable in the circulatory system and producing fewer toxic side effects.

[0007] Amanita toxins, mainly including amatoxin, phalloidin and amanita phalloidins (Wieland, T., Faulstich, H., CRC Crit. Rev. Biochem. 1978, 5(3): 185-260; Vetter, J., Toxicon 1998, 36(1): 13–24; Weiland, T., and Faulstich, H. 1983, Peptide toxins from Amanita, 585-635; Handbook of Natural Toxins, Volume I Plant and Fungal Toxins, ed. RF Keeler and AT Tu, Marcel Dekker, Inc. New York, NY; Wieland, T., Int J. Pept. Protein Res., 1983, 22, 257-76) are effective cytotoxic agents for antibody conjugates (Zhao, R. et al., WO2017046658). The amatoxin conjugate of the present invention contains a long-branched linker, which can prolong the half-life of the conjugate during targeted delivery and minimize exposure to non-target cells, tissues and organs. The drug has lower off-target toxicity and a larger therapeutic window. SUMMARY OF THE INVENTION

[0009] The present invention describes a conjugate of an amatoxin homologue and an antibody connected by a branched linker, and also describes a method of using the linker to conjugate an amatoxin homologue and an antibody. In one aspect of the invention, the conjugate containing the branched linker is represented by structural formula (I):

[0010]

[0011] in

[0012] “-” represents a single bond; n is 1 to 30;

[0013] T is a cell binding agent or molecule selected from antibodies, single chain antibodies, antibody fragments that bind to target cells, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments that bind to target cells, chimeric antibodies, chimeric antibody fragments that bind to target cells, domain antibodies, domain antibody fragments that bind to target cells, adnectin class antibodies, DARPin proteins, lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules (transferrin), and cell binding peptides, proteins or small molecules attached to albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles or (viral) capsids;

[0014] L1 and L2 are chain structures composed of atoms such as C, N, O, S, Si and P, preferably with 0-500 atoms, covalently linking W and V1, V1 and V2. The atoms constituting L1 and L2 can be combined in any chemical manner, for example, to form alkylene, alkenylene and alkynylene groups, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, diaminoureas, alkoxyamines, carbamates, amino acids, peptides, acyloxyamines, hydroxamic acids or combinations thereof. Preferably, L1 and L2, the same or different, are independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3'), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O; such as polyethyleneoxy of the following structural formula: (OCH2CH2) p OR3, or (OCH2CH(CH3)) p OR3, or NH(CH2CH2O) p R3, or NH(CH2CH(CH3)O) p R3, or N[(CH2CH2O) p R3]-[(CH2CH2O) p' R3'] or (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3, wherein p and p' are independently selected from integers ranging from 0 to about 1000, or combinations thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa) r , r = 1-12 (1 to 12 amino acid units), including natural or unnatural amino acids, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units of the same or different sequences;

[0015] W is a stretcher unit, typically a self-immolative spacer, a polypeptide unit, a hydrazone, a disulfide, a thioether, an ester or an amide bond; w is 1 or 2 or 3;

[0016] V1 and V2 are independent spacer units selected from O, NH, S, C1-C8 alkyl; C2-C8 heteroalkyl, alkenyl or alkynyl; C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylalkyl, heteroalkylcycloalkyl, or alkylcarbonyl; or (Aa) r, r = 1-12 (1-12 amino acid units), including natural or unnatural amino acids, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units of the same or different sequences; or (CH2CH2O) p , p is 0-1000; v1 and v2 are independently 0, 1 or 2, but v1 and v2 are not 0 at the same time. When v1 or v2 is 0, it means that the side chain Q1 or Q2 fragment is absent.

[0017] Q1 and Q2 are independently represented by formula (I-q1):

[0018]

[0019] in is the position connected to L1 or L2, G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, (Aa) r , (r = 1-12); X1 and X2 are independently O, CH2, S, NH, N(R 12 ), + NH(R 12 ), + N(R 12 )(R 13 ), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y2 is O, NH, NR1, CH2, S; G3 is OH, SH, OR1, SR1, OC(O)R1, NHC(O)R 12 、C(O)R 12 、CH3、NH2、NR 12 , + NH(R 12 ), + N(R 12 )(R 13 ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 13 , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2)q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH、OC(O)NH(CH2) q1 C(O)OH、NHCO(CH2) q1 P(O)(OH)2、NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2、NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH、OS(O)2NH(CH2) q1 C(O)OH、NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR1, Ar, ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 or (Aa) q1 , p1, p2 and p3 are independently 0-100, but not 0 at the same time, q1 and q2 are independently 0-24;

[0020] Preferably Q1 and Q2 are independently C2-C 90 Polycarboxylic acid or C-2-C 90 Polyalkylamine, C6-C 90 Oligosaccharides or polysaccharides, C6-C 90Zwitterions containing quaternary ammonium cations and carboxylic acid anions or zwitterions containing quaternary ammonium cations and sulfonic acid anions (PSB), biodegradable polymers, such as polylactic acid / glycolic acid (PLGA), poly(acrylates), chitosan, copolymers of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(ethylene glycol)-modified peptides, poly(ethylene glycol)-modified liposomes, poly(ethylene glycol)-modified alkyl carboxylic acids, poly(ethylene glycol)-modified alkylamines, hyaluronic acid (HA) (glycosaminoglycans), heparin or heparan sulfate (HSGAG), chondroitin sulfate or chondroitin sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfates, poly(ethylene glycol)-modified alkyl phosphates, or poly(ethylene glycol)-modified alkyl quaternary ammonium salts;

[0021] D is an amatoxin homologue having the following structural formula (II):

[0022]

[0023] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0024] Where ----- is independently a site to which W is attached;

[0025] A single bond on an aromatic (indole) ring means that it is attached to any carbon on the aromatic ring;

[0026] Indicates an optional single key or a key that can be omitted;

[0027] R1 and R2 are independently selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH3)CH2OH, CH(OH)CH3, C1-C8 alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (ester), –OC(=O)OR 12 -(carbonate), –OC(=O)NHR 12 (Carbamate); C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl;

[0028] R3 and R4 are independently selected from H, OH, –OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OCOOR 12(carbonate), –OC(=O)NHR 12 (Carbamate), -OP(O)(OR 12 )(OR 12 ')(phosphate), OP(O)(NHR 12 )(NHR 12 ')(phosphoramide), O-SO3 - , or O-glycosides;

[0029] R5 is selected from H, OH, NH2, NHOH, NHNH2, –OR 12、 –NHR 12 NHNHR 12 ,–NR 12 R 12 '、N(H)(R 12) R 13 CO(Aa) r , (amino acid or peptide, wherein Aa is an amino acid or a polypeptide, and r is 0-100);

[0030] R6 is selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH2OH)2, CH(CH3)OH, CH2CH2OH, PrOH, BuOH, C1~C8 alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic;

[0031] R7, R8 and R9 are independently selected from H, OH, CH3, CH(CH3)2, CH(CH3)CH2CH3, CH2OH, CH(OH)CH2OH, CH2CH(OH)CH2OH, CH(CH2OH)2, CH2C(OH)(CH2OH)2, CH2C(OH)(CH3)(CH2OH), CH2C( OH)(CH(CH3)2)(CH2OH), CH2CH2OH, PrOH, BuOH, CH2COOH, CH2CH2COOH, CH(OH)COOH, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2CH2CH2NHC(=NH)NH2, C1~C8 alkyl, CH2Ar, CH2SH, CH2SR 12 、CH2SSR 12 , CH2SSAr, CH2CH2SCH3, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic;

[0032] R 10 and R 11 Independently selected from H, NH2, OH, SH, NO2, halogen, -NHOH, -N3 (azido); -CN (cyano); C1-C8 alkyl, C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic or carbocyclic; -OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OC(O)OR 12 (carbonate), -OC(O)CH(R 12 )NHAa (Aa is an amino acid group), -NR 12 R 12 '(amine), -NR 12 COR 12 '(amide), -R 12 NHCOR 12’ (alkylamide), -R 12 NHR 12’ (Amine), -NHR 12 NHR 12 'NHR 12 ” (amine); –R 12 NCONR 12 '(urea); –R 12 NCOOR 12 '(Carbamate); –OCONR 12 R 12 '(Carbamate); –NR 12 (C=NH)NR 12 'R 12 ” (guanidine); –R 12 NHCO(Aa) p ,–R 12 NH R 12 'CO(Aa) p ,–NR 12 CO(Aa) p , (amino acid or peptide, where Aa is an amino acid or peptide, and p is 0-6); –N(R 12 )CONR 12 'R 12 ” (urea); –OCSNHR 12 (Thiocarbamate); -R 12 SH (sulfhydryl); –R 12 SR 12 '(sulfide); –R 12 SSR 12 '(disulfide); –S(O)R 12 (sulfoxide); –S(O2)R12 (sulfone); –SO3, HSO3, HSO2, or HSO3 - 、SO3 2- or -HSO2 - Salts (sulfites); –OSO3 - ;-N(R 12 )SOOR 12 '(Sulfonamide); H2S2O5 or S2O5 2- Salts (pyrosulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2 or PO3S 3- PO2S2 3- POS3 3- , PS4 3- Salts (mono-, di-, tri-, and tetra-thiophosphates); (R 12 O)2POSR 12 '(phosphorothioate); HS2O3 or S2O3 2- Salt (thiosulfate); HS2O4 or S2O4 2- Salts (dithionites); (P(=S)(OR 12 )(S)(OH) or salts formed with cations (dithiophosphates); -N(R 12 )OR 12 '(Hydroxyamine derivative); R 12 C(=O)NOH or salts formed with cations (hydroxamates); HOCH2SO2 - or its salts (formaldehyde sulfoxylate); –N(R 12 )COR 12 '(amide); R 12 R 12 'R 12 "NPO3H (trialkylphosphoramidate or phosphoramidite); or ArAr'Ar"NPO3H (triaryl phosphate); OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1; O-glycoside (glucoside, galactoside, mannoside, glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1'R2'R3'; R1', R2' and R3' are independently H, C1~C8 alkyl; Ar, Ar', and Ar" are C3-C8 aryl or heteroaryl;

[0033] R 12、 R 12 ', and R 12" is independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heteroaryl, heterocycle or carbocycle; or omitted;

[0034] X is S, O, NH, SO, SO2, or CH2;

[0035] m' is 0 or 1; n is 1-30.

[0036] In another aspect of the present invention, the structural formula of the conjugate containing a branched linker is represented as follows (III):

[0037]

[0038] Wherein, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, n and T are defined as in formula (I);

[0039] In another aspect of the present invention, the structure of the branched linker compound is shown in (IV), which can be conveniently reacted with the cell binding molecule T to form a conjugate in formula (I):

[0040]

[0041] Wherein, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are defined as the same as in structural formula (I); Lv1 is a functional group described below.

[0042] In another aspect of the present invention, the structure of the branched linker compound is shown in (V), which can be conveniently reacted with the cell binding molecule T to form a conjugate in formula (III):

[0043]

[0044] Wherein, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are defined the same as in structural formula (I).

[0045] Lv1 and Lv2 are the same or different reactive functional groups, which can react with thiol, amine, carboxylic acid, selenol, phenol or hydroxyl groups on the cell-binding molecules. Lv1 and Lv2 are independently selected from hydroxyl (OH); fluorine (F); chlorine (Cl); bromine (Br); iodine (I); nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; trifluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; trifluoromethanesulfonyl; imidazolyl; dichlorophenol; trichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazolyl; toluenesulfonyl; mesyl; 2-ethyl-5-phenylisoxazole-3'-sulfonyl, anhydride or anhydride formed by reaction with other anhydrides, such as acetic anhydride and formic anhydride; or an intermediate generated by reaction with a polypeptide condensation reagent or a Mitsunobu reaction reagent.Examples of condensing agents are as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide methyl p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), oxy-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-oxy-(1H-benzotriazol-1-yl)-ammonium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethylamino)-hexafluorophosphate (BTP), OP), (benzotriazol-1-yloxy)tripyrrolidinium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidine hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethyl-imidazolium hexafluorophosphate (CIP), chloropyrrolidinium hexafluorophosphate (PyCloP), fluoro-N,N,N',N' -bis(tetramethylene)formamidine hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiourea hexafluorophosphate, oxy-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxo-2-pyridyl)N,N,N',N'-tetramethylthiourea tetrafluoroborate, oxy-[(ethoxycarbonyl)-cyanomethylamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylaminooxy)dimethylamino-morpholino-hexafluorophosphate (COMU), oxy-(benzotriazol-1-yl)- N,N,N',N'-bis(tetramethylene)hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexyl-carbodiimide (with or without polymer binding), dipyrrolidinyl(N-succinimidyloxy)carbonium hexafluorophosphate (HSPyU), chlorodipyrrolidinylhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy)dipiperidinylcarbonium hexafluorophosphate (HBPipU), oxy-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromo(dimethylamino)-hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA,. ), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-oxy-(N-succinimidyl) hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyano-methyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N' , N'-tetramethyl-oxy-(N-succinimidyl) uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)-dipiperidinium (ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD). In addition, Lv1 and Lv2 can be anhydrides, or anhydrides formed by reacting with other C1-C8 anhydrides.

[0046] The present invention further relates to a method for preparing the cell binding molecule-drug conjugates in (I) and (III), and the use of the conjugates in (I) and (III). BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 shows the general synthetic route of the amatoxin analog fragments.

[0048] Figure 2 shows the synthesis of amatoxin analog fragments.

[0049] Figure 3 shows the synthesis of amatoxin analogs.

[0050] Figure 4 shows the synthesis of amatoxin analogs.

[0051] Figure 5 shows the synthesis of amatoxin analogs containing branched linkers.

[0052] FIG6 shows the synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody.

[0053] FIG. 7 shows the synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody.

[0054] FIG8 shows the synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody.

[0055] Figure 9 shows the synthesis of amatoxin analogs containing branched linkers

[0056] FIG. 10 shows the synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody.

[0057] Fig.11 The synthesis of amatoxin analogs containing branched linkers is shown.

[0058] Fig.12 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0059] Fig.13 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0060] Fig.14 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0061] Fig.15 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0062] Fig.16 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0063] Fig.17 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0064] Fig.18 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0065] Fig.19 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0066] FIG. 20 shows the synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody.

[0067] Fig.21 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0068] Fig. 22 The synthesis of amatoxin analogs containing branched linkers is shown.

[0069] Fig.23 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0070] Fig.24 The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0071] Fig.25The synthesis of an amatoxin analog containing a branched linker and its conjugation to an antibody are shown.

[0072] Fig.26 The synthesis of amatoxin analogs containing branched linkers is shown.

[0073] Fig. 27 The comparison of the antitumor effects of conjugated compounds 78a, 146, 154, 167, 197, 198, 216, 240, S-2 and T-DM1 in the human gastric cancer N87 cell model at a single intravenous injection at a dose of 6 mg / kg is shown.

[0074] Fig.28 Shown is an acute toxicity study of ADC conjugates 154, 146, 216, S-2, and T-DM1 by observing changes in mouse body weight (BW) for 12 days. Summary of the invention

[0075] definition

[0076] "Alkyl" refers to an aliphatic hydrocarbon group or a monovalent group produced by removing one or two hydrogen atoms from an alkane. It can be straight or branched, with C1-C8 (1-8 carbon atoms) in the chain. "Branched" means that one or more lower carbon number alkyl groups, such as methyl, ethyl or propyl, are attached to a straight chain alkyl group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methyl-hexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. The C1-C8 alkyl group may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from -C1-C8 alkyl and aryl.

[0077] "Halogen" refers to fluorine, chlorine, bromine or iodine atoms; fluorine and chlorine atoms are preferred.

[0078] "Heteroalkyl" refers to a C2-C8 alkyl group in which 1 to 4 carbon atoms are independently replaced by heteroatoms selected from O, S and N.

[0079] "Carbocycle" refers to a saturated or unsaturated monocyclic ring containing 3 to 8 carbon atoms, or a saturated or unsaturated bicyclic ring containing 7 to 13 carbon atoms. Monocyclic carbocycles have 3 to 6 ring atoms, typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, forming a [4,5], [5,5], [5,6] or [6,6] bicyclic ring system, or have 9 or 10 ring atoms, forming a [5,6] or [6,6] bicyclic ring system. Representative C3-C8 carbocycles include, but are not limited to: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl and -cyclooctadienyl.

[0080] The C3-C8 carbocycle may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from -C1-C8 alkyl and aryl.

[0081] "Alkenyl" refers to a straight or branched aliphatic hydrocarbon group containing a carbon-carbon double bond, containing 2-8 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, hexenyl, heptenyl, octenyl.

[0082] "Alkynyl" refers to a straight or branched aliphatic hydrocarbon group containing a carbon-carbon triple bond, containing 2-8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexynyl, heptynyl and octynyl.

[0083] "Alkylene" refers to a saturated branched or straight chain or cyclic hydrocarbon group containing 1 to 18 carbon atoms and having two monovalent free radicals generated by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), etc.

[0084] "Alkenylene" refers to an unsaturated branched or straight chain or cyclic hydrocarbon group containing 2-18 carbon atoms and having two monovalent free radicals generated by removing two hydrogen atoms from the same or two different carbon atoms of the parent olefin. Typical alkenylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0085] "Alkynylidene" refers to an unsaturated branched or straight chain or cyclic hydrocarbon radical containing 2-18 carbon atoms and having two monovalent free radicals generated by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical alkynylene groups include, but are not limited to, acetylene, propargyl and 4-pentynyl.

[0086] "Aryl" or "aromatic group" refers to an aromatic or heteroaromatic group consisting of one or more rings, containing three to fourteen carbon atoms, preferably six to ten carbon atoms. The term "heteroaromatic group" refers to a group generated by replacing one or more carbon atoms, preferably one, two, three or four carbon atoms, on an aromatic group with oxygen (O), nitrogen (N), silicon (Si), selenium (Se), phosphorus (P) or (S), preferably with oxygen, sulfur and nitrogen. The term "aryl" or "aromatic group" also refers to an aromatic group in which one or more hydrogen atoms are independently replaced by -R', halogen, -OR', -SR', -NR'R", -N=NR', -N=R', -NR'R", -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O)2OR', -PR'R", -P(O)R'R", -P(OR')(OR"), -P(O)(OR')(OR") or -OP(O)(OR')(OR"), wherein R' and R" are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl or a pharmaceutically acceptable salt thereof.

[0087] "Heterocycle" refers to a ring structure in which one to four ring carbon atoms are independently replaced by heteroatoms such as O, N, S, Se, B, Si or P. Preferred heteroatoms are O, N and S. The relevant description of heterocyclic compounds is also on pages 225-226 of The Handbook of Chemistry and Physics, 78th Edition, CRC Press, Inc., 1997-1998, p. 225 to 226, which is hereby incorporated by reference. Preferred non-aromatic heterocycles include epoxy, aziridinyl, thipropyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, and condensed ring systems derived from the above groups with phenyl.

[0088] The term "heteroaryl" or "aryl heterocycle" refers to an aromatic heterocyclic ring containing 3 to 14, preferably 5 to 10 atoms, including monocyclic, bicyclic or polycyclic rings. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and condensed ring systems obtained by condensing the above groups with phenyl.

[0089] "Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocycle", etc. also include the corresponding "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene", "heterocycle", etc. For the convenience of discussion, this patent application does not make a complete distinction between them.

[0090] "Aralkyl" refers to a non-cyclic alkyl group in which one of the alkyl groups is attached to a carbon atom (usually a terminal or sp 3 In the embodiment of the present invention, the hydrogen atom bonded to the carbon atom of the present invention is replaced by an aryl group. Typical aralkyl groups include benzyl, 2-phenylethyl-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, 2-naphthylethyl-1-yl, naphthobenzyl, 2-naphthylphenyl-1-yl, etc.

[0091] "Heteroaralkyl" refers to a class of non-cyclic alkyl groups in which one of the alkyl groups is attached to a carbon atom (usually a terminal or sp 3The hydrogen atom bonded to the carbon atom of the present invention is replaced by a heteroaryl group. Examples of heteroarylalkyl groups are 2-benzimidazolylmethyl, 2-furylethyl.

[0092] Examples of the “hydroxy protecting group” include methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, tert-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate, substituted acetate, pivalate, benzoate, methanesulfonate and p-toluenesulfonate.

[0093] "Leaving group" refers to a functional group that can be replaced by another functional group. These leaving groups are well known in the art, and examples include halides (such as chlorides, bromides and iodides), methanesulfonyl, p-toluenesulfonyl and trifluoromethanesulfonyl. Preferred leaving groups are selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; trifluoromethanesulfonyl; imidazolyl; chlorophenol; tetrachlorophenol; 1-hydroxybenzotriazolyl; toluenesulfonyl; methanesulfonyl; 2-ethyl-5-phenylisoxazole-3'-sulfonyl, anhydride or anhydride formed by reaction with other anhydrides, such as acetic anhydride, formic anhydride; or intermediates generated by reaction with polypeptide condensation reagents and Mitsunobu reaction reagents.

[0094] The following abbreviations are used in the present invention and are defined as follows: Boc, tert-butoxycarbonyl; BroP, tetradecylphosphonium bromide hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DEAD, diethyl azodicarboxylate; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA or DEA, diisopropylethylamine; DEPC, diethylcyanophosphate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray ionization mass spectrometry; EtOAc, ethyl acetate; Fmoc, N-(9-fluorenylmethoxycarbonyl); HATU, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MeCN, acetonitrile; MeOH, methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzoic acid; PBS, phosphate buffer (pH 7.0-7.5); Ph, phenyl; Phe, L-phenylalanine; PyBrop, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC, thin layer chromatography; UV is ultraviolet light.

[0095] "Amino acid" can be natural or non-natural, preferably α-amino acid. Natural amino acids can be encoded by genetic code, and they are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine. Unnatural amino acids are derivatives of proteinogenic amino acids and include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (neurotransmitter), ornithine, citrulline, β-alanine (3-aminopropionic acid), γ-carboxyglutamate, selenocysteine ​​(present in many non-eukaryotic and most eukaryotic cells, but not directly encoded by DNA), pyrrolysine (found only in some archaea and one bacterium), N-formylmethionine (usually the first amino acid in proteins in bacteria, mitochondria and chloroplasts), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA) and O-phosphoserine. The term "amino acid" also includes amino acid homologs and mimetics. Homologs are compounds with the same structural formula H2N(R)CHCO2H as natural amino acids, where R is in the natural amino acid. Examples of homologs include homoserine, norleucine, methionine-sulfoxide and methionine methylsulfonium. More preferred are amino acid mimetics, which are compounds with chemical structures different from α-amino acids but with similar modes of action. Natural amino acids are mostly in the "L" stereochemical configuration, and "unnatural amino acids" are also used to represent amino acids in the "D" configuration. When 1 to 8 amino acids are used in this patent application, their sequence is preferably a sequence that can be recognized by proteolytic enzymes. Many hydrolase recognition sequences are known in the art and can be found in: Matayoshi et al. Science 247:954 (1990); Dunn et al. Meth. Enzymol. 241:254 (1994); Seidah et al. Meth. Enzymol. 244:175 (1994); Thornberry, Meth. Enzymol. 244:615 (1994); Weber et al. Meth. Enzymol. 244:595 (1994); Smith et al. Meth. Enzymol. 244:412 (1994); and Bouvier et al. Meth. Enzymol. 248:614 (1995); which are incorporated herein by reference.In particular, it is selected from the following sequences: Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Asp-Lys, Asp-Glu, Glu-Lys, Lys, Cit, Ser and Glu.

[0096] A "glycoside" is a molecule in which a sugar is bonded to another group at its anomeric carbon via a glycosidic bond. Glycosides can be linked via an O- (yielding O-glycosides), N- (yielding glycosylamines), S- (yielding thioglycosides), or C- (yielding C-glycosides) glycosidic bond, and its empirical formula is C m (H2O) n (wherein m may be different from n, m, n<36), the glycosides in the present invention include glucose (dextrose), fructose (levulose), allose, altrose, mannose, gulose, idose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfonylquinovose (6-deoxy-6-sulfonyl-D-pyranose glucose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide) and stachyose. It can be in the configuration of D or L, in the form of a 5-atom cyclic furanose, in the form of a 6-atom cyclic pyranose, or in the non-cyclic form, α-isomer (-OH of the anomeric carbon is below the Haworth projection carbon atom plane), or β-isomer (-OH of the anomeric carbon is above the Haworth projection carbon atom plane). In the present invention, it is also referred to as monosaccharide, disaccharide, polyol or oligosaccharide containing 3-6 sugar units.

[0097] "Antibody" in the present invention refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, such as a molecule containing an antigen binding site that immunospecifically binds to a target antigen or a portion of a target antigen, wherein the target includes, but is not limited to, a cancer cell or a cell that produces autoimmune antibodies associated with an autoimmune disease. The immunoglobulin in the present invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin. The immunoglobulin can be from any species, but preferably the immunoglobulin is derived from a human, mouse, or rabbit. The antibodies of the present invention are preferably monoclonal antibodies, including but not limited to polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single-chain antibodies, Fv, Fab fragments, F(ab') fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs and any epitope-binding fragments of the above structures that can immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens.

[0098] An "enantiomer", also called an "optical isomer", is one of two stereoisomers that are mirror images of each other and are not superimposable (identical), just like the left and right hands of a person are not superimposable unless flipped along a plane (you cannot make the hands superimposable by simply changing their orientation). A single chiral atom or similar structural feature in a compound gives the compound two possible structures that are not superimposable and are mirror images of each other. The presence of multiple chiral features in a compound increases the number of possible configurations, some of which may be mirror images of each other. An enantiomerically pure compound is one that has only one chirality within the capability of the detection method. In a symmetrical environment, the two enantiomers can rotate plane polarized light (+ / -) in opposite directions by equal amounts (polarized light can be considered an asymmetric medium), but otherwise have identical chemical and physical properties. For this reason, they are sometimes called optical isomers. A mixture of optically active isomers and equal amounts of their enantiomers is called a racemate, which has no net rotation of plane polarized light because every positive rotation (+) is exactly canceled by a negative rotation (-). Usually two enantiomers react differently to other enantiomers. Because many biological molecules are enantiomers, sometimes there are significant differences in the effects of the two enantiomers on biological organisms. For example, in drugs, usually only one enantiomer can produce the desired physiological effect, while the other enantiomer is either less active or inactive, and sometimes even produces adverse reactions. Based on such discoveries, drugs consisting of only one enantiomer ("enantiomerically pure") can be developed to enhance pharmacological efficacy and sometimes eliminate some side effects.

[0099] Isotopes are different nuclides of a particular chemical element that have the same number of protons but different numbers of neutrons. All isotopes of the same element have the same number of protons. An atomic number designates a specific element, but not an isotope; atoms of a particular element can have different numbers of neutrons. The number of nucleons (protons and neutrons) is the mass number of the atom, and each isotope of a particular element has a different mass number. For example, carbon-12, carbon-13, and carbon-14 are three isotopes of the element carbon, with mass numbers 12, 13, and 14, respectively. Carbon's atomic number is 6, meaning that each carbon atom has 6 protons, so the numbers of neutrons in these isotopes are 6, 7, and 8, respectively. Hydrogen atoms have three isotopes: protium (1H), deuterium ( 2 H) and tritium ( 3 H), deuterium has twice the mass of protium, and tritium has three times the mass of protium. Isotope substitution experiments can be used to determine the mechanism of chemical reactions through the kinetic isotope effect. Isotope substitution experiments can also be used to study how the body reacts to exogenous compounds after they enter the body through absorption and distribution mechanisms, metabolic changes in substances in the body (for example, through the action of metabolic enzymes such as cytochrome P450 or glucuronyl transferase), and the excretion pathways of drug metabolites, which are pharmacokinetic (PK) studies. Isotope substitution experiments can be used to study the biochemical and physiological effects of drugs, including the effects manifested in animals (including humans), microorganisms or biological combinations (such as infections), which are pharmacodynamic (PD) studies. The two (PK and PD) together determine the dosage, benefits and side effects of the drug. Isotopes can be a stable (non-radioactive) or unstable element. Isotope substitutions of drugs may also have different therapeutic effects than the original drug.

[0100] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans, as appropriate.

[0101] "Pharmaceutically acceptable solvate" or "solvate" refers to a combination of one or more solvent molecules and the compounds disclosed herein. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0102] "Pharmaceutically acceptable excipients" include any carrier, diluent, adjuvant or other, such as preservatives or antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. These media and agents are well known in the art for pharmaceutically active substances. Any conventional media or agents, unless incompatible with the active ingredient, may also be considered for use in the therapeutic composition. Supplementary active ingredients may also be added to the composition to form a suitable therapeutic combination.

[0103] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, obtained by preparing acid or base salts of the parent compound. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts formed by non-toxic inorganic or organic acids and the parent compound. For example, the conventional non-toxic salts include salts derived from inorganic acids (e.g., hydrochloric acid, hydrobromide, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.); and salts prepared from organic acids (e.g., acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, maleic acid and lactic acid, etc. Other addition salts include ammonium salts, such as salts of trimethylamine, meglumine, glycerol, etc., metal salts, such as sodium, potassium, calcium, zinc or magnesium salts.

[0104] The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. Generally speaking, these salts can be prepared by adding an equivalent amount of a suitable base or acid to the parent compound in water or an organic solvent or a mixture of the two. Generally speaking, ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred non-aqueous media. A list of suitable salts is found in Remington's Pharmaceutical Sciences, published by Mack Publishing Company in 1985, the disclosure of which is incorporated herein by reference.

[0105] "Administering" or "administering" refers to transferring, delivering, introducing or transporting a drug or other agent to a subject by any means. Such means include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous or intrathecal administration. The present invention also contemplates the use of a device or apparatus to administer the agent. Such a device may use active or passive transmission and may be a slow release or rapid release delivery device.

[0106] In the context of cancer, the term "treating" includes any or all of the following: preventing tumor or cancer cell growth, replication, reducing overall tumor mass, and ameliorating one or more symptoms associated with the disease.

[0107] With respect to autoimmune diseases, the term "treating" includes any or all of the following: preventing the replication of cells associated with the autoimmune disease, including but not limited to cells capable of producing autoimmune antibodies, reducing the amount of autoimmune antibodies and ameliorating one or more symptoms of the autoimmune disease.

[0108] With respect to an infectious disease, the term "treating" includes any or all of: preventing the growth, proliferation or replication of the pathogen that causes the infectious disease, as well as ameliorating one or more symptoms of the infectious disease.

[0109] Examples of "mammals" or "animals" include, but are not limited to, humans, mice, rats, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry.

[0110] The novel conjugate disclosed in the present invention uses a branched linker. Examples 1 to 26 show some suitable linkers and their synthesis.

[0111] A conjugate of a cell binding agent-cytotoxic molecule connected by a branched linker

[0112] In one aspect of the present invention, the structure of the conjugate containing a branched linker is represented by formula (I):

[0113]

[0114] “-” represents a single bond; n is 1 to 30;

[0115] T is a cell binding agent or molecule selected from an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody, a domain antibody fragment that binds to a target cell, an adnectin-like antibody, a DARPin protein, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule (transferrin) and / or a cell binding peptide, protein or small molecule attached to an albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle or a (viral) capsid;

[0116] L1 and L2 are chain structures composed of atoms such as C, N, O, S, Si and P, preferably with 0-500 atoms, covalently linking W and V1, V1 and V2. The atoms constituting L1 and L2 can be combined in any chemical manner, for example, to form alkylene, alkenylene and alkynylene groups, ethers, polyoxyalkylenes, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, diaminoureas, alkoxyamines, carbamates, amino acids, peptides, acyloxyamines, hydroxamic acids or combinations thereof. Preferably, L1 and L2, the same or different, are independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3'), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O; such as polyethyleneoxy of the following structural formula: (OCH2CH2) p OR3, or (OCH2CH(CH3)) p OR3, or NH(CH2CH2O) p R3, or NH(CH2CH(CH3)O) p R3, or N[(CH2CH2O) p R3]-[(CH2CH2O)p' R3'] or (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3, wherein p and p' are independently selected from integers ranging from 0 to about 1000, or combinations thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa) r , r = 1-12 (1 to 12 amino acid units), (Aa) r is a natural or unnatural amino acid, a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit of the same or different sequence;

[0117] W is a stretcher unit, typically a self-immolative spacer, a polypeptide unit, a hydrazone, a disulfide, a thioether, an ester or an amide bond; w is 1 or 2 or 3;

[0118] V1 and V2 are independent spacer units selected from O, NH, S, C1-C8 alkyl; C2-C8 heteroalkyl, alkenyl or alkynyl; C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylalkyl, heteroalkylcycloalkyl, or alkylcarbonyl; or (Aa) r , r = 1-12 (1-12 amino acid units), (Aa) r is a natural or non-natural amino acid, a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit of the same or different sequence; or (CH2CH2O) p , p is 0-1000; v1 and v2 are independently 0, 1 or 2, but v1 and v2 are not 0 at the same time. When v1 or v2 is 0, it means that the side chain Q1 or Q2 fragment is absent.

[0119] Q1 and Q2 are independently represented by formula (I-q1):

[0120]

[0121] in is the position connected to L1 or L2, G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, or (Aa)q1 ; G3 is OH, SH, OR 12 , SR 12 , OC(O)R 12 , NHC(O)R 12 , C(O)R 12 , CH3, NH2, NR 12 , + NH(R 12 ), + N(R 12 )(R 12’ ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 12’ , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH, OC(O)NH(CH2) q1 C(O)OH, NHCO(CH2) q1 P(O)(OH)2, NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2, NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH, OS(O)2NH(CH2) q1 C(O)OH, NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR 12、Ar、ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 , or (Aa) q1 ; (Aa) q1 is a peptide containing the same or different sequences of natural or unnatural amino acids; X1 and X2 are independently O, CH2, S, S(O), NHNH, NH, N(R 12 ), + NH(R 12 ), + N(R 12 )(R 12’ ), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y2 is O, NH, NR 12 , CH2, S, NHNH, Ar; p1, p2 and p3 are independently 0-100 but not 0 at the same time; q1 and q2 are independently 0-24; R 12 , R 12 '、R 13 and R 13 ' is independently H, C1-C8 alkyl; C2-C8 heteroalkyl, or heterocycle; C3-C8 aryl, aralkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocycle or alkylcarbonyl;

[0122] Preferably Q1 and Q2 are independently C2-C 100 Polycarboxylic acid or C-2-C 90 Polyalkylamine, C6-C 90 Oligosaccharides or polysaccharides, C6-C 100 Zwitterionic betaines or zwitterionic poly(sulfonyl betaines) (PSB) containing quaternary ammonium cations and sulfonate anions, C6-C 100 Biodegradable polymers such as polylactic acid / glycolic acid (PLGA), poly(acrylates), chitosan, copolymers of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(ethylene glycol)-modified peptides, poly(ethylene glycol)-modified liposomes, poly(ethylene glycol)-modified alkyl carboxylic acids, poly(ethylene glycol)-modified alkylamines, hyaluronic acid (HA) (glycosaminoglycans), heparin or heparan sulfate (HSGAG), chondroitin sulfate or chondroitin sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfates, poly(ethylene glycol)-modified alkyl phosphates, or poly(ethylene glycol)-modified alkyl quaternary ammonium salts;

[0123] The structure examples of Q1 and Q2 are as follows:

[0124]

[0125]

[0126]

[0127] Among them, R 25 and R 25 'Independently selected from H, HC(O), CH3C(O), CH3C(NH), C1-C 18 Alkyl, C1-C 18 Alkyl-Y1-SO3H, C1-C 18 Alkyl-Y1-PO3H2, C1-C 18 Alkyl-Y1-CO2H, C1-C 18 Alkyl-Y1-N + R1'R2'R3'R4', C1-C 18 Alkyl-Y1-CONH2, C2-C 18 Alkyl, C2-C 18 Esters, C2-C 18 Ether, C2-C 18 Amine, C2-C 18 Alkyl carboxamide, C3-C 18 Aryl, C3-C 18 Cycloalkyl, C3-C 18 Heterocycle, 1-24 amino acids, C2-C 18 Lipids, C2-C 18 Fatty acids or C2-C 18 Fatty ammonium lipids; X1 and X2 are independently selected from NH, N(R1'), O, CH2, S, C(O), S(O), S(O2), P(O)(OH), NHNH, CH=CH, Ar or (Aa) q1 , q1=0-24 (0-24 amino acids, q1=0 indicates default); X1, X2, X3, X4, Y1, Y2 and Y3 are independently selected from NH, N(R1'), O, C(O), CH2, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH, Ar or (Aa) q1 , X1, X2, X3, X4, Y1, Y2 and Y3 are independently optional; p1, p2 and p3 are independently 0-100, but cannot be 0 at the same time; q1, q2 and q3 are independently 0-24; R1', R2', R3' and R4' are independently selected from H and C1-C6 alkyl; Aa is a natural or non-natural amino acid; Ar or (Aa) q1 are the same or different peptide sequences; q1=0 means (Aa)q1 is absent;

[0128] D is an amatoxin homologue having the following formula (II):

[0129]

[0130] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0131] Among them ----- is the site connected to W;

[0132] A single bond on an aromatic (indole) ring refers to its attachment to any carbon of the aromatic ring;

[0133] Indicates an optional single key or optional default;

[0134] R1 and R2 are independently selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH3)CH2OH, CH(OH)CH3, C1-C8 alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (ester), –OC(=O)OR 12- (carbonate), –OC(=O)NHR 12 (Carbamate); C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.

[0135] R3 and R4 are independently selected from H, OH, –OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OCOOR 12 (carbonate), –OC(=O)NHR 12 (Carbamate), -OP(O)(OR 12 )(OR 12 ')(phosphate), OP(O)(NHR 12 )(NHR 12 ')(phosphoamine), O-SO3 - , or O-glycosides;

[0136] R5 is selected from H, OH, NH2, NHOH, NHNH2, –OR 12 , –NHR 12 NHNHR 12 ,–NR 12 R 12 '、N(H)(R 12) R 13 CO(Aa)r , (amino acid or peptide, wherein Aa is an amino acid or a polypeptide, r = 0-6);

[0137] R6 is selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH2OH)2, CH(CH3)OH, CH2CH2OH, PrOH, BuOH, C1~C8 alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic;

[0138] R7, R8 and R9 are independently selected from H, OH, CH3, CH(CH3)2, CH(CH3)CH2CH3, CH2OH, CH(OH)CH2OH, CH2CH(OH)CH2OH, CH(CH2OH)2, CH2C(OH)(CH2OH)2, CH2C(OH)(CH3)(CH2OH), CH2C( OH)(CH(CH3)2)(CH2OH), CH2CH2OH, PrOH, BuOH, CH2COOH, CH2CH2COOH, CH(OH)COOH, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2CH2CH2NHC(=NH)NH2, C1~C8 alkyl, CH2Ar, CH2SH, CH2SR 12 、CH2SSR 12 , CH2SSAr, CH2CH2SCH3, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic;

[0139] R 10 and R 11 Independently selected from H, NH2, OH, SH, NO2, halogen, -NHOH, -N3 (azido); -CN (cyano); C1-C8 alkyl, C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic or carbocyclic; -OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OC(O)OR 12 (carbonate), -OC(O)CH(R 12 )NHAa (Aa is an amino acid group), -NR 12 R 12 '(amine), -NR 12 COR 12 '(amide), -R 12NHCOR 12 '(alkylamide), -R 12 NHR 12 '(Amine), NHR 12 NHR 12 'NHR 12 ” (amine); –R 12 NCONR 12 '(urea); –R 12 NCOOR 12 '(Carbamate); –OCONR 12 R 12 '(Carbamate); –NR 12 (C=NH)NR 12 'R 12 ” (guanidine); –NR 12 CO(Aa) p , (amino acid or peptide, where Aa is an amino acid or peptide, and p is 0-6); –N(R 12 )CONR 12 'R 12 ” (urea); –OCSNHR 12 (Thiocarbamate); -R 12 SH (sulfhydryl); –R 12 SR 12 '(sulfide); –R 12 SSR 12 '(disulfide); –S(O)R 12 (sulfoxide); –S(O2)R 12 (sulfone); –SO3, HSO3, HSO2, or HSO3 - 、SO3 2- or -HSO2 - Salts (sulfites); –OSO3 - ;-N(R 12 )SOOR 12 '(Sulfonamide); H2S2O5 or S2O5 2- Salts (pyrosulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2 or PO3S 3- PO2S2 3- POS3 3- , PS4 3- Salts (mono-, di-, tri-, and tetra-thiophosphates); (R 12 O)2POSR 12 '(phosphorothioate); HS2O3 or S2O3 2- (Thiosulfate); HS2O4 or S2O4 2-Salts (dithionites); (P(=S)(OR 12 )(S)(OH) or salts formed with cations (dithiophosphates); -N(R 12 )OR 12 '(Hydroxyamine derivative); R 12 C(=O)NOH or salts formed with cations (hydroxamates); HOCH2SO2 - , or its salts (formaldehyde sulfoxylate); –N(R 12 )COR 12 '(amide); R 12 R 12 'R 12 "NPO3H (trialkylaminophosphoric acid or aminophosphoric acid); or ArAr'Ar"NPO3H (triarylphosphoric acid); OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1; O-glycoside (glucoside, galactoside, mannoside, glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1'R2'R3'; R1', R2' and R3' are independently H, C1~C8 alkyl; Ar, Ar', and Ar" are C3-C8 aryl or heteroaryl;

[0140] Where R 12 , R 12 ', and R 12 " is independently selected from H, C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atom ester, ether or amide; or (OCH2CH2) p or (OCH2CH(CH3)) p The polyoxyethylene units are selected from the group consisting of: polyoxyethylene units, wherein p is an integer from 0 to 1000, or a combination thereof, or is absent.

[0141] X is S, O, NH, SO, SO2 or CH2.

[0142] m' is 0 or 1; n is 1-20.

[0143] Examples of preferred amatoxin structures are as follows:

[0144]

[0145]

[0146] More preferably, the amatoxin structure is selected from:

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0153] Where Z2 is oxygen or a lone pair of electrons; R 15 YesH、NHR 12 , OR 12 , C1-C8 straight chain or branched alkyl or heteroalkyl; C2-C8 straight chain or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 straight chain or branched aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-R1C(O)OR 12 ), carbamate (-R 12 C(O)NR 12’ R 13 ); or a carboxylic acid ester, ester, ether or amide of 1 to 8 carbon atoms; or 1 to 8 amino acids; or such as (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000; Z1 is H, O, S, NH, NHNH, R 12 , or default; R 21 It is COR 12 、NHCOR 12 、COOR 12 ,CONHR 12 , R 12 , R 12 NH; R 22 YesR 12 , SR 12 、SCH(CH3)R 12 SC(CH3)2R 12 , X is O, S, NH, NHNH, or CH2. R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 12’ , R 13 The definitions of , and X1 are the same as above;

[0154] In addition, W, L1, L2, V1 and V2 can independently be composed of one or more of the following linker components: 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), p-aminobenzyloxy-carbonyl ("PAB"), 4-thiopentanoyl ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1 acyl ("MCC"), (4-acetyl)aminobenzoyl ("SIAB"), 4-thiobutyryl (SPDB), 4-thio-2-hydroxysulfonyl-butyryl (2-Sulfo-SPDB), the structure of which is shown below, or a natural or non-natural peptide containing 1-12 natural or non-natural amino acid units. The natural amino acids are preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, and alanine;

[0155] 6-Maleimidocaproyl (MC),

[0156] Maleimidopropionyl (MP),

[0157] Valine-citrulline (val-cit),

[0158] Alanine-phenylalanine (ala-phe), Lysine-phenylalanine (lys-phe), p-Aminobenzyloxy-carbonyl (PAB), 4-Thiopentanoyl (SPP), 4-Thiobutyryl (SPDB), 4-(N-Maleimidomethyl)cyclohexane-1-yl (MCC), Maleimidoethylamino (ME), 4-Sulfo-2-hydroxysulfonyl-butyryl (2-Sulfo-SPDB), Aryl sulfide group (PySS), (4-Acetyl)aminobenzoyl (SIAB), Oxybenzyl sulfide group, Aminobenzyl sulfide, Dioxybenzyl sulfide group, Diaminobenzyl sulfide group, Aminooxybenzyl sulfide, Alkoxyamino (AOA), Ethyleneoxy (EO), 4-Methyl-4-thio-pentanoyl (MPDP), Triazole, Disulfide, Alkylsulfonyl, Alkyl sulfonamides, Disulfonamide, Phosphodiamide, Alkylphosphonamide, Phosphonic acid, N-Methylalkylphosphonamide, N,N'-dimethylphosphoramide, Alkylphosphine diamide, Hydrazine, Acetamidine; Oxime, Diacetylhydrazine, Aminoethylamine, Aminoethyl-aminoethylamine

[0159] and L- or D-, natural or non-natural peptides containing 1-20 amino acids; wherein is a linking site; preferably, X2, X3, X4, X5 or X6 are independently selected from NH; NHNH; N(R 12 ); N(R 12 )N(R 12 '); O; S; C1-C6 alkyl; C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; CH2OR 12 、CH2SR 12 、CH2NHR 12 or 1 to 8 amino acids; 12 and R 12 ' is independently H; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; ester, ether or amide of 1-8 carbon atoms; or a polyoxyethylene unit of the formula (OCH2CH2)p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or a combination of the above.

[0160] W, L1, L2, V1 and V2 independently may contain self-destructive or non-self-destructive components, peptide units, hydrazone bonds, disulfide bonds, esters, oximes, amides or thioether bonds. Self-destructive units include, but are not limited to, aromatic compounds with electronic structures similar to p-aminobenzylcarbamoyl (PAB), such as derivatives of 2-aminoimidazole-5-methanol, heterocyclic PAB homologues, β-glucuronides, and o- or p-aminobenzyl acetals;

[0161] Preferred self-destructive linker components have one of the following structures:

[0162]

[0163] Wherein, * marks the attachment point of additional spacers or releasable linkers, or cytotoxic agents, and / or binding molecules (CBA); X 1 , Y 1 , Z 2 and Z 3 are independently NH, O or S; Z 1 H, NHR1, OR1, SR 1、 COX1R1, wherein X1 and R1 are as defined above; v is 0 or 1; U 1 H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5') or OCH2PO(OR5(OR5'), wherein R5 and R5' are independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl or amino acid; C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl or glycoside; or a pharmaceutically cationic salt;

[0164] W, L1, L2, V1 and V2 can independently be a non-self-immolative linker component comprising one of the following structures:

[0165]

[0166]

[0167] Wherein, * marks the attachment point of additional spacers or releasable linkers, or cytotoxic agents, and / or binding molecules (CBA); X 1 , Y 1 , U 1 , R5, R5' are as defined above; r is 0-100; m and n are independently 0-20;

[0168] More preferably, W, L1, L2, V1 and V2 are independently releasable linker components. The term "releasable" refers to a linker comprising at least one bond that can be broken under physiological conditions, such as pH, acid, base, oxidation, metabolism, biochemical or enzyme unstable bonds. It should be understood that the bond breakage is not necessarily caused by a biological or metabolic process, but may be a chemical reaction, such as hydrolysis or substitution reaction. Examples of such physiological conditions include: endosomes with a lower pH than the pH in the cytoplasm, and a large amount of glutathione in millimolar concentrations in malignant cells, which can undergo disulfide exchange reactions with intracellular thiols;

[0169] Examples of releasable components in W, L1, L2, V1, and V2 include, but are not limited to:

[0170] -(CR 15 R 16 ) m (Aa)r(CR 17 R 18 ) n (OCH2CH2) t -,-(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -,-(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -,-(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -,-(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -,-(CR 15 R16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -,-(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m (OCO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m -(CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -,-(CR 15 R16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -,-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -,-(CR 15 R 16 ) m -phenyl-CO(Aa) t- (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Furan-CO(Aa) t (CR 17 R 18 ) n -,-(CR 15 R6) m -Oxazole-CO(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t (CCR 17 R 18 ) n -,-(CR 15 R 16 ) t -Thiophene-CO(CR 17 R 18 ) n -,-(CR 15 R 16 ) t -imidazole-CO-(CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Morpholine-CO(Aa) t- (CR 17 R 18) n -,-(CR 15 R 16 ) t -Piperazine-CO(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -N-Methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -(Aa) t Phenyl-,-(CR 15 R 16 ) m -(Aa) t Furan-,-(CR 15 R 16 ) m -Oxazole (Aa) t -,-(CR 15 R 16 ) m -Thiazolyl(Aa) t -,-(CR 15 R 16 ) m -Thienyl-(Aa) t -,-(CR 15 R 16 ) m -Imidazole (Aa) t -,-(CR 15 R 16 ) m -Morpholine-(Aa) t -,-(CR 15 R 16 ) m -Piperazine-(Aa) t -,-(CR 15 R 16 ) m -N-Methylpiperazine-(Aa) t -,-K(CR 15 R 16 ) m (Aa)r(CR 17 R 18 ) n (OCH2CH2) t -,-K(CR 15 R16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -,-K(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -,-K(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -,-K(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r ,-K(CR 15 R 16 ) m (NR 11 CO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -,-K(CR5R6) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m (O-CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m (OCNR17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m (CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -,-K(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -,-K-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-K(CR 15 R 16 ) m -phenyl-CO(Aa) t (CR 17 R 18 ) n -,-K-(CR 15 R 16 ) m -furan-CO(Aa) t (CR17 R 18 ) n -,-K(CR 15 R 16 ) m -Oxazole-CO(Aa) t (CR 17 R 18 ) n -,-K(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t- (CR 17 R 18 ) n -,-K(CR 15 R 16 ) t -Thiophene-CO(CR 17 R 18 ) n -,-K(CR 15 R 16 ) t Imidazole-CO-(CR 17 R 18 ) n -, -K(CR5R6) t Morpholine-CO(Aa) t -(CR 17 R 18 ) n -,-K(CR 15 R 16 ) t -Piperazine-CO(Aa) t- (CR 17 R 18 ) n -,-K(CR 15 R 16 ) t -N-Methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -,-K(CR 15 R 16 ) m -(Aa) t Phenyl, -K-(CR 15 R 16 ) m- (Aa) t Furan-,-K(CR 15 R 16 ) m -Oxazole-(Aa) t-,-K(CR 15 R 16 ) m -Thiazolyl(Aa) t -,-K(CR 15 R 16 ) m -Thienyl-(Aa) t -,-K(CR 15 R 16 ) m -Imidazole (Aa) t -,-K(CR 15 R 16 ) m -Morpholine (Aa) t -,-K(CR 15 R 16 ) m Piperazine (Aa) t G, -K(CR5R6) m -N-Methylpiperazine (Aa) t -; where m, Aa, m, n, R 13 , R 14 and R 15 Definitions are as above; t and r are independently 0-100; R 16 , R 17 , R 18 , R 19 and R 20 independently selected from H, halogen, C1-C8 alkyl or heteroalkyl, C2-C8 aryl, alkenyl, alkynyl, ether, ester, amine or amide, C3-C8 aryl, optionally substituted with one or more halides, CN, NR 12 R 12’ , CF3, OR 12 , aryl, heterocyclic, S(O)R 12 , SO2R 12， -CO2H, -SO3H, -OR 12 , -CO2R 12 , -CONR 12 ,-PO2R 12 R 13 ,-PO3Hor P(O)R 12 R 12’ R 13 Replaced by; K is NR 12 , -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, Het (with C3-C 12or a peptide containing 1-20 identical or different amino acids.

[0174] More preferably, W, L1, L2, V1 and V2 are independently a straight chain alkyl group having 1 to 6 carbon atoms, or a structure such as (OCH2CH2) p The peptide may be a polyoxyethylene unit of p=1-5000, or a peptide containing 1-4 amino acid units (L or D form), or a combination of the above.

[0175] Or any one or more of W, Q1, Q2, L1, L2, V1 or V2 may be independently defaulted, but Q1 and Q2 cannot be defaulted at the same time.

[0176] On the other hand, in general, when V1 and / or V2 are linked to a cell binding molecule T, or when L1 and / or L2 are directly linked to a cell binding molecule (V1 and V2 are absent), one or more of the following structures are generated:

[0177]

[0178]

[0179]

[0180] Where R 20 and R 21 are independently C1-C8 alkyl, C2-C8 heteroalkyl or heterocycle, C3-C8 aryl, aralkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocycle or alkylcarbonyl, or C2-C 100 With the formula (CH2CH2O) p The polyalkylene glycol, p is as defined above.

[0181] On the other hand, Q1 and Q2 are preferably selected from C2-C 18 Lipids, or C2-C 18 Fatty acids or C2-C 18Polyalkylene glycol of fatty ammonium lipids. Polyalkylene glycol not only makes the conjugate more hydrophilic, but also prevents the conjugate linker from being hydrolyzed by hydrolases, such as proteases or esterases. The lipid on the conjugate can bind to albumin in mammalian blood, allowing the conjugate to be slowly released from the complex in the blood circulation. Therefore, the branched linker of the patent application can make the conjugate more stable in the blood circulation. The polyalkylene glycols herein include, but are not limited to, polyethylene glycol (PEG), poly(propylene glycol) and copolymers of ethylene oxide and propylene oxide, preferably PEG, more preferably a monofunctional activated hydroxy PEG (e.g., a hydroxy PEG with one terminal activated, including reactive esters of hydroxy PEG-carboxylic acid, hydroxy PEG-monoaldehyde, hydroxy PEG-monoamine, hydroxy PEG-monohydrazide, hydroxy PEG-monohydrazine, hydroxy PEG-monoiodoacetamide, hydroxy PEG-monomaleimide, hydroxy PEG-monoorthoformyl disulfide, hydroxy PEG-monoxime, hydroxy PEG-monophenyl carbonate, hydroxy PEG-monophenylglyoxal, hydroxy PEG-monothiazolidine-2-thione, hydroxy PEG-monothioester, hydroxy PEG-monothiol, hydroxy PEG-monotriazine and hydroxy PEG-monovinyl sulfone). The molecular weight of the polyalkylene glycol is about 10Da to about 200kDa, preferably in the range of about 88Da to about 40kDa, two branches, each with a molecular weight of about 88Da to about 40kDa; more preferably two branches, each with a molecular weight of about 88Da to about 20kDa. In a specific example, the polyalkylene glycol is polyethylene glycol and has a molecular weight of about 10kDa, 20 or 40kDa. In other specific examples, PEG is a linear or branched 10kDa PEG, a linear or branched 20kDa PEG, a linear or branched 40kDa PEG. Many U.S. patents have disclosed the preparation of linear or branched "non-antigenic" PEG polymers and their derivatives or conjugates, such as U.S. Patents 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,1 and 6,180,095.

[0182] An example of structural formula (I) is as follows:

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; wherein X8 is O, S, NH, NHNH, NHR 12 , SR 12 、SSR 12 SSCH(CH3)R 12 、SSC(CH3)2R 12 , or R 12 ;X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 The definitions of mAb are as described above; Aa is a natural or unnatural amino acid; r is 0-100; (Aa)r is a peptide containing the same or different amino acid sequence, when r>2; when r=0, (Aa)r is omitted.

[0192] In another aspect of the present invention, the structural formula of the conjugate containing a branched linker is represented as follows (III):

[0193]

[0194] wherein D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, n and T are the same as defined in formula (I).

[0195] An example of structural formula (III) is as follows:

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0203] Where X8 is O, S, NH, NHNH, NHR 12 , SR 12 、SSR 12 SSCH(CH3)R 12 、SSC(CH3)2R 12 , or R 12 ;X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 The definitions of , and mAb are as described above; Aa is a natural or unnatural amino acid; r is 0-100; (Aa)r is a peptide containing the same or different amino acid sequence, when r>2; r=0, (Aa)r is default.

[0204] In another aspect of the present invention, the structure of the branched linker compound is shown in (IV), which can be conveniently reacted with the cell binding molecule T to form a conjugate in formula (I):

[0205]

[0206] Wherein, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are defined as in formula (I);

[0207] Lv1 is a reactive functional group that can react with thiols, amines, carboxylic acids, selenols, phenols or hydroxyls on cell-binding molecules. It includes, but is not limited to, halogen (such as fluorine, chlorine, bromine, iodine); methanesulfonyl; toluenesulfonyl; trifluoromethanesulfonyl; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; trifluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; imidazolyl; monochlorophenol; dichlorophenol; trichlorophenol; tetrachlorophenol; N-(benzotriazolyl)oxy; 2-ethyl-5-phenylisoxazolyl-3′-sulfonyl; phenoxydiazolylsulfonyl; 2-ethyl-5-phenylisoxazolyl; phenoxydiazolyl; oxadiazolyl; unsaturated carbon (double or triple bonds between carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen or carbon-oxygen); or intermediates generated by the reaction with a Mitsunobu reaction reagent.Examples of condensing agents are as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide methyl p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-oxy-(1H-benzotriazol-1-yl)-ammonium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethylamino)-hexafluorophosphate (B OP), (benzotriazol-1-yloxy)tripyrrolidinium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidine hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethyl-imidazolium hexafluorophosphate (CIP), chloropyrrolidinium hexafluorophosphate (PyCloP), fluoro-N,N,N',N' -bis(tetramethylene)formamidine hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiourea hexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxo-2-pyridyl)N,N,N',N'-tetramethylthiourea tetrafluoroborate, O-[(ethoxycarbonyl)-cyanomethylamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylaminooxy)dimethylamino-morpholino-hexafluorophosphate (COMU), O-(benzotriazol-1-yl)- N,N,N',N'-bis(tetramethylene)hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexyl-carbodiimide (with or without polymer binding), dipyrrolidinyl(N-succinimidyloxy)carbonium hexafluorophosphate (HSPyU), chlorodipyrrolidinylhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy)dipiperidinylcarbonium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromo(dimethylamino)-hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA,. ), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-oxy-(N-succinimidyl) hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyano-methyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N' , N'-tetramethyl-oxy-(N-succinimidyl) uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)-dipiperidinium (ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD). In addition, Lv1 can be an anhydride, or an anhydride formed by reacting with other C1-C8 anhydrides;

[0208] Preferred Lv1 include: halides (such as fluoride, chloride, bromide and iodide), mesyl, tosyl, trifluoromethylsulfonyl, trifluoromethylsulfonate, nitrophenol, N-succinimide (NHS), phenol; dinitrophenol; pentafluorophenol, tetrafluorophenol, trifluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, 1H- Imidazol-1-yl, monochlorophenol, dichlorophenol, trichlorophenol, tetrachlorophenol, N-(benzotriazolyl)oxy, 2-ethyl-5-phenylisoxazole-3′-sulfonyl, phenyloxadiazole-sulfonyl, 2-ethyl-5-phenylisoxazolyl, phenyloxadiazole, oxadiazole, unsaturated carbon (double or triple bonds between carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen or carbon-oxygen), or one of the following structures:

[0209] disulfide; Haloacetyl; Acyl halides; N-hydroxysuccinimide ester; Maleimide; Monosubstituted maleimide; Disubstituted maleimide; Monosubstituted succinimides; Disubstituted maleimide; Substituted maleic acid; -CHO aldehyde; Ethylenesulfonyl; Acryloyl; 2-(Tosyloxy)acetyl; 2-(methylsulfonyloxy)acetyl; 2-(Nitrophenol)acetyl; 2-(Dinitrophenyl)acetyl; 2-(Fluorophenol)-acetyl; 2-(Difluorophenol)-acetyl; 2-((trifluoromethylsulfonyl)oxy)acetyl; Ketone or aldehyde, 2-(Pentafluorophenol)acetyl; Methylsulfone phenyl oxadiazole (ODA); Anhydride, Alkoxyamine; Azide, Alkynyl, or Hydrazide;

[0210] Wherein, X1' is F, Cl, Br, I or Lv3; X2' is O, NH, N(R1) or CH2; R3 is H, aryl or heteroaryl, wherein one or more H atoms can be independently substituted by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1 or -COOR1; Lv3 is a leaving group selected from F, Cl, Br, I; nitrophenyl; N-hydroxysuccinimide (NHS) ; phenol group; dinitrophenol group; pentafluorophenol group; tetrafluorophenol group; difluorophenol group; monofluorophenol group; pentachlorophenol group; trifluoromethanesulfonyl group; imidazolyl group; dichlorophenol group; tetrachlorophenol group; 1-hydroxybenzotriazolyl group; toluenesulfonyl group; methanesulfonyl group; 2-ethyl-5-phenylisoxazole-3′-sulfonyl group, anhydride or anhydride formed by reaction with other anhydrides, such as acetic anhydride, formic anhydride; or an intermediate formed by reaction with a polypeptide condensation reagent or a Mitsunobu reaction reagent.

[0211] An example of structural formula (IV) is as follows:

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0223] Where X8 is O, S, NH, NHNH, NHR 12 , SR 12 、SSR 12 SSCH(CH3)R 12 、SSC(CH3)2R 12 , or R 12 ;X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 The definitions of , and mAb are as described above; Aa is a natural or unnatural amino acid; r is 0-100; (Aa)r is a peptide containing the same or different amino acid sequence, when r>2; r=0, (Aa)r is default.

[0224] In another aspect of the present invention, the structure of the branched linker compound is shown in (V), which can be conveniently reacted with the cell binding molecule T to form a conjugate in formula (III):

[0225]

[0226] Wherein, the definitions of D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are the same as those in structural formula (I); wherein Lv1 and Lv2 are independently the same as those of Lv1 in formula (IV), and Lv1 and Lv2 may be the same or different.

[0227] An example of structural formula (V) is as follows:

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof;

[0237] Among them, X1, X2, X3, X4, X5, X 8、 Z2, Z3, p, p1, p2, p3, q1, q2, Lv1, Lv2, Lv3, Lv 3’ , m, n, R 12 , R 12 '、R 15 , R 25 , (Aa)r, and mAb are defined as above;

[0238] The present invention further relates to a method for preparing conjugates of amatoxin analogs and cell binding molecules in structural formula (I) and (III), and the use of these conjugates.

[0239] The cell-binding agent or cell-binding molecule T may be any type of molecule currently known or to be disclosed, which has therapeutic significance or can be modified by biological means, and can bind, complex or react with a part of a cell. Preferred cell-binding agents or cell-binding molecules are immunotherapeutic proteins, antibodies and single-chain antibodies; antibody fragments that bind to target cells; monoclonal antibodies; single-chain monoclonal antibodies; or monoclonal antibody fragments that bind to target cells; chimeric antibodies; chimeric antibody fragments that bind to target cells; domain antibodies; domain antibody fragments that bind to target cells; adnectin antibodies, DARPin proteins, lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules (transferrin) and / or cell-binding peptides, proteins or small molecules containing more than four amino acid fragments attached to albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles or (virus) capsids;

[0240] Preferably, Lv1, Lv2, Lv3 or Lv3' can react with a sulfhydryl group on a cell-binding agent or cell-binding molecule. The sulfhydryl group is optimally generated by reducing the interchain disulfide bond of the cell-binding agent, and the reducing agent is selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), di(2-carbonylethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA) and / or β-mercaptoethanol (β-ME, 2-ME). The sulfhydryl group on the cell-binding agent / molecule can also be generated by Traut's reagent or thiolactone, wherein the Traut's reagent or thiolactone reacts with an amino group on the cell-binding agent / molecule to form a sulfhydryl group, which can then be reacted with Lv1, Lv2, Lv3 or Lv3' simultaneously or sequentially:

[0241]

[0242] Preparation of conjugates of amatoxin analogs and cell-binding molecules connected by branched linkers

[0243] Figures 1-26 illustrate the preparation of conjugates of amatoxin homologues with cell-binding molecules of the present invention and the synthetic routes for coupling with branched linkers.

[0244] The conjugates of formula (I) and (III) can be prepared from the intermediate compounds of formula (IV) and (V), respectively. Generally, the amatoxin homologues in (IV) and (V) have functional groups of Lv1 and Lv2, which can be easily reacted with cell binding molecules or modified cell binding molecules. The preparation of the amatoxin homologues in (IV) and (V) and the preparation of the conjugates in parts (I) and (III) are illustrated in Figures 1-26.

[0245] Typically, to synthesize the conjugate of formula (I), the functional group Lv1 in formula (IV) reacts with one, two or more groups on the cell binding molecule, and the reaction is carried out in an aqueous medium at 0-60°C and pH 5-9, with or without the addition of 0-30% of a water-miscible organic solvent such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol or ethylene glycol, followed by dialysis or chromatography to obtain the conjugate of formula (I). The amino acid residues on the cell binding molecule (reactive functional groups for coupling) can be obtained by protein engineering methods.

[0246] The conjugate in formula (III) can also be obtained by reacting the functional groups Lv1 and Lv2 in formula (V) with two or more groups on the cell binding molecule, preferably a pair of free thiol groups generated by reducing disulfide bonds on the cell binding molecule, and the reaction occurs in an aqueous medium at 0-60°C and pH 5-9, with or without the addition of 0-30% of a water-soluble (miscible) organic solvent to form the conjugate molecule. The paired thiol groups are preferably generated by reducing the interchain disulfide bonds on the cell binding agent, and the reducing agent can be selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine (β-MEA), or / and β-mercaptoethanol (β-ME, 2-ME), and the reduction reaction is carried out in an aqueous medium at pH 4-9, with or without the addition of 0-30% of a water-soluble (miscible) organic solvent.

[0247] The reactive groups of Lv1 and Lv2 in formula (IV) and (V) can independently be the following groups: disulfide, thiol, thioester, maleimide, halogen-substituted maleimide, haloacetyl, azide, 1-alkyne, ketone, aldehyde, alkoxyamino, trifluoromethanesulfonyl, carbonyl imidazole, toluenesulfonyl, methanesulfonyl, 2-ethyl-5-phenylisoxazole-3′-sulfonyl, nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, anhydride or hydrazide group, ester or other acid derivatives. It can react with one, two or more groups on a cell binding molecule or a cell binding agent simultaneously or sequentially, in an aqueous medium at 0-60°C and a pH of 4-9.5, with or without the addition of 0-30% water-soluble (miscible) organic solvent, and after column chromatography or dialysis to obtain the conjugates of formula (I) and (III). The reactive groups of Lv1 and Lv2 of formula (IV) and (V) react with the modified cell binding molecule in different ways. For example, the preparation of the cell binding agent-amanitin analog conjugate containing a disulfide bond linker in formula (I) can be carried out by exchanging disulfide bonds between the disulfide bonds in the modified cell binding agent and Lv1 and Lv2 containing free sulfhydryl groups, or by exchanging the free sulfhydryl groups in the modified cell binding agent with the disulfide bonds on Lv1 and / or Lv2. In order to accelerate the disulfide exchange reaction, the disulfide group is usually selected from disulfide pyridine, dithionitropyridine, dithionitrobenzene, dithionitrobenzene benzoic acid or disulfide dinitrobenzene and the like.

[0248] In the conjugates of formula (I) and (III), the linkage containing a thioether bond is usually achieved by reacting a maleimido group or a haloacetyl group or an ethylsulfonyl group on the modified cell-binding agent or amatoxin homologue in (IV) and (V), respectively, with a free sulfhydryl group on the amatoxin homologue or a sulfhydryl group on the modified cell-binding agent; the linkage containing a hydrazone bond that is easily degraded by acid can be achieved by reacting a carbonyl group on the drug or cell-binding molecule in (IV) and (V) with a hydrazide group on the cell-binding molecule or on the drug in (IV) and (V). These methods are well known in the art (see, e.g., P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32, 548-55, 1959; P. Trail et al., Cancer Res. 54, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32, 548-55, 1959; P. Trail et al., Cancer Res. 54, 3336-34, 1993; Res., 57, 100-5, 1997); the connection containing a triazole bond can be achieved by reacting the alkyne group on the drug or cell binding molecule in (IV) and (V) with the corresponding azide through click chemistry (Huisgen cycloaddition) reaction (Lutz, JF. et al., 2008, Adv. Drug Del. Rev. 60, 958-70; Sletten, EM et al. 2011, Acc Chem. Research 44, 666-76). The connection containing an oxime bond can be achieved by reacting the ketone or aldehyde on the drug or cell binding molecule in (IV) and (V) with the corresponding hydroxylamine. The cell binding molecule containing a sulfhydryl group can react with the drug-linker molecule containing maleimide, haloacetyl or ethylsulfonyl substituents in formula (IV) and (V) in a buffer solution with a pH of 5.5-9.0 to obtain the conjugates connected by thioether in formula (I) and (III). Cell binding molecules containing thiol groups can also undergo disulfide exchange with drug-linkers with pyridyl disulfide groups in formula (IV) and (V) to obtain disulfide-linked conjugates. Cell binding molecules containing hydroxyl or thiol groups can react with drug-linkers with halogens, especially halogens at the α position of carboxylic acid esters in formula (IV) and (V) in the presence of mild bases, such as pH 8.0-9.5, to obtain conjugated drugs with ether or thioether bonds. Hydroxyl or amino groups on cell binding molecules can condense with drug-linkers with carboxyl groups in (IV) and (V) in the presence of condensing agents such as EDC or DCC to form ester linkages. Cell binding molecules containing amino groups can be condensed with carboxylates of the following groups on the drug-linker molecule to obtain conjugates containing amide bond linkers: NHS, imidazole, nitrophenol, phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonyl, dichlorophenol, tetrachlorophenol, 1-hydroxyphenyltriazole, toluenesulfonyl, methanesulfonyl, 2-ethyl-5-phenylisoxazolyl-3′-sulfonyl.

[0249] The synthesized conjugate can be purified by standard biochemical methods, such as gel filtration on a Sephadex G25 or Sephacryl S300 column, adsorption chromatography, ion exchange, or dialysis. In some cases, the cell binding agent is a small molecule compound (e.g., folic acid, melanocyte stimulating hormone, EGF, etc.), which can be purified by chromatography, such as HPLC, medium pressure column chromatography, or ion exchange chromatography, after conjugation with a small molecule drug.

[0250] In order to achieve a higher yield of the coupling reaction with a pair of free thiol groups on a cell-binding molecule, preferably an antibody, it may be necessary to add a small amount of a water-soluble organic solvent or a phase transfer agent to the reaction mixture. The cross-linker (linker) of formula (IV) or (V) can be dissolved in a polar organic solvent miscible with water, such as various alcohols (such as methanol, ethanol and propanol), acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) at a high concentration, such as 1-50 mg / ml. At the same time, the cell-binding molecule, such as an antibody, is dissolved in a buffer having a pH of 4 to 9.5, preferably pH 6 to 8.5, at a concentration of 1 to 50 mg / ml and then treated with 0.5 to 20 equivalents of TCEP or DTT for 20 minutes to 48 hours. After reduction, DTT can be removed by SEC chromatography purification. TCEP can also be removed by SEC chromatography or left in the reaction mixture without further purification, but it is best to neutralize TCEP with an azide (such as 4-azidobenzoic acid, 4-(azidomethyl)benzoic acid, azidopolyethylene glycol (such as 2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol). In addition, the reduction of antibodies or other cell binding agents with TCEP can be carried out in the presence of a drug of formula (IV) or (V), in which case the coupling of the drug to the cell binding molecule can be achieved simultaneously with the reduction of TCEP.

[0251] The aqueous solution for the reaction of modifying the cell binding agent is a buffer solution between pH 4 and 9, preferably between 6.0 and 7.5, and may contain any non-nucleophilic buffer salt suitable for use in this pH range. Typical buffers include phosphate, acetate, triethanolamine hydrochloride, HEPES and MOPS buffers, and may also contain other components, such as cyclodextrin, hydroxypropyl-β-cyclodextrin, polyethylene glycol, sucrose and other salts, such as NaCl and KCl. After the drug-linker in formula (IV) or (V) is added to the solution containing the reduced cell binding molecule, the reaction mixture is incubated at a temperature of 4°C to 45°C, preferably 15°C. The progress of the reaction can be monitored by measuring the decrease in absorbance at a specific ultraviolet wavelength (e.g., 252nm), or the increase in absorbance at a specific ultraviolet wavelength (e.g., 280nm), or other appropriate wavelengths. After the reaction is completed, the modified cell-binding agent can be isolated in a conventional manner, for example, using gel filtration chromatography, ion exchange chromatography, adsorption chromatography, silica gel or alumina column chromatography, crystallization, preparative thin layer chromatography or HPLC methods.

[0252] The degree of modification can be evaluated by measuring the ultraviolet absorbance of the nitropyridinethione, dinitropyridinedithione, pyridinethione, carboxyamidopyridinedithione and dicarboxyamidopyridinedithione groups generated by the reaction. The coupling reaction, modification or coupling reaction without chromophore molecules can be monitored by LC-MS, preferably HPLC-MS / MS, UPLC-QTOF mass spectrometry or capillary electrophoresis mass spectrometry (CE-MS). The branched linker of the present invention contains a variety of functional groups and can react with cell binding molecules, especially modified cell binding molecules with suitable substituents. For example, modified cell binding molecules with amino or hydroxyl groups can react with drugs with N-hydroxysuccinimide (NHS) esters, and modified cell binding molecules with sulfhydryl groups can react with drugs with maleimido or haloacetyl groups. In addition, modified cell binding molecules with carbonyl (ketone or aldehyde) obtained by protein engineering, enzymatic reaction or chemical modification can react with drugs with hydrazide or alkoxyamine. Based on the reactivity of the functional groups on the modified cell-binding molecule, one skilled in the art can readily determine which drug-linker molecule to use.

[0253] Cell Binding Agents

[0254] The cell binding agent of the present invention, including the cell binding agent in the conjugate and the modified cell binding agent, can be any type of molecule currently known or to be disclosed, which can bind, complex or react with cell fragments and has therapeutic significance or is biologically modified.

[0255] Cell binding agents include, but are not limited to, large molecular weight proteins, such as whole antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies, such as bispecific antibodies); single chain antibodies; antibody fragments such as Fab, Fab', F(ab')2, F v(Parham, J. Immunol. 1983, 131, 2895-2902); fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies; CDRs; bivalent antibodies; trivalent antibodies; tetravalent antibodies; minibodies; small immunoproteins; epitope-binding fragments of any of the above antibodies that can immunospecifically bind to cancer cell antigens, viral antigens, and microbial antigens; proteins produced by the immune system that can recognize and bind to specific antigens or have a desired biological activity (Miller et al. J. of Immunology 2003, 170, 4854-4861); interferons (such as type I, II, and III); polypeptides; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-16, IL-17, GM-CSF, interferon-γ (IFN-γ); hormones such as insulin, TRH (thyrotropin-releasing hormone), MSH (melanocyte-stimulating hormone), steroid hormones such as androgens and estrogens; growth factors and colony stimulating factors such as epidermal growth factor (EGF), granulocyte macrophage colony stimulating factor (GM-CSF), transforming growth factors (TGF) such as TGFα, TGFβ, insulin and insulin-like growth factor (IGF-I, IGF-II), G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 1984, 5, 155-158); vaccinia growth factor (VGF); fibroblast growth factor (FGF); small molecular weight proteins; polypeptides; peptides and peptide hormones, such as bombesin, gastrin, gastrin-releasing peptide; platelet-derived growth factor; interleukins and cytokines, for example, interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte macrophage colony stimulating factor (GM-CSF); vitamins, such as folic acid; apoproteins and glycoproteins, such as transferrin (O'Keefe et al, J.Biol.Chem. 1985 260 932-937); sugar-binding proteins or lipoproteins, such as lectins; cell nutrient delivery molecules; small molecule inhibitors, such as prostate-specific membrane antigen (PSMA) inhibitors and small molecule tyrosine kinase inhibitors (TKI), non-peptide or any other cell-binding molecule or substance, such as bioactive polymers (Dhar, et al. al, Proc.Natl.Acad.Sci.2008,105,17356-61), fusion proteins, kinase inhibitors, gene targeting agents, bioactive dendrimers (Lee, et al, Nat.Biotechnol.2005,23,1517-26; Almutairi, et al; Proc.Natl.Acad.Sci.2009, 106, 685-90), nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12), liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9) and viral capsids (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93).

[0256] In general, monoclonal antibodies are preferred as cell surface binding agents if appropriate monoclonal antibodies are available. Antibodies can be murine, human, humanized, chimeric or derived from other species.

[0257] The production of antibodies used in the present invention includes in vivo or in vitro methods or a combination thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well known in the art, such as described in U.S. Patent No. 4,493,795. Monoclonal antibodies are usually prepared by fusing myeloma cells with spleen cells from mice that have been immunized with the desired antigen. G.; Milstein, C. Nature 1975, 256: 495-497). The detailed process is described in "Antibodies--A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988)", which is cited here for reference. Specifically, the target antigen, such as an antigen separated from a target cell, a complete virus, an inactivated whole virus and a viral protein, can be used to immunize mice, rats, hamsters or any other mammals for preparation. Splenocytes are usually fused with myeloma cells using polyethylene glycol (PEG) 6000. Fusion cells are screened by sensitivity to HAT (hypoxanthine-aminopterin-thymidine). Hybridomas implementing the monoclonal antibodies of the present invention can be determined by their ability to immunoreact with specific receptors or their ability to inhibit receptor activity on target cells.

[0258] The production of monoclonal antibodies used in the present invention is carried out in a monoclonal hybridoma culture, which contains a nutrient medium and a hybridoma that can secrete antibody molecules with appropriate antigen specificity. The culture is maintained under suitable conditions for a period of time sufficient to allow the hybridoma to secrete the antibody molecules into the culture medium. The culture medium containing the antibody is then collected. The antibody molecules are further separated using known techniques, such as protein A affinity chromatography, anionic, cationic, hydrophobic or size exclusion chromatography (particularly by protein A affinity chromatography and size exclusion chromatography), centrifugation, differential solubility or any other standard technique for purifying proteins.

[0259] The culture media that can be used to prepare these compositions are well known in the art and are commercially available, including synthetic culture media. An example of a synthetic culture medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 1959, 8, 396) supplemented with 4.5 g / ml glucose, 0-20 mM glutamine, 0-20% fetal bovine serum, several ppm of heavy metals such as Cu, Mn, Fe or Zn or / and heavy metal salts, and defoaming agents such as polyoxyethylene-polyoxypropylene block copolymers.

[0260] In addition, antibody-producing cell lines can also be obtained by techniques other than fusion, such as transplantation of oncogenic DNA into B lymphocytes, or transfection with an oncogenic virus, such as Epstein-Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated herpesvirus (KSHV), as described in U.S. Patents 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. Monoclonal antibodies can also be prepared by using anti-receptor peptides or peptides containing a terminal carboxyl group, which are well known in the art, and can be referenced to Niman et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 4949-4953; Geysen et al., Proc. Natl. Acad. Sci. USA, 1985, 82: 178-182; Lei et al. Biochemistry 1995, 34 (20): 6675-6688. Generally, as an immunogen for producing anti-receptor peptide monoclonal antibodies, the anti-receptor peptide or peptide homologue can be used alone or linked to an immunogenic carrier.

[0261] The monoclonal antibodies used as binding molecules in the present invention can also be obtained by other techniques known in the art. Particularly useful are methods for making fully human antibodies. One method is phage display technology, which uses affinity enrichment to select human antibodies that can specifically bind to antigens. Phage display technology is also described in detail in the literature, and the construction and screening of phage display libraries are also well known in the art. References can be made to Dente et al, Gene. 1994, 148 (1): 7-13; Little et al, Biotechnol Adv. 1994, 12 (3): 539-55; Clackson et al., Nature 1991, 352: 264-628; Huse et al., Science 1989, 246: 1275-1281.

[0262] Monoclonal antibodies produced by hybridomas fused with non-human, such as mouse cells, can be humanized to avoid the production of human anti-mouse antibodies. Common antibody humanization methods are complementary determining region transplantation techniques, which have also been described in detail, such as U.S. Patents 5,859,205 and 6,797,492; Liu et al, Immunol Rev. 2008, 222: 9-27; Almagro et al, Front Biosci. 2008, 13: 1619-33; Lazar et al, Mol Immunol. 2007, 44 (8): 1986-98; Li et al, Proc. Natl. Acad. Sci. USA. 2006, 103 (10): 3557-62, cited herein as reference. Complete human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys or other mammals carrying most of the heavy and light chains of human globulins with an immunogen. Examples of these mice include: Xenomouse (Abgenix / Amgen), HuMAb-Mouse (Medarex / BMS) and VelociMouse (Regeneron), see U.S. Patents 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149 and 5,569,825. When used for human therapy, the variable region of the mouse and the constant region of the human can also be fused to form a "chimeric antibody", which has significantly lower immunogenicity in humans than mouse monoclonal antibodies (Kipriyanov et al, Mol Biotechnol. 2004, 26: 39-60; Houdebine, Curr Opin Biotechnol. 2002, 13: 625-9). In addition, site-directed mutagenesis in the variable region of an antibody can result in antibodies with higher affinity and specificity (Brannigan et al, Nat Rev Mol Cell Biol. 2002, 3:964-70; Adams et al, J Immunol Methods. 1999, 231:249-60), and changes in the constant region of an antibody can improve its effector function in mediating binding and cytotoxicity.

[0263] Antibodies specific for malignant tumor cell antigens can also be obtained from commercial sources or produced by any known method, such as chemical synthesis or recombinant expression technology. Nucleotide sequences encoding antibodies specific for malignant tumor cell antigens can be obtained commercially, such as from the GenBank database or similar databases, literature publications, or from conventional cloning and sequencing.

[0264] In addition to antibodies, a peptide or protein that interacts with an epitope or corresponding receptor on a target cell (binding, blocking, targeting or other types of effects) can also be used as a binding molecule. These peptides or proteins may be any random peptides or proteins that have affinity for the epitope or corresponding receptor and do not necessarily have to be members of the immunoglobulin family. These peptides can be isolated by techniques similar to phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23 (4): 307-49). Peptides obtained from random peptide libraries can be used similarly to antibodies and antibody fragments. Peptide or protein binding molecules can be coupled or connected to macromolecules or other substances, including but not limited to albumin, polymers, liposomes, nanoparticles, dendrimers, as long as such a connection can retain the antigen binding specificity of the peptide or protein.

[0265] Examples of antibodies linked to drug molecules via the linker of the present invention in conjugates for treating cancer, autoimmune diseases and / or infectious diseases include, but are not limited to, 3F8 (anti-GD2), abamomab (anti-CA-125), abciximab (anti-CD41 (integrin α-IIb), adalimumab (anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), afelimomab (anti-TNF-α), afutuzumab (anti-CD20), alacizumab (anti-VEGFR2), ALD 518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Azithromycin (anti-CEA), Aseluzumab (anti-L-selectin CD62L), Atlizumab (tocilizumab, Actemra, R oActemra, anti-IL-6 receptor), Atorolimub (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid), Basiliximab (Simulect, anti-CD25 (alpha chain of IL-2 receptor)), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Benlysta (LymphoStat-B, anti-BAFF), Benralizu mab (anti-CD125), Bertilimumab (anti-CCL11 (eotaxin-1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II β chain), Bivatuzumab (anti-CD44v6), Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30 TNRSF8), Briakinumab (anti-IL-12, IL-23), Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4), Certolizumab (Cimzia anti-TNF-α), Cetuximab (Erbitux,IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4), Clivatuzu-mab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (IL-2 receptor alpha chain)), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B lymphoma cells) , Dorlimomab, Dorlixizumab, Ecoromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11a)), Efungumab (Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab (anti-I CAM-1 (CD54)), Epitumomab (anti-episialin), Eptazumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2 / neu, CD3), Eptazumab (Abegrin, anti-integrin αvβ3), Exbivirumab (anti-HBsAg), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory Syncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80), Gantenerumab (anti-β-amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatumumab (CR011,anti-GPNMB), Golimumab (Simponi, anti-TNF-α), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF- α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (IL-2 receptor α chain), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab ( Anti-NCA-90 (granulocyte antigen), Lerdelimumab (anti-TGFβ2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-HBsAg), Lintuzumab (anti-CD33), Lumitumumab (anti-CD40), Lumitumumab (anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Massimoumab (anti-T-cell receptor), Matuzumab (anti-EGFR), Mep Olizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGFβ1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside), Morolimumab (anti-rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrin α4), Nebazumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-CD20), Afolimomab (anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20),Olaratumab (anti-PDGF-Rα), Omalizumab (Xolair, anti-IgE Fc region) Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa, anti-Pseudomonas aeruginosa, anti- aeruginosa)), Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2 / neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-D4), Pritumumab (anti-vimentin), PRO140 (anti-CCR5) Racotumomab (1E10, anti-N-glycolylneuraminic acid (NeuGc, NGNA)-ganglioside GM3)), Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), reslizumab (anti-IL-5), rilotumumab (anti-HGF), rituximab (MabThera, rituxanmab, anti-CD20), robatumumab (anti-IGF-1 receptor), rontalizumab (anti-IFN-α), rovelizumab (leukAr-rest, anti-CD11, CD18), ruplizumab (Antova, anti-CD154 (CD40L)), satumomab (anti-TAG-72), sevirumab (anti-cytomegalovirus), sibrotuzumab (anti-FAP), sifalimumab (anti-IFN-α), siltuximab (anti-IL-6), siplizumab (anti-CD2), Smart MI95 (anti-CD33), Solanezumab (anti-β-amyloid), Sonepcizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab (anti-myostatin),Sulesomab (LeukoScan, anti-NCA-90 (granulocyte antigen)), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin αIIbβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-aggregating factor A)), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40), Teplizumab (anti-CD3), TG N1412 (anti-CD28), Ticilimumab (Tremelimumab, anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, anti-HER2 / neu), Tremelimumab (anti-CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab (anti-integrin α4β7), Veltuzumab (anti-CD20), Vepalilimomab (anti-AOC3 (VAP-1)), Visilizumab (Nuvi on, anti-CD3), Vitaxin (anti-vascular integrin avb3), Volociximab (anti-integrin α5β1), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFR, Zanolimumab (HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab (anti-CD5), Etanercept, Alefacept Abatacept Rilonacept (Arcalyst), 14F7 (anti-IRP-2 (iron regulatory protein 2)), 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst., for the treatment of melanoma and solid tumors), J591 (anti-PSMA, from Weill Cornell Medical College, for the treatment of prostate cancer), 225.28S (anti-HMW-MAA (high molecular weight melanoma associated antigen), SorinRadiofarmaci SRL (from Milan, Italy, for the treatment of melanoma), COL-1 (anti-CEACAM3, CGM1, from NatCancer Inst. for the treatment of colorectal cancer and gastric cancer), CYT-356 ( treatment of prostate cancer), HNK20 (OraVax Inc. for respiratory syncytial virus infection), ImmuRAIT (from Immunomedics, for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm), MAK-195F (anti-TNF (tumor necrosis factor, TNFA, TNF-α, TNFSF2, from Abbott / Knoll, for sepsis and toxic shock), MEDI-500 (T10B9, anti-CD3, TRαβ (T cell receptor α / β), from MedImmune Inc, for graft-versus-host disease), RING SCAN (anti-TAG 72 (tumor-associated glycoprotein 72), from Neoprobe Corp., for breast, colon and rectal cancer), Avicidin (anti-EPCAM (epithelial cell adhesion molecule)), anti-TACSTD1 (tumor-associated calcium signaling 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 (epithelial glycoprotein 2), anti-KSA, KS1 / 4 antigen, M4S, tumor antigen 17-1A, CD326 (from NeoRx, for colon, ovarian, prostate and NHL), LymphoCide (from Immunomedics), Smart ID10 (from ProteinDesign Labs), Oncolym (from Techniclone Inc), Allomune (from BioTransplant), anti-VEGF (from Genentech), CEAcide (from Immunomedics), IMC-1C11 (from ImClone Systems) and Cetuximab (from ImClone).

[0266] Other antibodies that can serve as cell-binding molecules / ligands include, but are not limited to, antibodies to the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer), CA15-3 (various cancers), CA19-9 (various cancers), L6 (various cancers), Lewis Y (various cancers), Lewis X (various cancers), alpha-fetoprotein (various cancers), CA242 (colorectal cancer), placental alkaline phosphatase (various cancers), prostate-specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (various cancers), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3ε (T cell cellular lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemias, lymphomas, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemias, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphomas, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumors, multiple myeloma), CD66e (various cancers), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD80 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD90 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD100 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD110 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD111 (metastatic melanoma, sarcoma), CD112 (leukemia), CD113 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD114 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD115 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD116 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD117 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD118 (metastatic renal cell carcinoma and non-Hodgkin's =Chin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen, CEA, CD66e) (breast, colorectal and lung cancer), DLL4, EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, blood tumors, solid tumors), Endoglin (CD1 05, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head and neck cancer, colon cancer, NHL prostate cancer, ovarian cancer), ERBB2 (epidermal growth factor receptor 2, lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (various cancers), G-28 (cell surface antigen glycolipid, melanoma), GD3 idiotype (respective cancers), heat shock protein (various cancers), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (various cancers),IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemias and lymphomas), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman's disease, interleukin 6-dependent tumors), integrins (αvβ3, α5β1, α6β4, αllβ3, α5β5, αvβ5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers), MAGE 4 (various cancers), anti-transferrin receptor (various cancers), p97 (melanoma), MS4A1 (transmembrane 4 domains subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and alpha gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domains subfamily A member 1, small cell lung cancer, NHL), Nucleolin, Neu oncogene products (respective cancers), P21 (various cancers), anti-(N-glycolylneuraminic acid) antibody binding site (breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumors), PSA (prostate cancer), ROBO4, TAG 72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (various cancers), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, various cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-a, VEGF-2 (CD309) (various cancers). Other tumor-associated antigens that can be recognized by antibodies have been summarized and reviewed (Gerber, et al, mAbs 2009, 1:3, 247-253; Novellino et al, Cancer Immunol Immunother. 2005, 54(3), 187-207; Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76).

[0267] The cell binding agent, preferably an antibody, can be against the following cells: tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes. More specifically, the cell binding agent can be any agent / molecule that can be against one of the following antigens or receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31 , CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, C D54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, C D83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133 , CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166, .CD168, CD174, CD180 、CD184、CDw186、CD194、CD195、CD200、CD200a、CD200b、CD209、CD221、CD227、CD235a、CD240、CD262、CD271、CD274、CD276(B7-H3)、CD303、CD304、CD309、CD326、4-1BB、5AC、5T4(Trophoblast glycoprotein, TPBG, WNT-activation inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, promoter receptor kinase 1, AFP, AKAP-4, ALK,α integrin, αvβ6, aminopeptidase N, amyloid β, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin protective antigen, antimetastatic protein receptor, AOC3 (VAP-1), B7-H3, anthrax Bacillus, BAFF (B cell activation factor), B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CA LLA, CanAg, canine lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC fragment chemokine 11), CCR4 (CC chemokine receptor 4, CD194), CCR5, CD3E (ε), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), cluster factor A, CRIPTO, FCSF1R (colony Stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), CXC chemokine receptor 4, cyclic ADP ribonuclease, cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, Dabigatran, DLL3 (Delta ligand 3), DLL4 (Delta ligand 4) , DPP4 (dipeptidyl-peptidase 4), DR5 (death receptor 5), Escherichia coli shiga toxin type-1, Escherichia coli shiga toxin type-2, ED-B, EGFL7 (EGF domain-containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, frizzled receptor, fucose GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypican3, N-glycolylneuraminic acid, GM3, GMCSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB),GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase C receptor, heat-stable enterotoxin receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (hepatocyte growth factor / scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF–1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2R (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-16, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, αIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or glycosyl inhibitory factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemoattractant protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1, MYCN, myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neuronal apoptosis regulating protease 1, NOGO-A, Notch receptor, nucleolin,Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, PAP, anti-(N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1, CD279), PDGF-Rα (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate co-transporter, PMEL 17. Polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), somatomedin C, SIP (sphingosine 1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TN F-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signal sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR-2, vimentin, WT1, XAGE 1, cells expressing insulin growth factor receptor, or cells expressing epidermal growth factor receptor.

[0268] In another specific embodiment, the cell binding molecule can be a ligand or receptor agonist selected from the following: folic acid derivatives (binding to the folic acid receptor, a protein overexpressed in ovarian cancer and other malignancies) (Low, PS et al. 2008, Acc. Chem. Res. 41, 120-9); glutamate urea derivatives (binding to prostate specific membrane antigen, a surface marker of prostate cancer cells) (Hillier, SM et al., 2009, Cancer Res. 69, 6932-40); somatostatin (also known as growth hormone inhibiting hormone (GHIH) or growth hormone release inhibitory factor (SRIF) or growth hormone release inhibitory hormone) and its homologues, such as octreotide (Sandostatin) and lanreotide (Somatuline) (especially for neuroendocrine tumors, GH-producing pituitary adenomas, paragangliomas, non-functional pituitary adenomas, pheochromocytomas (Ginj, M., et al., 2006, Proc. Natl. Acad. Sci. USA 103, 16436-41), somatostatin receptor subtypes (sst1, sst2, sst3, sst4 and sst5) in the following tumors: GH-secreting pituitary adenomas (Reubi JC, L and Olt AM 1984 J. Clin. Endocrinol Metab 59: 1148-51; Re ubi JC, L and Olt AM 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al., J Clin Endocrinol Metab 61: 98-103), gastroenteropancreatic tumors (Reubi JC, et al., 1987 J Clin Endocrinol Metab 65: 1127-34; Reubi, JC, et al., 1990 Cancer Res 50: 5969-77), pheochromocytoma (Epel-baum J, et al. 1995 J Clin Endocrinol Metab 80: 1837–44; Reubi JC et al., 1992 J Clin Endocrinol Metab 74: 1082–9), neuroblastoma (Prevost G, 1996 Neuroendocrinology 63: 188–197; Moertel, CL, et al., 1994 Am J Clin Path 102: 752–756), medullary thyroid carcinoma (Reubi, J.C, et al. 1991 Lab Invest 64:567–573), small cell lung cancer (Sagman U, et al. 1990 Cancer 66:2129–2133), meningioma, medulloblastoma or glioma (Reubi JC, et al. 1986 J Clin Endocrinol Metab 63:433–8; Reubi JC, et al. 1987 Cancer Res 47:5758–64; Fruhwald, MC, et al. 1999 Pediatr Res 45:697–708), breast cancer (Reubi JC, et al. 1990 Int J Cancer 46:416–20; Srkalovic G, et al. 1990 J Clin Endocrinol Metab 70:661–669), lymphoma (Reubi JC, et al. 1992, Int J Cancer50:895–900), renal cell carcinoma (Reubi JC, et al. 1992, Cancer Res52:6074–6078), mesenchymal tumors (Reubi JC, et al. 1996 Cancer Res 56:1922–31), prostate cancer (Reubi J C, et al. 1995, J. Clin. Endocrinol Metab 80:2806–14; et al. 1989, Prostate 14:191–208; Halmos G, et al. J. Clin. Endo-crinol Metab 85:2564–71), ovarian cancer (Halmos, G, et al., 2000 J Clin Endocrinol Metab 85:3509–12; Reubi JC, et al. 1991 Am J Pathol 138:1267–72), gastric cancer (Reubi JC, et al. 1999, Int J Cancer 81:376–86; Miller, GV, 1992 Br J Cancer 66:391–95), liver cancer (Kouroumalis E, et al. 1998 Gut 42:442–7; Reubi JC, et al. 1999 Gut 45:66–774) and nasopharyngeal carcinoma (Loh K.S, et al. 2002 Virchows Arch 441:444–8); aromatic sulfonamides (carbonic anhydrase IX specific) (markers of hypoxia and renal cell carcinoma) (Neri, D., et al., Nat. Rev. Drug Discov.2011, 10, 767-7); pituitary adenylate cyclase-activating peptide (PACAP) (PAC1) (for pheochromocytoma and paraganglioma); vasoactive intestinal peptide (VIP) and its receptor subtypes (VPAC1, VPAC2); α-melanocyte stimulating hormone (α-MSH) receptor; cholecystokinin (CCK) or gastrin receptor and its receptor subtypes (CCK1 (formerly CCK-A) and CCK2; vasoactive intestinal peptide (VIP) and its receptor subtypes (VPAC1, VPAC2); α-melanocyte stimulating hormone (α-MSH) receptor; cholecystokinin (CCK) or gastrin receptor and its receptor subtypes (CCK1 (formerly CCK-A) and CCK2); cholecystokinin (CCK) or gastrin receptor and its receptor subtype (CCK); bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) or gastrin releasing peptide (GRP) and its receptor subtypes (BB1, GRP receptor subtype (BB2), BB3 and BB4) (Ohlsson, B., et al., 1999, Sc and J. Gastroenterology 34(12):1224–9; Weber, HC, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1):66–71, Gonzalez N, et al., 2008, Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64); neurotensin receptors and their receptor subtypes (NTR1, NTR2, NTR3); substance P receptors and their receptor subtypes (e.g. NK1 receptor of glial tumors, Hennig IM, et al. 1995 Int. J. Cancer 61, 786–792); neuropeptide Y (NPY) receptors and their receptor subtypes (Y1–Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g. cRGDfV) (Laakkonen P, Vuorinen K.2010, Integr Biol(Camb).2(7–8):326–337; ChenK, Chen Research, 19(2A), 959-968; Thumshirn, G, et al., 2003 Chem. Eur.J.9, 2717-2725), and TAASGVRSMH or LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (a 31 amino acid peptide that binds to nucleolin receptors expressed on the cell surface) (Zitzmann, S., 2002 Cancer Res., 62, 18, 5139-5143; Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, 2 (7-8), 326-337; MA Burg, 1999 Cancer Res., 59 (12), 2869-2874; K. Porkka et al. 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9); cell penetrating peptides (CPPs) (Nakase I, et al., 2012, J. Control Release.159(2), 181–188); peptide hormones, such as agonists and antagonists of luteinizing hormone-releasing hormone (LHRH), gonadotropin-releasing hormone (GnRH) agonists, which act by targeting follicle-stimulating hormone (FSH) and luteinizing hormone (LH) as well as testosterone production, such as 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-Bn)-Leu-Arg-Pro-NHEt), leuprorelin (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), nafarelin, desloline, aberelin (Ac-D-2 Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-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-hydroor otyl)-D-4-aminoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2) and gannel (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) (Thundimadathil, J., J. AminoAcids, 2012, 967347; Boccon-Gibod, L.; et al., 2011, Therapeutic Advances in Urology 3(3):127–140; Debruyne, F., 2006, Future Oncology, 2(6), 677–696; Schally AV; Nagy, A. 1999 Eur J Endocrinol 141:1–14; Koppan M, et al. 1999 Prostate 38:151–158); pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectins and nodular receptors (NLRs) (Fukata, M. et al., 2009, Semin. Immunol. 21, 242–253; Maisonneuve, C. et al., 2014, Proc. Natl. Acad. Sci. USA 111, 1–6; Botos, I. et al., 2011, Structure 19, 447–459; Means, TK et al., 2000, Life Sci. 68, 241–258), with molecular weights ranging from small molecules (imiquimod, guanine and adenosine homologues) to large and complex biomacromolecules such as lipopolysaccharide (LPS), nucleic acids (CpG DNA, polyI:C) and lipopeptides (Pam3CSK4) (Kasturi, SP et al., 2011, Nature 470, 543–547; Lane, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dudek, AZ et al., 2007, Clin. Cancer Res.13, 7119–25); calcitonin receptor, which is a 32-amino acid neuropeptide that is involved in the regulation of calcium levels mainly through its effects on osteoclasts and kidneys (Zaidi M, et al., 1990 Crit Rev Clin Lab Sci 28, 109–174; Gorn, AH, et al., 1995 J Clin Invest 95: 2680–91); integrin receptors and their receptor subclasses (such as αVβ1, αVβ3, αVβ5, αVβ6, α6β4, α7β1, αLβ2, αIIbβ3, etc.), usually play an important role in angiogenesis and are expressed on the surface of various cells, especially osteoclasts, endothelial cells and tumor cells (Ruoslahti, E. et al., 1994 Cell 77, 477-8; Albelda, SM et al., 1990 Cancer Res., 50, 6757-64). Short peptides, GRGDSPK and cyclic RGD pentapeptides, such as cyclo(RGDfV)(L1) and its derivatives (cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N(Me)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me)V-)(cilengitide)) have high affinity for integrin receptors (Dechantsreiter, MA et al., 1999 J. Med. Chem. 42, 3033-40, Goodman, SL, et al., 2002 J. Med. Chem. 45, 1045-51). .

[0269] The cell binding molecules or ligands or cell receptor agonists can be Ig-based and non-Ig-based protein scaffold molecules. The Ig-based scaffold can be selected from, but not limited to, nanobodies (derivatives of VHH (camelid Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-97); domain antibodies (dAb, derivatives of VH or VL domains) (Holt, LJ, et al., 2003, Trends Biotechnol. 21, 484-90); bispecific T cell adaptors (BiTE, bispecific diabodies) (Baeuerle, PA et al., 2009, Curr. Opin. Mol. Ther. 11, 22-30); dual affinity retargeting agents (DART, bispecific diabodies) (Moore PA P et al. 2011, Blood 117 (17), 4542-51); tetravalent tandem antibodies (T and Ab, dimeric bispecific diabodies) (Cochlovius, B et al. 2000, Cancer Res. 60(16):4336–4341).The non-Ig scaffold can be selected from, but not limited to, Anticalin (derivatives of Lipocalins) (Skerra A. 2008, FEBS J., 275 (11): 2677-83; Beste G et al., 1999 Proc. Nat. Acad. USA. 96 (5): 1898-903; Skerra, A. 2000 Biochim Biophys Acta, 1482 (1-2): 337-50; Skerra, A. 2007, Curr Opin Biotechnol. 18 (4): 295-304; Skerra, A. 2008, FEBS J. 275 (11): 2677-83); Adnectin (10th FN3 (fibronectin)) (Koide, A et al., 1998 J. Mol. Biol., 284 (4): 1141-51; Batori V, 2002, Protein Eng. 15(12): 1015–20; Tolcher, AW, 2011, Clin. Cancer Res. 17(2): 363–71; Hackel, BJ, 2010, Protein Eng. Des. Sel. 23(4): 211–19); designed ankyrin repeat proteins (DARPins) (derivatives of ankyrin repeat (AR) proteins) (Boersma, YL et al., 2011 Curr Opin Biotechnol. 22(6): 849–57), such as DARPin C9, DARPin Ec4 and DARPin E69_LZ3_E01 (Winkler J et al., 2009 Mol Cancer Ther. 8(9), 2674–83; Patricia MK.M. et al., Clin Cancer Res. 2011; 17(1): 100–10; Boersma YL et al., 2011 J. Biol. Chem. 286(48), 41273–85); high affinity multimer (domain A / low-density lipoprotein (LDL) receptor) (Boersma YL, 2011 J. Biol. Chem. 286(48): 41273–41285; Silverman J et al., 2005 Nat. Biotechnol., 23(12): 1556–61).

[0270] Examples of the structures of the small molecule cell binding molecules / ligands or cell receptor agonists of the present patent application are as follows: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, somatostatin homolog), LB08 (lanreotide, somatostatin homolog), LB09 (vapreotide (Sanvar), somatostatin homolog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin releasing peptide receptor (GRPr), M BA), LB13 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (cobalamin, vitamin B12 homolog), LB17 (cobalamin, vitamin B12 homolog), LB18 (for αvβ3 integrin receptor, cyclic RGD pentapeptide), LB19 (heterobivalent peptide ligand for VEGF receptor), LB20 (neuromedullin B), LB21 (bombesin, acts on G protein-coupled receptors), LB22 (TLR2, Acts on Toll-like receptors), LB23 (acts on androgen receptors), LB24 (cilengitide or cyclic (-RGDfV-) conjugates, acts on αv integrin receptors) LB25 (rifabutin homologues), LB26 (rifabutin homologues), LB27 (rifabutin homologues), LB28 (fludrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclomethasone propionate), LB32 (triamcinolone acetate), LB33 (prednisolone), LB35 (methylprednisolone), LB36 (betamethasone), LB37 (irinotecan homologues), LB LB38 (crizotinib homologue), LB39 (bortezomib homologue), LB40 (carfilzomib homologue), LB41 (carfilzomib homologue), LB42 (leuprolide homologue), LB43 (triptorelin homologue), LB44 (clindamycin), LB45 (liraglutide homologue), LB46 (semivincristine homologue), LB47 (retapaline homologue), LB48 (dibulol homologue), LB49 (vinblastine homologue), LB50 (lixisenide homologue), LB51 (osimidine homologue), LB52 (nucleoside homologue), LB53 (erlotinib homologue) and LB54 (lapatinib homologue), whose structures are shown below:

[0271] LB01 (Folic acid conjugate),

[0272] LB02 (PMSA ligand conjugate),

[0273] LB03 (PMSA ligand conjugate),

[0274] LB04 (PMSA ligand),

[0275] LB05 (somatostatin),

[0276] LB06 (somatostatin),

[0277] LB07 (octreotide, a somatostatin homologue),

[0278] LB08 (Lantide, somatostatin homologue),

[0279] LB09 (aminopeptide (Sanvar), somatostatin homologue),

[0280] LB10 (CAIX ligand),

[0281] LB11 (CAIX ligand),

[0282] LB12 (Gastrin-releasing peptide receptor (GRPr), MBA),

[0283]

[0284] LB13 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone (GnRH) ligand),

[0285]

[0286] LB14 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone (GnRH) ligand),

[0287]

[0288] LB15 (GnRH antagonist, abarelix),

[0289] LB16 (cobalamin, a vitamin B12 homologue),

[0290] LB17 (cobalamin, a vitamin B12 homologue),

[0291]

[0292] LB18 (cyclo-RGD pentapeptide, acting on α v β3 integrin receptor),

[0293]

[0294] LB19 (heterologous bivalent peptide ligand conjugate, acting on vascular endothelial growth factor VEGF receptor),

[0295] LB20 (neuromedullin B),

[0296]

[0297] LB21 (bombesin conjugate, acting on G protein-coupled receptors),

[0298] LB22 (TLR2 conjugate, acting on Toll-like receptors),

[0299] LB23 (androgen receptor),

[0300] LB24 (Cilengitide / cyclic (-RGDfV-) conjugate, acting on α v Integrin receptors

[0301] LB25 (rifabutin homologue),

[0302] LB26 (rifabutin homologue),

[0303] LB27 (rifabutin homologue),

[0304] LB28 (fludrocortisone),

[0305] LB29 (dexamethasone),

[0306] LB30 (fluticasone propionate),

[0307] LB31 (beclomethasone propionate),

[0308] LB32 (triamcinolone acetonide),

[0309] LB33 (prednisone),

[0310] LB34 (prednisolone),

[0311] LB35 (methylprednisolone),

[0312] LB36 (beflumethasone),

[0313] LB37 (irinotecan homologue),

[0314] LB38 (crizotinib homologue),

[0315] LB39 (bortezomib homologue), wherein Y5 is N, CH, C(Cl), C(CH3) or C(COOR1); R1 is H, C1-C6 alkyl, C3-C8 aryl;

[0316] LB40 (carfilzomib homologue),

[0317] LB41 (carfilzomib homologue),

[0318] LB42 (leuproreli homologue),

[0319] LB43 (triptorelin homologue),

[0320] LB44 (clindamycin),

[0321] LB45 (liraglutide homologue),

[0322] LB46 (semaglutide homologue),

[0323] LB47 (retapalene homologue),

[0324] LB48 (Dingbull homologue),

[0325] LB49 (vinblastine homologue),

[0326] LB50 (Lisisen peptide homologue),

[0327] LB51 (oscitinib homologue),

[0328] LB52 (nucleoside homologue),

[0329] LB53 (erlotinib homologue),

[0330] LB54 (lapatinib homologue),

[0331] in is the attachment position of the branch linker; X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH2, C(O)NHNHC(O) and C(O)NR1; X1 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR1, C(O)R1 or C(O)O; X5 is H, CH3, F, or Cl; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, N(R1) 12 R 12’ R 13 R 13’ );R 12 , R 12’ , R 13 and R 13’ The definition is the same as that of formula (I).

[0332] Application of conjugates

[0333] In a specific embodiment, the cell-binding agent-drug conjugate connected by the branched linker of the present patent can be used as a cancer targeted therapy. Target cancers include, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumors (brainstem glioma, cerebellar astrocytoma, brain astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic gliomas), breast cancer, carcinoid tumors, gastrointestinal cancer, unknown small cell carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of tumors (PNET), intracranial germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer (gastric cancer), extragonadal germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell), lymphoma (AIDS-related, central nervous system, cutaneous T-cell, Hodgkin disease, non-Hodgkin's disease), malignant mesothelioma, melanoma, Merkel cell carcinoma, metastatic squamous neck cancer and occult primary cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplasia, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumor, low-grade tumor), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer , pheochromocytoma, pituitary tumors, plasma cell tumors, prostate cancer rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sessery syndrome, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine tumors, soft tissue sarcomas, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer, unusual juvenile cancers, vaginal tumors, vulvar tumors and Wilms' tumor.

[0334] In another specific embodiment, the cell-binding agent-drug conjugates connected by the branched linker of the present invention can be used as a composition and method for treating or preventing autoimmune diseases. Autoimmune diseases include, but are not limited to, Achlorhydra, autoimmune active chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, azoospermia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM / TBM nephritis, antiphospholipid syndrome, anti-abnormal enzyme syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome I, II and III type, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo disease / Balo concentric sclerosis, Bechets syndrome, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, Chagas disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, celiac disease, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis , CREST syndrome, Crohns disease (idiopathic inflammatory bowel disease), Cushing syndrome, cutaneous leukocytosis vasculitis, DeGorge's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse cutaneous systemic sclerosis, Dressler syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, Eosinophilic fasciitis, epidermolysis bullosa, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans, fibromyalgia, fibrosing myositis, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Good Paschal syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, hepatitis of pregnancy, hidradenitis suppurativa, Hughes syndrome (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating polyneuritis, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenia gravis syndrome, leukocytoclastic vasculitis, lichen planus, scleroderma,Linear IgA disease (LAD), Lou Gehrig's disease (also known as amyotrophic lateral sclerosis), lupus hepatitis, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyarteritis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mohammed-Habermann disease, Macaulay syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neuromyotonia, eyelid cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), Paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, ParryRomberg syndrome, Parsonnage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, anemia, peripheral encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, gangrenous dermatitis, pure red cell aplasia, Rasmussen encephalitis, Raynaud's phenomenon, relapsing polychondritis, Raynaud's Syndrome, restless legs syndrome, posterior neuropathy, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthropathies, viscosemia, Still's disease, stiff-man syndrome, subacute bacterial endocarditis, Susac syndrome, Sweet syndrome, chorea, sympathetic anemia, Takayasu arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathies, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome.

[0335] In another specific embodiment, in the conjugate for treating or preventing autoimmune diseases, the binding molecules connected to the drug molecules through the branched linker of the present patent include, but are not limited to, anti-elastin antibodies, Abys anti-epithelial cell antibodies, anti-basement membrane type IV collagen antibodies, anti-nuclear antibodies, anti-ds DNA, anti-ss DNA, anti-cardiolipin antibodies IgM, IgG, anti-celiac disease antibodies, anti-phospholipid antibodies IgK, IgG, anti-SM antibodies, anti-mitochondrial antibodies, thyroid antibodies, microsomal antibodies, T cell antibodies, thyroglobulin antibodies, anti-SCL-70, anti-Jo, anti-U.sub.1RNP, anti-La / SSB, anti-SSA, anti-SSB, anti-parietal cell antibodies, anti-histone, anti-RNP, C-ANCA, P-ANCA, anti-centromere, anti-fibrinogen, anti-GBM antibodies, anti-ganglioside antibodies, anti-Desmogein 3 antibody, anti-p62 antibody, anti-sp100 antibody, anti-mitochondrial (M2) antibody, rheumatoid factor antibody, anti-MCV antibody, anti-topoisomerase antibody, anti-neutrophil cytoplasmic (cANCA) antibody.

[0336] In certain preferred embodiments, the binding molecules on the conjugates of this patent can bind to receptors or receptor complexes expressed on activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex comprises an immunoglobulin gene superfamily member (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD79b, CD90, CD125, CD147, CD152 / CTLA-4, PD-1 or ICOS), a TNF receptor superfamily member (e.g., CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin (type C, type S or type I) or a complement control protein.

[0337] In another specific embodiment, the available cell binding ligand with immunospecificity to viral or microbial antigens is a humanized or human monoclonal antibody. "Viral antigens" include, but are not limited to, any viral peptides, polypeptide proteins (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV Tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen) that can elicit an immune response. "Microbial antigens" include, but are not limited to, any microbial peptides, polypeptides, proteins, sugars, polysaccharides or lipid molecules (e.g., bacteria, fungi, pathogenic protozoa or yeast polypeptides, including, for example, LPS and capsular polysaccharides) that can elicit an immune response. Examples of antibodies that can be used to treat viral or microbial infections include, but are not limited to: palivizumab, a humanized anti-respiratory syncytial virus monoclonal antibody used to treat RSV infection; PRO542, a CD4 fusion antibody used to treat HIV infection; oseltamivir, a human antibody used to treat hepatitis B virus; PROTVIR, a humanized IgG1 antibody used to treat cytomegalovirus, and anti-LPS antibodies.

[0338] The cell binding molecule-drug conjugates prepared by the branched linker of the present invention can be used to treat infectious diseases. These infectious diseases include, but are not limited to, acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amebiasis, anaplasmosis, anthrax, hemolytic Yersinia infection, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, Bacteroides infection, bagworm disease, ascariasis, BK virus infection, black hair nodular disease, human blastocyst infection, blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, calicivirus infection ( Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (candidiasis, thrush), Cat Scratch Disease, Cellulitis, Chagas Disease (American Trypanosomiasis), Ascocystis, Chickenpox, Chlamydia, Chlamydia Pneumoniae Infection, Cholera, Chromatoblastoma, Clonorchis sinensis, Clostridium difficile Infection, Coccidioidomycosis, Colorado Tick Fever, Common Cold (Acute Viral Rhinopharyngitis, Acute Rhinitis), Creutzfeldt-Jakob Disease, Crimean-Congo Hemorrhagic Fever, Cryptococcosis, Cryptosporidiosis, Cutaneous Larva Migrans, Cyclosporiasis, Enterobacter Infection, Enterovirus Infection, Epidemic Typhus, Erythema Infectiosum (Fifth Disease), Acute Eruption, Fasciola Hepatica, Fasciola Hepatica, Fatal Familial Insomnia, Filariasis, Parturition Clostridium perfringens food poisoning, free-living amebic infection, fusobacterium infection, gas gangrene (clostridial myonecrosis), geotrichomoniasis, Gerstmann-Strassler-Scheckel disease syndrome, giardiasis, glanders, gonorrhea, granulomatous diarrhea (fifth STD), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot and mouth disease (HFMD), Hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworm infection, human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis, human Metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, hymenolepidium infection, Epstein-Barr virus infectious mononucleosis (mononucleosis), influenza, isosporiasis, Kawasaki disease, keratitis, Kingella infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leishmaniasis, Lyme disease, lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever, measles, melioidosis (Wyeth's disease), meningitis, meningococcal disease, metagonomiasis, microsporidiosis, molluscum contagiosum, mumps, murine typhus (endemic typhus), mycoplasma pneumonia, mycetoma, myiasis,Neonatal conjunctivitis (neonatal eye disease), variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness), paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, head lice, body lice, pubic lice, pelvic inflammatory disease, whooping cough, plague, pneumococcal infection, pneumocystis pneumonia, pneumonia, poliomyelitis, Prevotella infection, primary amebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat-bite fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsialpox, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, Shigella Bacillaceae disease (Bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporotrichosis, staphylococcal food poisoning, infection with Staphylococcus aureus, strongyloidiasis, syphilis, tapeworm disease, tetanus, Barber's itch, scalp ringworm, body ringworm, tinea cruris, hand ringworm, palm black ringworm, pedis, onychomycosis (onychomycosis), tinea versicolor, toxocariasis (ocular larva migrans), toxocariasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomoniasis, whipworm disease (whipworm infection), tuberculosis, rabbit fever, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white hair nodule disease (white ringworm), Yersinia pseudotuberculosis, Yersinia pestis enteropathy, yellow fever, zygomycosis.

[0339] The pathogenic strains that the conjugates of the present invention are preferably able to resist include, but are not limited to, Acinetobacter baumannii, Actinomyces israelii, Actinomyces and Propionibacterium, Trypanosoma brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma, Bacillus anthracis, Vibrio hemolyticus, Junin virus, Ascaris, Aspergillus, Astroviridae, Babesia, Bacillus cereus, various bacteria, Bacteroides, Escherichia coli, Ascaris, BK virus, Nodularia, Blastocystis hominis, Blastomyces dermatitidis, Machupo virus, Borrelia, Clostridium botulinum, Qingfengteng, Brucella, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae, Campylobacter, usually Candida albicans and other Candida species genus, Bartonella henselae, group A Streptococci and Staphylococci, Trypanosoma cruzi, Haemophilus ducreyi, VZV, Chlamydia trachomatis, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., Ancylostoma brasiliensis, various parasites, Cyclospora, Taenia solium, Cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4)-Flavivirus, Bifidobacterium fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Guinea worm, Ebola virus, Echinococcus spp., Ehrlichia enterococci, Enterovirus spp., Rickettsia prowazekii, Parvovirus B19, human herpesvirus 6 and 7, Fasciola hepatica, and Giant Fasciola gigantea, FFI prions, Filariales superfamily, Clostridium perfringens, Fusobacterium, other Clostridia, Geotrichum candidum, GSS prions, Giardia intestinalis, Burkholderia, Bacillus spinulosus and Gram-negative Candida, Neisseria gonorrhoeae, Klebsiella granulomatosa, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, enteroviruses, mainly Coxsackie A virus and enterovirus 71, unnamed virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, duodenal adenoma and ampullary carcinoma Haemophilus influenzae, human bocavirus, Ebola virus Rihidia, Haemophilus phagocytophilus, human metapneumovirus, Ehrlichia chaffeensis, human papillomavirus, human parainfluenza virus, Hymenoleuca microti and Hymenoleuca minima, Epstein-Barr virus, Orthomyxoviridae family, Isospora belli, Kingella, Klebsiella pneumoniae, Klebsiella, Legionella pneumophila, Legionella pneumophila, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium tuberculosis, Leptospira, Listeria monocytogenes, Borrelia burgdorferi and other Borrelia species, Trichinella bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV) Plasmodium, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitidis, Metagonium yokogawa, Microsporida,Molluscum contagiosum virus (MCV), mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, various bacterial and fungal parasitic fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prions, Nocardia and other Nocardia species, Onchocerca spp., Discoidea subfamily, Parasaurolophus sempervirens and other Parasaurolophus species, Pasteurella species, head lice, human lice, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumococcus, poliovirus, Prevotella species, Nassaurus, JC virus, Chlamydia psittaci, Coxiella burnetii, rabies virus, Streptococcus and spirochetes, respiratory syncytial virus, rhinovirus Sporozoites, rhinovirus, Rickettsia, Rickettsia spp., Rift Valley fever virus, Rickettsia spp., rotavirus, rubella, Salmonella, SARS coronavirus, human scabies, Schistosoma, Somatic cell, Shigella, Varicella zoster virus, Smallpox major or Smallpox minor, Sporothrix schenckii, Staphylococcus aureus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, Treponema pallidum, Tapeworm, Tetanus, Ringworm, Ringworm, Epidermophyton floccosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton rubrum, Wernicke's Exophiala, Trichophyton, Cell death, Toxoplasma or Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichomonas sanguinis, Mycobacterium tuberculosis, Francesca tularensis, Urea and equine encephalitis virus, Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, Trichomonas beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales (mucormycosis) and Entomophthora (entomycosis), Mucorales Pseudomonas aeruginosa, Campylobacter (Vibrio), Aeromonas, Ehrlich, Yersinia, Shigella dysenteriae, Shigella, Shigella, Salmonella, Salmonella typhi, Borrelia yawsii, Borrelia vincensis, Borrelia burgdorferi Spirochetes, Leptospira, Pneumocystis carinii, Brucella abortus, Brucella, Brucella, Mycoplasma, Rickettsia prowazekii, Rickettsia tsutsugamushi, Chlamydia, Pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum), Protozoa (Entamoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trypanosoma gambiae, Trypanosoma rhodesiense, Leishmania roegneri, Leishmania tropicalis, Leishmania braziliensis, Pneumocystis pneumoniae, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria), or Helminiths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Hookworm).

[0340] Other conjugates for use in the treatment of viral diseases include, but are not limited to, antibodies against the following pathogenic viral antigens: poxvirus, herpes virus, adenovirus, parvovirus, enterovirus, picornavirus, parvovirus, reovirus, retrovirus, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, arbovirus, rhabdovirus, salmonella, non-a / non-b hepatitis virus, rhinovirus, coronavirus, rotovirus, oncogenic viruses such as HBV (hepatocellular carcinoma), human papillomavirus (cervical cancer, anal cancer), Kaposi's sarcoma-related herpes virus, Herpes virus (Kaposi's sarcoma), human herpes virus type 4 (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma), oncovirus (Merkel cell carcinoma), SV40 (simian virus 40), HCV (hepatocellular carcinoma), HTLV-1 (adult T-cell leukemia / lymphoma), immune disorder causing viruses, such as human immunodeficiency virus (AIDS), central nervous system viruses, such as JCV (progressive multifocal leukoencephalopathy), hepatitis C virus (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), Yabo virus encephalitis , Orthomyxovirus (encephalitis encephalitis), RV (rabies), Proboscis virus, Herpes meningitis, Ramsay Hunt syndrome type II, Poliovirus (poliovirus, post-polio syndrome), HTLV-1 (tropical paralytic paralysis), Cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis), Cardiovascular viruses, such as CBV (pericarditis, myocarditis), Respiratory system / acute viral endonasal inflammation / viral pneumonia, such as Epstein-Barr virus (EBV infection / infectious mononucleosis), Cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome) or orthomyxovirus, influenza virus a / b / c (flu / avian flu), paramyxovirus, human parainfluenza virus, RSV (human respiratory syncytial virus), hMPV, digestive system viruses (mumps virus, cytomegalovirus (cytomegalovirus esophagitis), adenovirus (adenovirus infection), rotavirus, norovirus, astrovirus, coronavirus, hepatitis B virus, CBV, hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, HGV), urogenital viruses such as BK virus, MuV (mumps).

[0341] Furthermore, the present invention also includes a composition of the conjugate of the present invention and an acceptable carrier, diluent or excipient to treat cancer, infection or autoimmune disease. Methods for treating cancer, infection and autoimmune disease can be implemented in vitro, in vivo or ex vivo. Examples of in vitro uses include treating cell cultures with it to kill all cells except variants that do not express the target antigen; or killing variants that express unwanted antigens. Examples of ex vivo use include treating hematopoietic stem cells (HSC) before transplantation (HSCT) to kill diseased or malignant cells. For example, tumor cells or lymphocytes are removed from the bone marrow before autologous transplantation in cancer treatment or in the treatment of autoimmune diseases, or T cells and other lymphocytes are removed from allogeneic bone marrow or tissue before transplantation to prevent graft-versus-host disease. Such clinical ex vivo treatment can be carried out as follows: bone marrow is harvested from a patient or other individual, and then incubated in a serum-containing culture medium at about 37°C for about 15 minutes to about 48 hours, and the conjugate of the present invention is added to the culture medium at a concentration ranging from about 1 pM to 0.1 mM. The specific drug concentration and incubation time should be determined by a professional clinician. After incubation, the bone marrow cells are washed with a serum-containing medium and injected intravenously into the patient according to known methods. If the patient receives other treatments (such as ablative chemotherapy or whole-body radiation therapy) between bone marrow collection and re-infusion of therapeutic cells, the processed bone marrow cells should be frozen and stored in liquid nitrogen using standard medical equipment.

[0342] Chemotherapeutic or cytotoxic drugs for synergistic effects

[0343] Chemotherapeutic drugs that can work synergistically with the present invention are small molecule drugs including cytotoxic agents. "Small molecule drugs" in the present invention generally refer to organic, inorganic or metal organic compounds with a molecular weight of 100 to 2500, preferably 200 to 2000. These small molecule drugs have been fully described in the literature in the art, such as WO05058367A2 and U.S. Patent 4,956,303, as well as Chessum, N., et al., Prog Med Chem. 2015, 54: 1-63; Eder, J., et al., Nat Rev Drug Discov. 2014, 13 (8): 577-87; Zhang, M.-Q., et al., Curr Opin Biotechnol. 2007, 18 (6): 478-88; cited herein as reference. Small molecule drugs include known drugs and drugs to be disclosed.

[0344] Known drugs include but are not limited to:

[0345] 1) Chemotherapeutic drugs: a) Alkylating agents, such as nitrogen mustards: chlorpheniramine, chlorprenazine, cyclophosphamide, dacarbazine, estradiol nitrogen mustard, ifosfamide, nitrogen mustard, dimethoxamine hydrochloride, nitrogen oxide mustard, amlodipine hydrochloride, mycophenolic acid, dulcitol, guapobroman, new nitrogen mustard, phenylephrine, phenylephrine, thiotetracycline, trofosfamide, uracil; CC-1065 (including its adolesin, carbenicillin, oxadiazine, pyrrolebenzodiazepine (PBD) or dimers of tomemycin, indolebenzodiazepine, imidazolebenzothiadiazepine or oxazolidinebenzodiazepine; nitrosoureas (carmustine, lomustine, clostridium dichloride, fotemustine, nimodipine); a) Plant alkaloids: such as vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine, norvinblastine); taxanes (paclitaxel, docetaxel and their homologues); maytansines (DM1, DM2, DM3, DM4, maytansine, ansamycin and their homologues); cryptophycins (especially cryptophycin 1 and cryptophycin 2); 8); epothilone, soft coral alcohol, dimolide, grass bryozoan lactone, dolastatin, auristatin, cephalostatin; pancratistatin; sarcodictyin; sponge inhibitor; c) DNA topoisomerase inhibitors, such as etoposide (9-aminocamptothecin, camptothecin, clenatol, doramycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, noxol, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000)); mitomycin (mitomycin C); d) antimetabolites, such as antifolates, DHFR inhibitors preparations (methotrexate, tramecil, dimethoate, pteropterin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazofurine, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine analogs, uracil analogs (ancitabine, azacitidine, 6-azauracil, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, 5-fluorouracil, floxuridine, ratitrexed (Tomudex); cytosine analogs (cytarabine, cytosine arabinoside, fludarabine);Purine analogs (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine); folic acid supplements, such as florin acid; e) hormone therapy agents, such as receptor antagonists, antiestrogens (megestrol, raloxifene, tamoxifen), LHRH stimulants (gostaline, leuprolide acetate); antiandrogens (bicalutamide, flutamide, calustratone, betandrosterone propionate, epiandrostenone, goserelin, leuprolide, metilidine, nilutamide, endorphins esters, trilostane and other androgen inhibitors); retinoids, vitamin D3 homologues (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A); cytokines (interferon-α, interferon-γ, tumor necrosis factor (TNF), human TNF-containing proteins); f) kinase inhibitors, such as BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, guagatanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SK g) poly (ADP-ribose) polymerase (PARP) inhibitors, such as olaparib, niraparib, iniparib, tarazoparib, veliparib, CEP 9722 (Cephalon), E7016 (Eisai), BGB-290 (Beigene), 3-aminobenzamide; h) antibiotics, such as enediyne antibiotics (calicheamicin, in particular calicheamicin γ1, δ1, α1 and β1 (see J. Med. Chem. 1996, 39 (11), 2103-2117;Angew Chem Intl. Ed. Engl. 1994, 33: 183-186), dynemycins, including dynemycin A and deoxymycin, esperamicin, kataramicin, C-1027, maduropeptin, neocarbazin and related chromoprotein enediyne antibiotics), aclacinomysins, actinomycins, anthramycins, azaserine, bleomycin, kanonomycin, karanomycin, carminomicin, carmomycin, chromomycin, dazuramycin, rouximycin, Erythromycin, desidarubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholine-doxorubicin, cyanomorpholine-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin, epirubicin, aclarubicin, idarubicin, marcomycin, nitomycin, mycophenolic acid, nogamycin, olivemycin, peplomycin, potfiromycin, puromycin, quinamycin, rhodoxomycin, streptomycin, streptozotocin, tuberculin, ubenimex , nestatin, doxycycline; i) others, such as polyketides (annona), especially bullatacin and bullatacinone; gemcitabine, cyclooxygenase (such as carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, alloceptin-7, Xegev a, Provenge, Yervoy, prenylation inhibitors (such as lovastatin), dopaminergic neurotoxins (such as staurosporine), actinomycins (such as actinomycin D, dactinomycin), bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), anthracyclines (such as daunorubicin), amatoxin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, doxycycline, mitoxantrone, MDR inhibitors (such as verapamil), Ca; 2+ATPase inhibitors (such as 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; Antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane, aceglucuronolide, aldehyde phosphamide, aminolevulinic acid, amsacrine, arabinoside, bestrabucil, bisantrene, edatraxate, defofamine, mecocin, diazocine, eflornithine (DFMO), elfomithine, elliptonium acetate, etoglu, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguanidine, mitoxantrone, moguadol, diamine nitracrine, pentostatin, methamine nitrogen mustard, pirarubicin, podophyllic acid, 2-ethylhydrazine, methylbenzalkonium; Piperazinedione propane; rhizoxin; siroxicam; spirogermanium; tricrosporin; triimine quinone; trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosporin A, baculosporin A and anguidine), polyurethanes, siRNA, antisense drugs and nucleases.

[0346] 2) Autoimmune disease drugs, including but not limited to, cyclosporine, cyclosporin A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus.

[0347] 3) Anti-infective drugs, including but not limited to a) aminoglycosides: amikacin, astamicin, gentamicin (netilmicin, sisomicin, isopamicin), hygromycin B, kanamycin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methyl zithromycin; b) amides: chloramphenicol azide, chloramphenicol, florfenicol, thiamphenicol; c) ansamycins: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem / simepentylene Statins, meropenem, panipenem; e) Cephem: carbacephem (loracabib), ceftriaxone, clomiphene, cephradine, cefoperazone, cefuroxime, cefuroxime, cephalosporin, cephalothin or cephalothin, cephalexin, cefuroxime, cefadroxil, cefamandole, cefpirin, hydroxylamine cephalosporin, fluazole cephalosporin, cefadroxil, cephalosporin, cefoperazone, cefoperazone, cefoperazone, cefuroxime, cefoperazone, cefoperazone, cefuroxime, cefoperpiroxil, cefoperazone, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefoperazone, cefoperazone, cefoperazone, cefuroxime ... oxime, thiamethoxam cephalosporin, cefazolin, cefalexin, cefimizolin, cefpiramide, cefpirome, cefpodoxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefoperazone, cefoperazone, cefoperazone, cefoperazone, cephalosporin (cefoxitin, cefotetan, cefuroxime), oxygen (carbon) cephems (floxacin, latamoxef); f) glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g) glycylcycline: such as tigecycline; h) β-lactamase inhibitors: penicillins (sulbactam, tazobactam), oxygen Penicillamine (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA); k) Macrolides: azithromycin, clotrimazole, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolide (telithromycin, cypermethrin), midecamycin, micamycin, oleandomycin, rifamycins (isoniazid, rifampicin, rifabutin, rifapentine), ropitomycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; l) Monocyclic amines: aztreonam, tigemonam; m) Oxazolidinones: linezolid;n) Penicillins: amoxicillin, ampicillin (pivacillin, herocillin, bacampicillin, ampicillin, doxorubicin), azlocillin, azlocillin, benzylpenicillin, benzathine penicillin benzylpenicillin, benzathine penicillin phenoxymethylpenicillin, clocillin, procaine penicillin (meticillin), mezlocillin, methicillin, nafcillin, oxacillin, acetylpenicillin, penicillin, pheneticillin, guanacillin, ampicillin, sulfenicillin, temocillin, ticarcillin; o) Peptides: bacitracin, mucosin, p) Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinas, danofloxacin, difloxacin, enoxacin, enrofloxacin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, levofloxacin, lomefloxacin, mabofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, toloxacin, trovafloxacin; q) Streptogramins: pristinamycin, quinupristin / Dafopristin; r) Sulfonamides: amitraz, sulfazosulfonamide, sulfadiazine, sulfamethoxazole, sulfadiazine, sulfapyridine, sulfisoxazole, trimethoprim, sulfamethoxazole (compound sulfamethoxazole); s) Steroid antibiotics: such as fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, clomicycline, demeclocycline, ramoxicline, mexicycline, methacycline, minocycline, oxytetracycline, panmethocycline, pyrrolidinomethyltetracycline, tetracycline, glycylcycline (such as tigecycline); u) Other types of antibiotics: cypermethrin , arsphenamine, bacterial terpene alcohol inhibitors (bacitracin), DANAL / AR inhibitors (cycloserine), dictyostatin, sphaerolide, sphaerolide, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthesis inhibitors (such as fosfomycin), nitrofurantoin, paclitaxel, pramoxetine, pyrazinamide, quinupristin / dalfopristin, rifampicin, tazobactam, tinidazole, rutin. ;

[0348] 4) Antiviral drugs: a) Entry / fusion inhibitors: apavirol, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b) Integrase inhibitors: raltegravir, elvite-gravir, globoidnan A; c) Maturation inhibitors: bevirimat, vivecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: abacavir, aciclovir, adefovir, amosivir, abciximab, brivudine, cidofovir, clavudine, dexamethasone, didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluracillin (5-FU), 3'-fluorouracil Substituted 2',3'-deoxynucleoside homologues, such as 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, 9-guanine, iodine, lamivudine (3TC), 1-nucleosides (e.g., β-1-thymidine and β-1-2'-deoxycytidine), penciclovir, racivir, ribavirin, ditidine, stavudine (d4T), viramidine, telbivudine f) Non-nucleoside: amantadine, atipiridine, caprovirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, amivirine, efavirenz, foscarnet (phosphocarboxylic acid), imiquimod, pegylated interferon, loviride, lodadecytidine, methylthiazone, nevirapine, NOV-205, long-acting interferon α, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), acetamide adamantane; g) protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) other types of antiviral drugs: antibody enzyme, arbidol, calanolide a, ceragenin, cypermethrin-n, diaryl pyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, mixed inhibitors, ribavirin, seliciclib.

[0349] 5) Radioisotopes used in radiation therapy. Examples of radioisotopes (radionuclides) are 3 H. 11 C. 14 C.18 F. 32 P. 35 S. 64 Cu, 68 Ga, 86 Y. 99 Tc, 111 In, 123 I. 124 I, 125I, 131 I. 133 Xe, 177 Lu, 211 At or 213 Bi. Radioactive isotope-labeled antibodies can be used for receptor targeting imaging experiments, or can be used for targeted therapy such as the antibody-drug conjugate of the present invention (Wu et al. Nature Biotechnology 2005, 23 (9): 1137-1146). Cell binding molecules, such as antibodies, can be labeled by linking ligand reagents through the linker of this patent. The ligand can be combined, chelated or complexed with the radioactive metal using the methods described in the literature (Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. (1991)). Chelating ligands that can complex metal ions include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, TX) and the like.

[0350] 6) Another cell binding molecule-drug conjugate as a synergist. Preferred cytotoxic molecules on the synergistic conjugate are: amatoxin analogs, maytansinoid alkaloid homologues, paclitaxel (taxane) homologues, CC-1065 homologues, daunorubicin and doxorubicin compounds, amatoxin homologues, benzodiazepine dimers (such as dimers of pyrrolebenzodiazepines (PBD, tomamycin, anthramycin, indolebenzothiadiazepines, imidazolebenzothiadiazepines or oxazolidinebenzodiazepines), calicimycin and enediyne antibiotic compounds, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen, idarubicin, sea halys. toxins, auristatins (such as MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB) and EFP (AEFP)), duocarmycin, geldanamycin, methotrexate, thiotepa, vinblastine, vincristine, hemiasterlin, nasalamide, chlortetracycline, radiosumin, alterniflorin, microsclerodermin, thiophenecarboxamide, espamycin, PNU-159682, and their homologues and derivatives of the above substances.

[0351] 7) Pharmaceutically acceptable salts, acids or derivatives of any of the above drugs.

[0352] In another embodiment, the immunotoxin can be coupled to a cell-binding molecule as a synergistic drug. The immunotoxin of the present invention is a macromolecular drug, usually a cytotoxic protein derived from bacteria or plant proteins, such as diphtheria toxin (DT), cholera toxin (CT), trichosanthin (TCS), dianthin, Pseudomonas exotoxin A (ETA), erythrocyte toxin, diphtheria toxin, AB toxin, type III exotoxin, etc. It can also be a highly toxic bacterial pore-forming protoxin that is activated by protease hydrolysis. An example of a protoxin is prohemolysin and its genetically modified form topsalysin. Topsalysin is a modified recombinant protein that is selectively activated by enzymes in the prostate after modification, causing local cell death and tissue destruction without damaging adjacent tissues and nerves.

[0353] In another synergistic immunotherapy, antibodies to the following molecules: checkpoint inhibitors, TCR (T cell receptor) T cells, CAR (chimeric antigen receptor) T cells, B cell receptor (BCR), natural killer (NK) cells, or CD3, CD4, CD8, CD16 (FcγRIII), CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD37, CD38, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57bright, CD70, CD79, CD123, CD138, TNFβ, fatty acid synthase ligand, MHC class I molecules (HLA-A, B, C), VEGF, or NKR-P1 are preferably used in synergistic therapy with the conjugates of the present invention.

[0354] Preparation and application

[0355] The conjugate of the present patent application is formulated into a liquid or a form suitable for freeze-drying, and is reconstituted into a liquid preparation after freeze-drying. The conjugate in the liquid formulation or the formulated freeze-dried powder is the main component of the formulation, accounting for 0.01%-99% by weight. Generally speaking, the liquid formulation that can be administered to patients and does not have a high level of antibody aggregation contains, in addition to 0.1 g / L-300 g / L of the conjugate active ingredient, one or more polyols (such as sugars), a buffer with a pH value of 4.5 to 7.5, a surfactant (such as polysorbate 20 or 80), an antioxidant (such as ascorbic acid and / or methionine), a strengthener (such as mannitol, sorbitol or sodium chloride), a chelating agent (such as EDTA), a metal complex (such as a zinc-protein complex), a biodegradable polymer (such as a polyester), a preservative (such as benzyl alcohol) and / or free amino acids.

[0356] Suitable buffers for formulations include, but are not limited to, organic acid salts, such as sodium, potassium, ammonium or trihydroxyethylamine salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, tromethamine hydrochloric acid, sulfuric acid or phosphate buffers. In addition, amino acid cations can also be used as buffers. These amino acids include, but are not limited to, arginine, glycine, glycylglycine and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The pH of the buffer is 4.5 to pH 7.5, preferably about 4.5 to about 6.5, more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is about 10 mM to about 500 mM.

[0357] "Polyols" optionally contained in the formulation are substances with multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonic agents in liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation. Cosolvents that are best excluded increase the effective surface tension of the solvent at the protein interface, and the most energetically favorable structural conformations are those with the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. "Reducing sugars" refer to sugars containing hemiacetal groups that can reduce metal ions or react with lysine and other amino groups in proteins, and "non-reducing sugars" refer to sugars that do not have the properties of reducing sugars. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbitol, succinate, and raffinose. Sugar alcohols are selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, and glycerol. Sugar acids include L-gluconate and its metal salts. The polyol content in the liquid formulation or lyophilized preparation is 0.0%-20% by weight. Non-reducing sugars, sucrose or trehalose, preferably at a concentration of about 0.1% to 15% in the formulation, are preferred, with trehalose being preferred due to its solution stability.

[0358] The surfactant that can be selected in the preparation can be selected from polysorbates (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.); Poloxamers (such as poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407 or polypropylene glycol-propylene glycol, etc.); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; dodecyl, myristoyl, linoleyl or stearyl sulfonyl betaine; dodecyl, myristoyl, linoleyl or stearyl sulfonyl betaine; Myristoyl, linoleyl or stearyl sarcosine; linoleic, myristyl or cetyl betaine; lauramidopropyl, cocamidopropyl, linoleamidopropyl, myristoylpropyl, palmitoylpropyl or isostearamidopropyl-betaine (e.g. lauroyl); myristamidopropyl, palmitoylpropyl or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; lauryl betaine, lauryl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycerate; MONAQUAT TM Series (such as isostearyl ethyliminium ethyl sulfate); polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol (such as Pluronic, PF68, etc.). Preferred surfactants are polyoxyethylene sorbitol fatty acid esters, such as polysorbate 20, 40, 60 or 80 (Tween20, 40, 60 or 80). The concentration range of the surfactant in the preparation is 0.0% to about 2.0% by weight. In certain specific embodiments, the surfactant concentration is about 0.01% to about 0.2%. In one embodiment, the surfactant concentration is about 0.02%.

[0359] Optional "preservatives" in the formulation are compounds that can fundamentally reduce bacteria therein. Examples of preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, wherein the alkyl group is a long-chain alkyl group) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl esters, catechol, resorcinol, cyclohexanol, 3-pentanol and meta-cresol. The preservative content in the liquid formulation or freeze-dried powder is 0.0%-5.0% by weight. In one embodiment, the preservative used is benzyl alcohol.

[0360] Suitable free amino acids used as fillers or tension agents or osmotic pressure regulators in the formulation are selected from, but not limited to, one or more of arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid or aspartic acid. Preferably, the basic amino acids are arginine, lysine and / or histidine. If histidine is included in the composition, it can act as a buffer and a free amino acid, but when a histidine buffer is used, a non-histidine free amino acid, such as lysine, is usually also included. The amino acids can exist in D- and / or L- form, but the L- form is more common. The amino acids can exist in the form of any suitable salt, such as arginine hydrochloride. The amino acid content in the liquid formulation or lyophilized powder is 0.0%-30% by weight.

[0361] Optionally, the preparation further comprises methionine, glutathione, cysteine, cystine or ascorbic acid as antioxidants, with a concentration of up to about 5 mg / ml in the liquid formulation and a content of 0.0%-5.0% by weight in the lyophilized powder; optionally, the preparation comprises a metal chelator, such as EDTA, EGTA, etc., with a concentration of about 2 mM in the liquid formulation and a content of 0.0%-0.3% by weight in the lyophilized powder.

[0362] The final preparation can be adjusted to a preferred pH value with a buffering agent (such as an acid including HCl, H2SO4, acetic acid, H3PO4, citric acid, etc., or a base such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, tromethamine, etc.). The preparation should also be adjusted to "isotonic", that is, the target preparation has an osmotic pressure substantially the same as human blood. The osmotic pressure of an isotonic preparation is generally 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing type osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate or NaCl. Typically, the buffer salt and isotonic agent are present in the preparation at a maximum of 30% (by weight).

[0363] Other excipients that can be used in the liquid or lyophilized preparation of the present invention include: fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, Pluoronic F-127, cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40 and / or 70 kD), polydextrose, maltodextrin, polysucrose, gelatin, hydroxypropyl methylcellulose, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerol, EDTA, cresol, benzyl alcohol, phenol, polyols, and sugars whose carbonyl groups are hydrogenated and reduced to primary or secondary hydroxyl groups.

[0364] Other excipients that can be used in the liquid preparations of the present patent application also include: flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acid lipids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such as sodium, etc. These and other known pharmaceutical excipients and / or additives suitable for the preparations of the present invention are well known in the art, such as listed in the fourth edition of "The Handbook of Pharmaceutical Excipients" compiled by Rowe et al. of the American Pharmaceutical Association; and the 21st edition of "Remington: the Science and Practice of Pharmacy" compiled by Gennaro et al., published by Wilkins Publishing Company (2005).

[0365] The pharmaceutical container or device containing the conjugate preparation in the present patent application includes: vials, bottles, prefilled syringes, prefilled or automatic syringes. The liquid formulation can be freeze-dried in borosilicate or soda-lime glass vials, or drum-dried, into cake or powder form. Solid powders can also be prepared by effective spray drying and then packaged in vials or pharmaceutical containers for storage and distribution.

[0366] In a further embodiment, the present invention provides a method for preparing a formulation, comprising the following steps: (a) freeze-drying a liquid containing a conjugate, an excipient and a buffer system; (b) re-dissolving the freeze-dried mixture in step (a) in a medium to stabilize the reconstituted formulation. The liquid in step (a) may further contain a stabilizer and one or more excipients selected from the aforementioned fillers, salts, surfactants and preservatives. The reconstitution medium may be water, such as sterile water, bacteriostatic water for injection (BWFI), acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, an acidic solution of sodium chloride, an acidic solution of magnesium chloride or an acidic solution of arginine, in an amount of about 10 to about 250 mM.

[0367] The liquid preparation of the conjugate of the present patent application should have various set characteristics. One of the main issues to be considered is its stability, because proteins / antibodies often form soluble and insoluble aggregates during manufacture and storage. In addition, various chemical reactions (deamidation, oxidation, shearing, isomerization, etc.) may occur in the solution, resulting in increased levels of degradation products and / or loss of biological activity. The conjugate in the liquid or lyophilized preparation should preferably have a shelf life of more than 6 months at 25°C. The conjugate in the more preferred liquid or lyophilized preparation should have a shelf life of more than 12 months at 25°C. The most preferred liquid preparation should have a shelf life of about 24 to 36 months at 2-8°C, and the lyophilized powder should have a shelf life of up to about 60 months at 2-8°C. Liquid preparations and lyophilized preparations should have a shelf life of at least two years at -20°C or -70°C.

[0368] In certain embodiments, the formulation is stable after freezing (e.g., -20°C or -70°C) and thawing, such as after 1, 2, or 3 cycles of freezing and thawing. Stability can be qualitatively and / or quantitatively assessed in a variety of ways, including assessing the drug / antibody (protein) ratio and the formation of multimers (e.g., using UV absorption spectroscopy, size exclusion chromatography, measuring turbidity, and / or by visual inspection); assessing charge heterogeneity (using cation exchange chromatography, image capillary isoelectric focusing (ICIEF), or capillary electrophoresis); amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry or MALDI / TOF MS or HPLC-MS / MS analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS--C) analysis; assessing the biological activity or antigen binding ability of the antibody, etc. Formulation instability may involve one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shearing or hydrolysis or cleavage (e.g., hinge region cleavage), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, and differences in glycosylation.

[0369] If the biological activity of the conjugate remains about 20%, preferably about 10% (within the error range of the assay) of the activity at the time of preparation over a given period of time (e.g., 12 months), then the stable conjugate should "retain its biological activity" in a pharmaceutical formulation. The biological activity can be determined, for example, by antigen binding and / or in vitro cytotoxicity assays.

[0370] For clinical use, the branched linker conjugate of the present invention will be provided in the form of a solution or a lyophilized solid, which can be redissolved in sterile water for injection. The exemplary scheme for administering the conjugate is as follows: daily, weekly, biweekly, triweekly or monthly, intravenous push for 8-54 weeks. Add the drug to 50 to 1000 ml of normal saline, optionally, add human serum albumin (e.g., 0.5 to 1 ml of concentrated human serum albumin solution, 100 mg / ml). The weekly dosage is about 50 μg to 20 mg / kg body weight, intravenous injection (10 μg to 200 mg / kg body weight per injection). Within 4-54 weeks after the end of treatment, the patient can receive a second course of treatment. The specific clinical protocol for the route of administration, excipients, diluents, dosage, time, etc. can be determined by a skilled clinician.

[0371] Examples of diseases that may be treated by killing specific cells either in vivo or ex vivo include malignancies of any type of cancer, autoimmune diseases, transplant rejection, and infections (viral, bacterial or parasitic).

[0372] The amount of conjugate required to achieve the desired therapeutic effect depends on many factors, including the chemical properties of the conjugate, potency and bioavailability, the type of disease, the species of the patient, the disease state of the patient, and the route of administration, which determine the required drug dose, administration method and dosing schedule.

[0373] In general, the conjugate of the present invention can be formulated into a physiological buffer solution containing 0.1 to 10% w / v conjugate for injection. Typical dosage ranges are from 1 μg / kg to 0.1 g / kg (body weight), daily, weekly, biweekly, every three weeks or monthly. The preferred dosage range is 0.01 mg / kg to 25 mg / kg body weight / day, weekly, biweekly, every three weeks or monthly, or a toddler equivalent dose. The preferred dose of the drug to be administered may depend on factors such as the type and extent of progression of the disease or condition, the overall health of a particular patient, the relative biological efficacy of the selected compound, the formulation of the drug, the route of administration (intravenous, intramuscular or other), the pharmacokinetic properties of the drug's designated delivery route, and the rate of administration (bolus or continuous infusion) and dosing regimen (number of repetitions within a given time).

[0374] The conjugate of the present invention can also be administered in a unit dosage form, wherein the term "unit dose" refers to a single dose that can be given to a patient and can be easily disposed and packaged, while the active conjugate itself or as a pharmaceutically acceptable composition as described below, maintains a physically and chemically stable unit dose. Typical daily / weekly / biweekly / monthly total dose ranges from 0.01 to 100 mg / kg body weight. As a general guideline, the unit dose range for humans is 1 mg to 3000 mg daily / weekly / biweekly / three weeks / monthly. The unit dose is preferably 1 to 900 mg, once to four times a month, more preferably 1 mg to 500 mg, once a week / biweekly / three weeks. The conjugate provided herein can be formulated into a pharmaceutical composition by mixing with one or more pharmaceutically acceptable excipients. Such unit dosage compositions can be administered orally, such as in the form of tablets, simple capsules or soft gel capsules; or intranasally, such as in the form of powders, nasal drops or aerosols; or dermally, such as using topical ointments, creams, lotions, gels or sprays or by a transdermal patch.

[0375] In another embodiment, a pharmaceutical composition comprising a therapeutically effective dose of a conjugate of formula (I) or (III), or any other conjugate described herein, can be co-administered with other therapeutic agents, such as chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, agents for treating autoimmune disorders, anti-infective agents or other conjugates to effectively treat or prevent cancer or autoimmune diseases or infectious diseases.The synergist is preferably selected from one or more of the following drugs: abatacept, acetaminophen / hydrocodone, adalimumab, afatinib maleate, alemtuzumab, alitretinoin, trastuzumab, amphetamine mixed salt (amphetamine / dextroamphetamine), anastrozole, aripiprazole, atazanavir, atezolizumab, atorvastatin, axitinib, belin, bevacizumab, cabazitaxel, cabozantinib, bexarotene, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, budesonide, budesonide / formoterol, buprenorphine, capecitabine, carfilzomib, celecoxib, ceritinib, cetuximab, cyclosporine, cinacalcet, crizotinib, dabigatran, dabrafenib, darabetin alfa, darunavir, imatinib mesylate, dasatinib, denileukin, denosumab, divalproex sodium, dexlansoprazole, dexmethylphenidate, dinutuximab, doxycycline, duloxetine, emtricitabine / rilpivirine / tenofovir disoproxil fumarate, emtricitabine / tenofovir / efavirenz, enoxaparin , enzalutamide, epoetin alfa, erlotinib, esomeprazole, ezetimibe, ezetimibe / simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone / salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, imatinib, ibritumomab tiuxetan, ibrutinib, insulin aspart, insulin detemir, insulin glargine, insulin lispro, interferon beta 1a, interferon beta 1b, lapatinib, ipilimumab, ipratropium bromide / salbutamol, lanreotide acetate, lenalidomide, combined with xylazine, letrozole, levofloxacin Thyroxine, levothyroxine, lidocaine, linezolid, liraglutide, MEDI4736, memantine, methylphenidate, metoprolol, modafinil, mometasone, nilotinib, nivolumab, ofatumumab, olivaccin, pazopanib, pembrolizumab, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, quetiapine, rabeprazole, radium 223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxolitinib phosphate, salbutamol, sevelat The invention relates to an agent comprising the following: amin, sildenafil, siltuximab, sitagliptin, sitagliptin / metformin, solifenacin, sorafenib, sunitinib, tadalafil, tamoxifen, tiprapvir, temsirolimus, tenofovir / emtricitabine, testosterone gel, thalidomide (Talidex), tiotropium bromide, toremifene, trametinib, trastuzumab, Tretinoin, ustekinumab, valsartan, vandetanib, vemurafenib, vorinostat, aflibercept, Zostavax and homologues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients thereof, or combinations thereof.

[0376] The drug / cytotoxic agent coupled by the branched linker of the present invention can be any homologue and / or derivative of the amatoxin described in the present invention. Professionals and technicians in the field of drugs / cytotoxic agents can understand that the amatoxin described in the present invention can be modified as long as the obtained compound still retains the specificity and / or activity of the starting compound. Those skilled in the art should understand that these compounds can be used to replace the amatoxin homologues described in the present invention. Therefore, the amatoxin homologues of the present invention also include these derived and modified compounds.

[0377] All literature references cited in this specification and in the examples that follow are incorporated by reference in their entirety. Example

[0378] The present invention is further illustrated by the following examples, the contents of which are not intended to limit the scope of the invention. In the examples, cell lines, except where otherwise specified, are stored under conditions specified by the American Standard Culture Collection (ATCC), the German Strain Collection (DSMZ) or the Shanghai Cell Culture Center of the Chinese Academy of Sciences. Except where otherwise specified, cell culture reagents are from Invitrogen. All anhydrous reagents are commercially available and stored in Sure-Seal sealed bottles. Other reagents and solvents are purchased in accordance with the highest specifications and are not further processed when used. Preparative HPLC separation was performed using a Varain PreStar HPLC. NMR spectra were obtained on a Bruker 500MHz instrument, with chemical shifts in ppm, tetramethylsilane as reference (0ppm), and coupling constants (J) in Hz. Mass spectral data were obtained on a WatersXevo G2 QTof mass spectrometer (connected to a Waters Acquity UPLC high performance liquid chromatograph and a TUV detector). Typically, UPLC separations were run on a C8 column with mobile phase A consisting of 1% formic acid and phase B consisting of 100% CH3CN.

[0379] Example 1. Fmoc-Hyp(O t Bu)-Ile-O t Synthesis of Bu(1-1).

[0380]

[0381] At 0°C, H-Ile-O t To a solution of Bu·HCl (25.0 g, 0.11 mol) in DMF (300 mL), Fmoc-Hyp(O t Bu)-OH (45.9 g, 1.0 eq), HOB t(16.7 g, 1.1 eq), EDC (23.7 g, 1.1 eq) and DIPEA (48.7 mL, 2.5 eq). The reaction mixture was stirred at 10-25 ° C for 4 hours, diluted with water (500 mL), and extracted with ethyl acetate (300 mL×3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product. The crude product was purified by silica gel column (petroleum ether / ethyl acetate from 10:1 to 1:1) to give 44.5 g (yield 68.8%) of compound 1-1 as a white solid. ESI m / z C 34 H 47 N2O6[M+H] + : Calculated value 578.33; measured value 578.35.

[0382] Example 2. H-Hyp(O t Bu)-Ile-O t Synthesis of Bu(2).

[0383]

[0384] Piperidine (40 mL) was added to a DMF (200 mL) solution of compound 1-1 (44.5 g, 76.9 mmol), and the mixture was stirred at 10-25 ° C for 1 hour. DMF was removed under high vacuum to obtain a solid crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain the title compound 27.2 g (yield 93.7%) as a colorless oil. ESI m / z C 19 H 37 N2O4[M+H] + : Calculated value 357.2753; measured value 357.2768.

[0385] Example 3. Fmoc-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(2-1).

[0386]

[0387] To a solution of Fmoc-Asn(Trt)-OH (50 g, 1.1 eq) and NMM (27.2 mL, 3.0 eq) in THF (500 mL) was added dropwise at 0°C. iBuO2CCl (10.9mL, 1.1eq), the mixture was stirred at 0°C for 30 minutes, continued to stir at room temperature for 3 hours, and then added dropwise to a stirred solution of compound 2 (27.2g, 1.0eq) in THF (200mL) at 0°C. Stirring was continued at room temperature for 16 hours, water (500mL) was added, and the mixture was extracted with ethyl acetate (300mL×3). The combined organic phase was washed with brine (300mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by a silica gel column (10:1 to 1:1 petroleum ether / ethyl acetate) to give 52.3g of compound 2-1 (yield 70.2%) as a white solid. ESI m / zC 57 H 67 N4O8[M+H] + : Calculated value 935.4882; measured value 935.4895.

[0388] Example 4. H-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(3).

[0389]

[0390] Piperidine (20 mL) was added to a DMF (100 mL) solution of compound 2-1 (20 g, 21.4 mmol). The mixture was stirred at 10-25 ° C for 1 hour and concentrated under high vacuum to remove DMF to obtain a solid crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain the title compound 14.0 g (yield 92.3%) as a colorless oil. ESI m / z C 42 H 57 N4O6[M+H] + : Calculated value 713.4279; measured value 713.4285.

[0391] Example 5. Fmoc-Cys(Trt)-Asn(Trt)-Hyp( t Bu)-Ile-O t Synthesis of Bu(3-1).

[0392]

[0393] A mixture of compound 3 (7.3 g, 10.2 mmol), Fmoc-Cys(Trt)-OH (6.0 g, 1 eq) and EDC (9.7 g, 5.0 eq) in dichloromethane (80 mL) was stirred continuously for 16 hours at room temperature. Water (500 mL) was added and extracted with ethyl acetate (300 mL×3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (10:1 to 1:1 petroleum ether / ethyl acetate) to give 3-1 (9.8 g, yield 75.2%) as a white foamy solid. ESI m / z C 78 H 84 N5O9S[M+H] + : Calculated value 1266.5990; measured value 1266.5980.

[0394] Example 6. H-Cys(Trt)-Asn(Trt)-Hyp( t Bu)-Ile-O t Synthesis of Bu(4).

[0395]

[0396] To a solution of compound 3-1 (9.0 g, 7.03 mmol) in DMF (50 mL) was added piperidine (10 mL), and the mixture was stirred at 10-25 ° C for 1 hour. After removing DMF under high vacuum, the crude product was purified by silica gel column (1: 1 petroleum ether / ethyl acetate to 10: 1 dichloromethane / methanol) to obtain 7 g (yield 95.2%) of the title compound as a colorless oil. ESI m / zC 64 H 76 N5O7S[M+H] + : Calculated value 1058.5466; measured value 1058.5460.

[0397] Example 7. Cbz-Ile-Gly-O t Synthesis of Bu(5-1).

[0398]

[0399] To a solution of Cbz-L-Ile-OH (15 g, 57.1 mmol, 1.0 eq) in DMF (120 mL) was added H-Gly-O t Bu·HCl (10.5 g, 1.0 eq), HOB t(9.3 g, 1.2 eq), EDC (13.2 g, 1.2 eq) and DIPEA (25 mL, 2.5 eq). The reaction was stirred at 10-25 ° C for 16 hours. Water (300 mL) was then added and the reaction mixture was extracted with ethyl acetate (200 mL×3). The organic phases were combined and washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (10:1 to 1:1 petroleum ether / ethyl acetate) to give 5-1, 19.5 g (90.1% yield) as a white solid. ESI m / z C 20 H 31 N2O5[M+H] + : Calculated value 379.2234; measured value 379.2248.

[0400] Example 8. H-Ile-Gly-O t Synthesis of Bu(6).

[0401]

[0402] Pd / C (10 wt %, 2.0 g, containing 64.2% water) was added to a methanol mixture of compound 5-1 (19.5 g). The mixture was stirred under a hydrogen balloon (1 atm) for 16 hours, then filtered and the filtrate was concentrated to give compound 6 (11.5 g, yield 93.2%) as a colorless oil. ESI m / z C 12 H 25 N2O3[M+H] + : Calculated value 245.1866; measured value 245.1860.

[0403] Example 9. Fmoc-Gly-Ile-Gly-O t Synthesis of Bu(6-1).

[0404]

[0405] To a solution of compound 6 (3.0 g, 12.3 mmol, 1.0 eq) in DMF (30 mL) was added Fmoc-Gly-OH (3.6 g, 1.0 eq), HOB t(1.99 g, 1.2 eq), EDC (2.82 g, 1.2 eq) and DIPEA (3.2 mL, 1.5 eq) were added. The reaction mixture was stirred at 10-25 ° C for 2.5 hours. Water (50 mL) was added and the mixture was extracted with ethyl acetate (30 mL×3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (10: 1 to 1: 1 petroleum ether / ethyl acetate) to give 6-1 (6.6 g, yield 100%) as a waxy solid. ESI m / zC 29 H 38 N3O6[M+H] + : Calculated value 524.2761; measured value 524.2778.

[0406] Example 10. Synthesis of Fmoc-Gly-Ile-Gly-OH (7a).

[0407]

[0408] TFA (25 mL) was added to a solution of compound 6 (6.6 g) in dichloromethane (25 mL). The reaction mixture was stirred at room temperature for 16 hours, then concentrated, and co-concentrated with toluene three times to obtain a waxy solid compound 7a (8.2 g, crude product). ESIm / zC 25 H 30 N3O6[M+H] + : Calculated value 468.2135; measured value 468.2147.

[0409] Example 11. Fmoc-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp( t Bu)-Ile-O t Synthesis of Bu(7a-1).

[0410]

[0411] At 0°C, compound 4 (9.0 g, 1.0 eq), EDC (7.2 g, 5.0 eq) and DIPEA (6.8 mL, 3.5 eq) (pH 7.5) were added to a solution of compound 7a (8.2 g, crude product, 1.1 eq) in dichloromethane (100 mL). The reaction mixture was stirred at 10-25°C for 2.5 hours. Water (500 mL) was added, and the mixture was extracted with ethyl acetate (300 mL×3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to give 6.8 g (yield 53.1%) of 7a-1 as a light yellow oil. ESIm / z C 89 H 103 N8O 12 S[M+H] + : Calculated value 1507.7417; measured value 1507.7442.

[0412] Example 12. H-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp( t Bu)-Ile-O t Synthesis of Bu(9).

[0413]

[0414] To a solution of compound 7a-1 (6.8 g) in DMF (30 mL), piperidine (6 mL) was added, and the mixture was stirred at 10-25 ° C for 1 hour. DMF was then removed under high vacuum to give a solid crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to give 5.2 g (yield 91.2%) of the title compound as a colorless oil. ESI m / z C 74 H 193 N8O 10 S[M+H] + : Calculated value 1285.6736; measured value 1285.6750.

[0415] Example 13. Synthesis of 6-nitro-D-tryptophan (10a).

[0416]

[0417] Under vigorous stirring, a solution of fuming nitric acid (7.5 mL) in glacial acid (30 mL) was added to a suspension of D-tryptophan (40.8 g, 0.20 mol) and urea (0.50 g) in glacial acetic acid (500 mL). The solid dissolved and turned into a yellow solution. With continued stirring, the solution turned into a suspension. A solution of fuming nitric acid (17.5 mL) in glacial acetic acid (70 mL) was slowly added to the suspension at 10°C. The solid dissolved and the solution turned from yellow to brown. After the addition was complete, the solution was stirred continuously at room temperature for 22 hours. The reaction mixture was concentrated to about 100 mL, and then water (200 mL) was added to form a yellow precipitate which was collected by filtration and washed with a small amount of water and ethyl acetate. The solid was dried in air and weighed 16.8 g. The filtrate was further concentrated to dryness and recrystallized from 5% nitric acid to obtain another batch of product (10 g). ESIMS m / z 11 H 12 N3O4[M+H] + : Calculated value 250.0829; measured value 250.0835.

[0418] Example 14. Synthesis of 6-nitro-N,N′-bis(tert-butoxycarbonyl)-D-tryptophan (11).

[0419]

[0420] At 0°C, sodium hydroxide (9.7 g, 10 eq), Bu4NHSO4 (1.6 g, 0.2 eq) and a solution of Boc2O (20.2 g, 3.5 eq) in dichloromethane (30 mL) were added to a mixture of compound 10a (6.0 g, 24.1 mmol) in dichloromethane (50 mL). The reaction mixture was stirred at 10-25°C for 16 hours, then water (60 mL) was added and extracted with dichloromethane (300 mL×3). The combined organic phases were washed with brine (300 mL), dried over anhydrous sodium sulfate and concentrated to give the title compound 7.5 g (yield 69.4%) as a light yellow oil. ESIMS m / z 21 H 28 N3O8[M+H] + : Calculated value 450.0908; measured value 450.0930.

[0421] Example 15. Synthesis of 6-nitro-N,N′-bis(tert-butoxycarbonyl)-D-tryptophanyl ester (12).

[0422]

[0423] A mixture of compound 11 (7.1 g, 15.8 g, 1.0 eq), potassium carbonate (4.3 g, 2.0 eq), BnBr (3.5 mL, 1.1 eq) and acetone (80 mL) was refluxed for 2.5 h, then cooled to room temperature, added with water (30 mL), and extracted with dichloromethane (30 mL×3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (10:1 to 3:1 petroleum ether / ethyl acetate) to give 8.1 g of light red oil 12 (yield 95.2%). ESIMS m / z 28 H 34 N3O8[M+H] + : Calculated value 540.2347; measured value 540.2360.

[0424] Example 16. Synthesis of 2-benzyl 8-di-tert-butyl 3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (13).

[0425]

[0426] Preparation of DMDO: Distilled water (20 mL), acetone (30 mL) and sodium bicarbonate (24 g, 0.285 mol) were mixed in a 1-liter round-bottom flask, cooled in an ice / water bath and magnetically stirred. After 20 minutes, stirring was stopped and Oxone (25 g, 0.0406 mol) was added in one portion. The flask was loosely covered and stirred vigorously for 15 minutes while maintaining an ice bath. The flask containing the reactants was then connected to a rotary concentrator with a room temperature water bath. The buffer ball (250 mL) was cooled in a dry ice / acetone bath and vacuumed (165 mTorr) by a benchtop diaphragm pump. After 15 minutes, the water bath temperature was raised to 40°C in 10 minutes. When the water bath temperature reached 40°C, the vacuum was released and the flask was raised from the heated hot water bath, and the distillation was immediately stopped. The light yellow acetone solution of DMDO was poured from the buffer bulb directly into a graduated cylinder to measure the total volume of the solution (approximately 25 mL), and the solution was then dried over sodium sulfate.

[0427] Sodium sulfate was removed by filtration and the filter cake was washed with 5 mL of acetone. The prepared DMDO solution can be titrated according to the method of Adam et al. (Adam, W.; Chan, YY; Cremer, D.; Gauss, J.; Scheutzow, D.; Scheutzow, D.; Schindler, MJ Org. Chem. 1987, 52, 2800-2803). The results consistently showed 2.1–2.3 mmol of DMDO in the solution. The DMDO solution was used immediately after titration.

[0428] At 0°C, the above-mentioned cooled DMDO solution was added dropwise to a stirred mixture of compound 12 (0.60 g, 1.11 mmol) in acetone. The mixture was stirred at 0°C for 2 hours, then stirred at room temperature for 16 hours, concentrated to give a crude product, water (20 mL) was added, and the resulting solution was extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (10:1 to 3:1 petroleum ether / ethyl acetate) to give 0.41 g of a gray solid 13. ESIMS m / z 28 H 34 N3O9[M+H] + : Calculated value 556.2296, measured value 556.2320.

[0429] Example 17. Synthesis of 1,8-bis(tert-butoxycarbonyl)-3a-hydroxy-6-nitro-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (14).

[0430]

[0431] To a mixture of compound 13 (0.31 g, 0.56 mmol) in THF (15 mL) was added an aqueous solution (8 mL) of sodium hydroxide (0.089 g, 4.0 eq). The mixture was stirred at 40 ° C for 16 hours. Then the THF was concentrated to remove the residue, and the residue was diluted with water (15 mL) and washed with ethyl acetate (30 mL×2). The resulting aqueous phase was adjusted to pH 3 with 1N HCL and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to give an oil 14 (83.8% yield). ESIMS m / z 21 H 28 N3O9[M+H] + : Calculated value 466.1826; measured value 466.1840.

[0432] Example 18. Synthesis of N-phenylselenophthalimide (15).

[0433]

[0434] To a mixture of potassium phthalimide (4.03 g, 21.8 mmol) and phenylselenochloride (5.00 g, 26.1 mmol) was added anhydrous n-hexane (20 mL) under nitrogen. Stirring was continued for 3 hours at room temperature, anhydrous dichloromethane (100 mL) was added, and the light red solution was filtered to remove solid matter. The filtrate was concentrated to about 20 mL and diluted with dry n-hexane (80 mL). The resulting precipitate was collected by filtration, washed with dry n-hexane, and dried under vacuum to give a light yellow solid (4.55 g, 69% yield). ESIMS m / z C 14 H 10 NO2Se[M+H] + : Calculated value 303.9878; measured value 303.9890.

[0435] Example 19. Synthesis of 6-nitro-D-tryptophan methyl ester (16).

[0436]

[0437] To a methanol solution of 10a (2.00 g, 0.803 mmol) was added dichlorothionyl (0.58 mL, 8.03 mmol) dropwise. The mixture was then heated to reflux and stirred for 2 hours. After cooling to room temperature, the reaction mixture was diluted with water (60 mL), and the pH was adjusted to 8.0 using 10% sodium hydroxide, and then extracted with ethyl acetate (60 mL×3). The organic phases were combined and dried over sodium sulfate, filtered, and concentrated to give a light red solid (1.50 g, 71.4%). ESIMS m / z 12 H 14 N3O4[M+H] + : Calculated value 264.0985; measured value 264.0942.

[0438] Example 20. Synthesis of 6-nitro-N,N′-bis(tert-butoxycarbonyl)-D-tryptophanyl methyl ester (17).

[0439]

[0440] At 0°C, sodium hydroxide (1.09 g, 10 eq), Bu4NHSO4 (183 mg, 0.2 eq) and a dichloromethane (5 mL) solution of Boc2O (2.3 g, 3.5 eq) were added to a mixture of compound 16 (720 mg, 2.74 mmol) at 0°C. The reaction mixture was stirred at 10-25°C for 8 hours, then water (30 mL) was added and extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (10:1 to 3:1 petroleum ether / ethyl acetate) to give 8.1 g of the title compound as a light yellow oil (yield 64.2%). ESIMS m / z 22 H 30 N3O8[M+H] + : Calculated value 464.2034; measured value 464.2058.

[0441] Example 21. Synthesis of (2S)-1,8-di-tert-butyl 2-methyl 6-nitro-3a-(phenylseleno)-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (18).

[0442]

[0443] Sodium sulfate (1.8 g) and 4-toluenesulfonic acid pyridinium salt (68 mg, 0.2 eq) and N-phenylselenophthalimide (715 mg, 1.5 eq) were added to a solution of compound 17 (700 mg, 1.51 mmol) in dichloromethane (20 mL). The reactant was stirred at room temperature overnight, then diluted with water (30 mL) and extracted with ethyl acetate (30 mL×3). The organic phases were combined and dried over anhydrous sodium sulfate, concentrated to give a crude product, which was purified by silica gel column (10:1 to 3:1 petroleum ether / ethyl acetate) to give 400 mg (yield 43.0%) of the title compound as a light yellow oil, while recovering part of the starting material. ESIMS m / zC 28 H 34 N3O8Se[M+H] + : Calculated value 620.1512; measured value 620.1545.

[0444] Example 22. Synthesis of (2S)-1,8-di-tert-butyl 2-methyl 3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (19).

[0445]

[0446] Potassium carbonate (2.00 g, 5.0 eq) and m-CPBA (85%, 1.48 g, 2.5 eq) were added to a solution of compound 18 (1.80 g, 2.90 mmol) in dichloromethane (30 mmol) at 0°C. The reactants were heated to room temperature and stirred overnight, then diluted with dichloromethane (100 mL) and filtered. The filtrate was washed with saturated sodium bisulfite (30 mL×3) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified on a silica gel column (20:1 to 4:1 petroleum ether / ethyl acetate) to give 0.94 g (yield 67.7%) of the title compound as a white foam. ESIMS m / z 22 H 30 N3O9[M+H] + : Calculated value 479.1983; measured value 479.1995.

[0447] Example 23. Synthesis of 1,8-bis(tert-butoxycarbonyl)-3a-hydroxy-6-nitro-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (14).

[0448]

[0449] To a solution of compound 19 (0.90 g, 1.88 mmol) in THF / methanol / water (2:2:1) was added LiOH (0.23 g, 5.0 eq). The solution was stirred at room temperature overnight, then concentrated and purified by silica gel column (1:20 to 1:3 methanol / dichloromethane) to give the title compound (0.85 g, 97.3% yield). ESIMS m / z 21 H 28 N3O9[M+H] + : Calculated value 466.1826; measured value 466.1845.

[0450] Example 24. Synthesis of (S)-3-(1H-indol-2-yl)-2-(tritylamino)propanoic acid (32).

[0451]

[0452] Chlorotrimethylsilane (3.4 mL, 26.9 mmol) was slowly added to a suspension of L-tryptophan (5.00 g, 24.5 mL) in dichloromethane (40 mL) at room temperature. The mixture was stirred continuously for 4.5 hours, and triethylamine (6.8 mL, 49.0 mmol) was added, followed by a solution of trityl chloride (7.17 g, 25.7 mmol) in dichloromethane (20 mL). The mixture was stirred at room temperature for 20 hours and then quenched with methanol (25 mL). The reaction was concentrated to dryness and redissolved in dichloromethane, washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was further dissolved in dichloromethane and filtered through a pad of celite, and the filtrate was concentrated to give a light white foam (11.8 g), which was used directly in the next step. ESIMS m / z 446.30 ([M+H] + ).

[0453] Example 25. Synthesis of (S)-methyl 2-(3-(1H-indol-2-yl)-2-(tritylamino)propanamido)acetate (33).

[0454]

[0455] To a solution of 32 (9.27 g, 30.7 mmol) in THF (30 mL) were added glycine methyl ester hydrochloride (2.85 g, 22.8 mmol) and HOB t (3.08 g, 22.8 mmol). The mixture was cooled to 0 °C and triethylamine (7.4 mL, 51.9 mmol) was added, followed by EDC·HCl (4.38 g, 22.8 mmol) added in portions. The mixture was warmed to room temperature and stirred for 20 hours, then concentrated and redissolved in dichloromethane, washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was slurried with ethyl acetate and the white solid was collected by filtration (6.46 g, 65% yield for two steps). ESIMS m / z 518.20 ([M+H] + ).

[0456] Example 26. Synthesis of methyl 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamido)acetate (34).

[0457]

[0458] To a solution of Trt-Trp-Gly-OMe (0.80 g, 1.54 mmol) in dichloromethane (20 mL) was added a solution of DMDO in acetone (2.25 mmol) at -78°C. After 1 hour, the mixture was concentrated to dryness under reduced pressure at room temperature. Purification by column chromatography (n-hexane / ethyl acetate / triethylamine 70:30:1 to 30:70:1) gave a light yellow foamy mixture as a mixture of two diastereoisomers (0.58 g, 70% yield). ESIMS m / z 534.22 ([M+H] + ).

[0459] Example 27. Synthesis of 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamido)acetic acid (35).

[0460]

[0461] To a solution of Tr-Hpi-Gly-OMe (diastereoisomer mixture) (0.80 g, 1.50 mmol) in dioxane / water (30 mL, v / v 2:1) was added LiOH (0.63 g, 15.0 mmol) and stirred at room temperature for 30 minutes (TLC showed the disappearance of the starting material (dichloromethane / methanol, 9:1)). The reaction mixture was evaporated to dryness and the residue was purified by passing through a short silica gel column, eluting with dichloromethane / methanol / triethylamine (90:10:1). The components were combined and concentrated to give a light yellow solid, which was the triethylamine salt of the two diastereomers (0.89 g, 95% yield).

[0462] Example 28. Synthesis of (2S)-di-tert-butyl 2-((5S,8R,14S)-5-((2S,4R)-4-(tert-butoxy)-2-((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-14-((S)-sec-butyl)-3,7,10,13,16-pentaoxo-1,1,1-triphenyl-8-((tritylthio)methyl)-2,6,9,12,15-pentaazaheptadecan-17-yl)carbamoyl)-3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,8(2H,8aH)-dicarboxylate (36).

[0463]

[0464] To a solution of compound 14 (0.27 g, 0.58 mmol) in DMF (5 mL) were added compound 9 (0.75 g, 1.0 eq), EDC (0.17 g, 1.5 eq), HOBt (0.12 g, 1.5 eq) and DIPEA (0.25 mL, 2.5 eq) at 0 °C. The reaction mixture was stirred at 10-25 °C for 16 hours. Water (20 mL) was added and the mixture was extracted with ethyl acetate (15 mL×3). The combined organic phases were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to give 0.5 g (yield 52.3%) of 36 as a light yellow oil. ESIMS m / z 95 H 118 N 11 O 18 S[M+H] + : Calculated value 1732.8378; measured value 1732.8405.

[0465] Example 29. Synthesis of (2S,3S)-2-((2S,4R)-1-((S)-4-amino-2-((3R,9S,15S)-15-amino-9-((S)-sec-butyl)-19-nitro-5,8,11,14-tetraoxo-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,21-hexadecylhydro-[1,4,7,10,13]thiatetraazacyclooctadeca[18,17-b]indole-3-carboxamido)-4-oxobutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-methylpentanoic acid (37).

[0466]

[0467] To a solution of compound 36 (200 mg) in dichloromethane (1.0 mL) was added TFA (2.0 mL), and the reaction mixture was stirred at room temperature for 16 hours. It was then concentrated and co-concentrated with toluene three times. The crude product was purified by preparative HPLC (acetonitrile / water) to give compound 37 (50 mg, yield 47.1%) as a light yellow oil. ESIMS m / z 918.40 ([M+H] + ).

[0468] Example 30. Synthesis of compound 38.

[0469]

[0470] At 0°C, EDC (124.0 mg, 4.0 eq), HOBt (88.0 mg, 4.0 eq) and DIPEA (128 μL, 4 eq) were added to a solution of compound 37 (150 mg, 0.16 mmol) in DMF (20 mL). The reaction mixture was stirred at 10-25°C for 16 hours, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to give a crude product 38 (92.0 mg, yield 64.6%) as a yellow oil, which was used in the next step without further purification. ESIMS m / z 900.36 ([M+H] + ).

[0471] Example 31. Synthesis of compound 39.

[0472]

[0473] Pd / C (10 wt %, 200 mg, containing 64.2% water) was added to a solution of compound 38 (50.0 mg, 55.5 μmol) in methanol (50 mL). The mixture was stirred under a hydrogen balloon (1 atm) for 2 hours, then filtered and the filtrate was concentrated. Compound 39 (45.0 mg, yield 93.3%) was obtained as a colorless oil. ESIMS m / z 870.39 ([M+H] + ).

[0474] Example 32. Synthesis of compound 40.

[0475]

[0476] To a solution of compound 38 (90.0 mg, 100.0 μmol) in dichloromethane (20 mL) was added m-CPBA (85%, 24 mg, 1.2 eq) at 0°C, and the reaction was then warmed to room temperature and stirred for 2 hours. The reactant was diluted with dichloromethane (50 mL), quenched with saturated sodium bisulfate (30 mL), and washed with saturated sodium bicarbonate (30 mL) and brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative HPLC (acetonitrile / water) to give the title compound (57.1 mg, 62% yield) as a white foam. ESIMS m / z 916.30 ([M+H] + ).

[0477] Example 33. Synthesis of compound 42.

[0478]

[0479] Pd / C (10 wt %, 200 mg, containing 64.2% water) was added to a solution of compound 40 (57.0 mg, 62.3 μmol) in methanol (50 mL). The mixture was stirred under a hydrogen balloon (1 atm) for 2 hours, then filtered and the filtrate was concentrated. Compound 42 (50.0 mg, yield 90.7%) was obtained as a white foam. ESIMS m / z 886.56 ([M+H] + ).

[0480] Example 34. Solid phase synthesis of monocyclic octapeptide 63.

[0481] Fmoc-Ile-OH was attached to 2-chlorotrityl chloride resin according to the following scheme:

[0482] Fmoc-Ile-OH (0.35 g, 1.0 mmol) and DIPEA (0.70 mL, 4.0 mmol) are dissolved in anhydrous dichloromethane (10 mL). The resulting solution is added to 2-chlorotrityl resin (1.0 g, 0.911 mmol / g, GL Biochem), and the mixture is shaken under nitrogen for 1.5 hours. Methanol (2 mL) is subsequently added, and shaking is continued for 30 minutes. Under vacuum, the liquid is drained, and the resin is washed with dichloromethane (15 mL), DMF (10 mL) and methanol (10 mL), and dried under vacuum.

[0483] The coupling was performed according to the following scheme:

[0484] The resin was placed in a column and swelled in DMF (10 mL) for 30 minutes. The solvent was drained under vacuum and the N-terminal Fmoc protecting group was cleaved by reaction with 20% piperidine / DMF for 30 minutes. After deprotection, the resin was washed with DMF (3×10 mL), dichloromethane (3×10 mL), and DMF (3×10 mL) in sequence. The next Fmoc-protected amino acid (Fmoc-Xaa-OH, 5 eq) was coupled to the resin under the action of a DMF (10 ml) solution of coupling agent HBTU (5 eq) and DIPEA (10 eq) and the reaction was carried out for 2 hours. The resin was then washed thoroughly with DMF (3×10 mL), dichloromethane (3×10 mL), and DMF (3×10 mL) in sequence. A small amount of sample was taken and reacted with hexafluoroisopropanol (HFIP) and dichloromethane for 5 minutes to cleave the peptide from the resin, and then detected by mass spectrometry. When coupling with non-commercially available amino acids (eg, Trt-Hpi-Gly-OH), fewer equivalents (3 eq.) of amino acid and longer reaction times (3 h) were used.

[0485] When all couplings were completed, the peptide bound to the resin was transferred to a round-bottom flask, TFA (10 mL) was added and stirring was continued at room temperature for 5 hours. TFA treatment can simultaneously cleave acid-labile protecting groups. The resin was filtered and washed with dichloromethane (10 mL) and methanol (10 mL). The filtrate was concentrated and partitioned between water and ethyl acetate. The aqueous phase was purified by preparative HPLC (water / acetonitrile) to give the monocyclic octapeptide 63 as a white solid (40.3 mg, 5% yield). ESIMS m / z 888.38 ([M+H] + ).

[0486] Example 35. Ile 3 -Synthesis of S-deoxyamanitin (64).

[0487]

[0488] To a solution of the monocyclic octapeptide (257 mg, 0.289 mmol) in anhydrous DMF (50 mL) was added EDC·HCl (277 mg, 1.45 mmol), HOBt (390 mg, 2.89 mmol) and DIPEA (0.25 mL, 1.45 mmol). The reaction was stirred at room temperature for 20 hours, then concentrated and purified by preparative HPLC (water / acetonitrile) to give white solid compound 64 (90.1 mg, 36% yield). ESIMS m / z 870.40 ([M+H] + ).

[0489] Example 36. Synthesis of compound 65.

[0490]

[0491] To a solution of compound 64 (50.0 mg, 0.0575 mmol, 1.0 eq) in THF (10 mL) at 0°C was added t-BuONO (70 μL, 0.575 mmol). The reaction was stirred for 1 hour and then at room temperature for 20 hours. After the addition of water (50 mL), the reaction mixture was concentrated and purified by preparative HPLC (water / acetonitrile) to give a white solid (26.2 mg, 50% yield). ESIMS m / z 915.38 ([M+H] + ).

[0492] Example 37. Synthesis of compound 68.

[0493]

[0494] A mixture of nitro compound (26 mg, 0.0284 mmol) and Pd / C (10 wt%, 100 mg) in methanol (20 mL) was catalytically hydrogenated (1 atmosphere of hydrogen) at room temperature for 1 hour. The mixture was then filtered through diatomaceous earth (filter aid). The filtrate was concentrated to give a white solid (25 mg, 99% yield). ESIMS m / z 885.38 ([M+H] + ).

[0495] Example 38. Synthesis of compound 69.

[0496]

[0497] To a solution of compound 68 (25 mg, 0.0282 mmol) in THF / methanol / water (2:2:1, 50.0 mL) was added LiOH (3.40 mg, 5 eq) in an ice-water bath. The reaction was stirred at 0°C for 30 minutes. It was then concentrated and purified by a short silica gel column (0-10% methanol / dichloromethane) to give a white foam (15.2 mg, 61% yield). ESIMS m / z 871.38 ([M+H] + ).

[0498] Example 39. Synthesis of compound 71.

[0499]

[0500] To a solution of compound 3-N,N-(2″-maleimidoethyl)(2′,5′,8′,11′,14′,17′,20′,23′,26′-nonaoxaoctacosane-28′-sulfoxide)aminopropionic acid (70) (14.1 mg, 0.0206 mmol) in dichloromethane (20 mL) were added NHS (2.8 mg, 0.0248 mmol) and EDC·HCl (4.9 mg, 0.0258 mmol), and the reaction mixture was stirred at 40° C. for 2 h, then diluted with dichloromethane (100 mL), and washed with water (20 mL) and brine (20 mL). The organic phase was concentrated to give the crude product, which was used directly without further purification.

[0501] The above crude product and compound 69 (15 mg, 0.0172 mmol) were dissolved in DMF (10 mL), and DIPEA (15 μL, 5 eq) was added thereto. The reaction was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (12.0 mg, 36% yield). ESIMS m / z 1523.68 ([M+H] + ).

[0502] Example 40. Synthesis of 2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-tert-butyl ester (136).

[0503]

[0504] NaH (60%, 8.0 g, 200 mmol) was added to a solution of 2,5,8,11,14,17,20,23-octaoxacosane 25-ol (38.4 g, 100 mmol) in THF (1.0 L). After stirring at room temperature for 30 minutes, tert-butyl 2-bromoacetate (48.8 g, 250 mmol) was added and stirred at room temperature for 1 hour. The mixture was then poured into ice water, extracted with dichloromethane, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. Compound 136 (27.6 g, 59% yield) was obtained by column chromatography (0% to 5% methanol / dichloromethane) as a yellow oil. ESIMS m / z 499.40 ([M+H] + ).

[0505] Example 41. Synthesis of 2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-oic acid (137).

[0506]

[0507] Compound 136 (35.6 g, 73.8 mmol) was dissolved in dichloromethane (400 mL), and then formic acid (600 mL) was added. The resulting solution was stirred at 25 °C overnight. All volatiles were removed in vacuo to give the title product as a yellow oil (31.0 g, theoretical yield). ESIMS m / z 443.45 ([M+H] + ).

[0508] Example 42. Synthesis of 2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-yl chloride (138).

[0509]

[0510] To a solution of compound 137 (31.0 g, 73.8 mmol) in dichloromethane (600 mL) were added (COCl)2 (100 mL) and DMF (52 g, 0.74 mmol). The resulting solution was stirred at room temperature for 4 hours. All volatiles were removed in vacuo to give the title product (32.8 g) as a yellow oil. ESIMS m / z 461.38 ([M+H] + ).

[0511] Example 43. Synthesis of (S)-34-((benzyloxy)carbonyl)amino)-28-oxo-2,5,8,11,14,17,20,23,26-nonaoxa-29-azapentatriacontane-35-oic acid (139).

[0512]

[0513] ZL-Lys-OH (41.4 g, 147.6 mmol), sodium carbonate (23.4 g, 221.4 mmol) and sodium hydroxide (5.9 g, 147.6 mmol) were dissolved in water (720 mL). The mixture was cooled to 0 °C, and a solution of compound 138 (32.8 g, 73.8 mmol) in THF (20 mL) was added thereto. The resulting mixture was stirred at room temperature for 1 hour. THF was removed under vacuum, and concentrated HCl was added to the aqueous solution under ice cooling until the pH reached 3. After extraction with dichloromethane, the organic layer was washed with brine, dried over sodium sulfate, and concentrated to give the title product as a yellow oil (50.0 g, 99% yield). ESIMS m / z 705.30 ([M+H] + ).

[0514] Example 44. Synthesis of (S)-tert-butyl 34-((benzyloxy)carbonyl)amino)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazepin-40-ester (140).

[0515]

[0516] A mixture of tert-butyl 4-aminobutyrate (1.03 g, 6.12 mmol) and compound 139 (3.91 g, 5.56 mmol) in DMF (18 mL) was cooled to 0 °C, and HATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol) were added sequentially. The reaction was stirred for 1 hour, then diluted with water (300 mL) and extracted with ethyl acetate (3×250 mL). The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (32:1 dichloromethane / methanol) to give compound 140 (5.10 g, 99% yield). ESIMS m / z 846.50 ([M+H] + ).

[0517] Example 45. Synthesis of (S)-tert-butyl 34-amino-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-ester (141).

[0518]

[0519] In a hydrogenation bottle, Pd / C (10 wt %, 0.10 g) was added to a solution of compound 140 (1.0 g, 1.18 mmol) in methanol (50 mL). The mixture was shaken for 2 hours, filtered through celite (filter aid), and the filtrate was concentrated to give compound 141 (0.93 g, yield>100%). ESIMS m / z 712.50 ([M+H] + ).

[0520] Example 46. Synthesis of (S)-tert-butyl 34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-ester (142).

[0521]

[0522] To a solution of compound 141 (0.93 g, 1.18 mmol) and N-succinimidyl 4-maleimidobutyrate (0.50 g, 1.77 mmol, 1.eq) in 95% ethanol (50 mL) was added sodium bisulfate solution (0.1 M, pH 5.0, 10 mL) at room temperature. The mixture was stirred overnight, then concentrated and diluted with water (50 mL), extracted with dichloromethane (80 mL×3), dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography (25:1 dichloromethane / methanol) to give the title compound (0.82 g, 80%) as a light yellow oil. ESIMS m / z 877.52 ([M+H] + ).

[0523] Example 47. Synthesis of (S)-34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oic acid (143).

[0524]

[0525] Compound 144 (0.82 g, 0.94 mmol) was dissolved in formic acid (10 mL) and stirred at room temperature for 1 hour. The reaction mixture was concentrated and co-concentrated with toluene twice, and the residue was placed on a vacuum pump to give compound 143 (0.80 g, crude product). ESIMS m / z 820.45 ([M+H] +).

[0526] Example 48. Synthesis of (S)-2,5-dioxopyrrolidin-1-yl 34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-ester (144).

[0527]

[0528] To a solution of compound 143 (0.80 g, crude product, 0.94 mmol) in DMA (5.0 mL) were added NHS (0.12 g, 1.03 mmol) and EDC·HCl (0.27 g, 1.41 mmol). Stirring was continued at room temperature for 2 hours, then diluted with water (15 mL) and extracted with ethyl acetate (3×10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column (10-50% ethyl acetate / petroleum ether) to give a colorless oil (0.67 g, 78% yield). ESIMS m / z 918.55 ([M+H] + ).

[0529] Example 49. Synthesis of Compound 145.

[0530]

[0531] To a solution of compound 42 (50 mg, 0.0565 mmol) and compound 144 (77 mg, 1.5 eq) in ethanol (10 mL), 0.1 M sodium dihydrogen phosphate (10 mL) was added and stirred for 30 minutes. The reaction was concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (44 mg, 46% yield). ESIMS m / z 1689.10 ([M+H] + ).

[0532] Example 50. Synthesis of tert-butyl (2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamate (147).

[0533]

[0534] A mixture of N-Boc-ethylenediamine (5.6 mL, 35.4 mmol, 1.1 eq) and saturated sodium bicarbonate (60 mL) was cooled to 0°C, and N-methoxycarbonylmaleimide (5.00 g, 32.2 mmol, 1.0 eq) was added in batches. After stirring at 0°C for 30 minutes, the reaction was warmed to room temperature and stirred for 1 hour. The precipitate was collected by filtration, washed with cold water, and then dissolved in ethyl acetate.

[0535] The mixture was washed with brine, dried over anhydrous sodium sulfate, and concentrated to give a white solid (6.69 g, 87% yield). ESIMS m / z 241.12 ([M+H] + ).

[0536] Example 51. Synthesis of tert-butyl (2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)carbamate (148).

[0537]

[0538] In a high pressure tube, a solution of compound 147 (6.00 g, 25.0 mmol) and furan (18.0 mL) in toluene (120 mL) was heated to reflux and stirred for 16 hours. During the reaction, the colorless solution turned yellow. The mixture was then cooled to room temperature and concentrated. The resulting white solid was slurried with ether to give compound 148 (6.5 g, 84% yield). ESIMS m / z 309.13 ([M+H] + ).

[0539] Example 52. Synthesis of 2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxindole-1,3(2H)-dione hydrochloride (149).

[0540]

[0541] A solution of compound 148 (9.93 g, 32.2 mmol) in dioxane (15 mL) was reacted with concentrated hydrochloric acid (15 mL) at room temperature for 3 hours. The reactant was concentrated, the solid was collected by filtration, and the filter cake was washed with ethyl acetate. The solid was dried in an oven (50°C) overnight to give compound 149 (6.94 g, 88% yield). ESIMS m / z 206.05 ([M+H] + ).

[0542] Example 53. Synthesis of compound 151.

[0543]

[0544] To a solution of compound 149 (1.22 g, 5 mmol) in THF (10 mL) at -10°C was added POCl3 (0.47 mL, 5 mmol). After stirring for 10 min, 2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-amine (2.14 g, 5 mmol) was added, followed by DIPEA (0.87 mL, 5 mmol). The reaction was warmed to 0°C and stirred for 3 h, then concentrated. The residue was diluted with dichloromethane (10 mL) and filtered through celite, and the filtrate was used directly in the next step. ESIMS m / z 716.29 ([M+H] + ).

[0545] Example 54. Synthesis of compound 111.

[0546]

[0547] A mixture of compound 42 (1.50 g, 1.69 mmol), 4-(((benzyloxy)carbonyl)amino)butyric acid N-succinimidyl ester (0.67 g, 1.2 eq), DIPEA (0.44 mL, 1.5 eq) and DMF (10.0 ml) was stirred at room temperature overnight, then concentrated and purified by short silica gel column (10-85% ethyl acetate / petroleum ether) to give a colorless oil (1.58 g, 85% yield). ESIMS m / z 1105.47 ([M+H] + The oil was dissolved in THF (5.0 mL) and stirred with Pd / C (10 wt %, containing 62% water, 50 mg) under a hydrogen balloon for 1 hour, then filtered on celite and concentrated to give a colorless oil (1.40 g, yield 100%), which was used without further purification. ESIMS m / z 971.43 ([M+H] + ).

[0548] Example 55. Synthesis of Compound 152.

[0549]

[0550] One fifth of the above 151 solution (1.0 mmol) was taken and DIPEA (0.17 mL, 1.0 mmol) was added thereto at 0°C, followed by a solution of 111 (0.97 g, 1.0 mmol) in dichloromethane (1.0 mL). The reaction was stirred at 0°C for 2.5 hours and then at 30°C overnight. After concentration, the residue was purified by silica gel column (1-50% ethyl acetate / petroleum ether and 0-10% methanol / dichloromethane) to give the title product 152 (0.99 g, 60%) as a colorless oil. ESIMS m / z 1650.74 ([M+H]+ ).

[0551] Example 56. Synthesis of Compound 153.

[0552]

[0553] A solution of compound 152 (0.99 g, 0.0006 mmol) in toluene / DMA (1:1, 2.0 mL) was heated at 100 °C for 2 h, then concentrated and purified by silica gel column (0-10% methanol / dichloromethane) to give the title compound (0.48 g, 52% yield) as a white foam. ESIMS m / z 1582.90 ([M+H] + ).

[0554] Example 57. Synthesis of methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (156).

[0555]

[0556] A mixture of dimethyl succinate (20.0 g, 136.9 mmol) and dihydroxyethylamine (7.20 g, 68.7 mmol) in anhydrous toluene (500 ml) and pyridine (50 ml) was heated at 150°C for 28 hours. The mixture was concentrated and purified on a silica gel column eluting with 5-25% ethyl acetate / dichloromethane to give the title compound (12.5 g, 83% yield). ESIMS m / z 242.42 [M+Na] + .

[0557] Example 58. Synthesis of methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (157).

[0558]

[0559] To a solution of methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (12.0 g, 49.56 mmol) in anhydrous pyridine (350 ml) was added methanesulfonyl chloride (20.0 g, 175.4 mmol). After stirring overnight, the mixture was concentrated, diluted with ethyl acetate (350 mL), washed with cold 1 M sodium dihydrogen phosphate (2×300 mL), and the organic phase was dried over magnesium sulfate, filtered, and concentrated to give the crude product (~18.8 g, >100% yield). The crude product was used in the next step without further purification. ESIMS m / z 376.06 ([M+H] + ).

[0560] Example 59.3, Synthesis of 6-endooxo-Δ-tetrahydrophthalimide (159).

[0561]

[0562] To a solution of maleimide (10.0 g, 103.0 mmol) in toluene (200 mL) was added furan (10.0 mL, 137.4 mmol). The mixture was heated in a 1 L pressure reactor at 100°C for 8 hours. After cooling to room temperature, the solid was washed with methanol, the filtrate was concentrated and crystallized in ethyl acetate / n-hexane to give 16.7 g (99%) of the title compound. 1H NMR (CDCl3): 11.12 (s, 1H), 6.68-6.64 (m, 2H), 5.18-5.13 (m, 2H), 2.97-2.92 (m, 2H). ESIMS m / z [M+Na] + 188.04.

[0563] Example 60. Synthesis of methyl 4-((2-((3aR,4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)(2-(((4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)amino)-4-oxobutanoate (160).

[0564]

[0565] To a solution of methyl 4-(bis(2-(((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (157, freshly prepared, 90% purity, 8.5 g, 20 mmol) in DMA (350 mL) was added 3,6-endo-[Delta]-tetrahydrophthalimide (10.2 g, 61.8 mmol), sodium carbonate (8.0 g, 75.5 mmol) and sodium iodide (0.3 g, 2.0 mmol). The mixture was stirred at room temperature overnight, concentrated, diluted with ethyl acetate (350 mL), washed with saturated sodium bicarbonate solution (300 mL), saturated NaCl (300 mL) and 1 M NaH2PO4 (300 mL). The organic layer was dried over sodium sulfate, filtered, concentrated, loaded onto a silica gel column and eluted with 10-30% ethyl acetate / n-hexane to give the title compound (7.9 g, 77% yield). ESIMS m / z[M+Na] + 536.4.

[0566] Example 61. Synthesis of 4-(bis(2-(2-,2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoic acid (161).

[0567]

[0568] A solution of compound 160 (3.0 g, 5.8 mmol) and trimethyltinanol (4.8 g, 26.4 mmol) in 1,2-dichloroethane (150 mL) was refluxed at 80 ° C for 8 hours, then cooled to room temperature and the residue was purified by passing through a short silica gel column, eluting with dichloromethane / methanol to remove excess trimethyltin hydroxide. The fractions were then combined, concentrated and diluted with DMA and toluene, heated to 120 ° C and stirred overnight. The reaction mixture was loaded onto a silica gel column and eluted with 5-10% methanol / dichloromethane to give the title compound (1.62 g, 76% yield). ESIMS m / z [M+Na] + 386.2.

[0569] Example 62. Synthesis of (S)-34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxo-29,36-diazatetracontane-40-tert-butyl ester (163).

[0570]

[0571] To a solution of compound 161 (1.62 g, 4.20 mmol) and compound 141 (2.71 g, 3.82 mmol) in DMA (20 mL) was added EDC·HCl (0.81 g, 4.20 mmol). The reactants were stirred at room temperature overnight, then poured into water (50 mL) and extracted with ethyl acetate (3×40 mL). The organic phases were combined, washed with brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (10-50% ethyl acetate / petroleum ether) to give a colorless oil (3.20 g, 80% yield). ESIMS m / z 1057.85 ([M+H] + ).

[0572] Example 63. Synthesis of (S)-34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontan-40-oic acid (164).

[0573]

[0574] A solution of compound 163 (3.20 g, 3.03 mmol) in formic acid (10 mL) was stirred at room temperature overnight. The solution was then concentrated and co-evaporated with toluene three times to give a colorless oil (3.00 g, crude product) which was used without further purification. ESIMS m / z 1001.50 ([M+H] + ).

[0575] Example 64. Synthesis of (S)-2,5-dioxopyrrolidin-1-yl 34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanamido)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxo-29,36-diazatetracontan-40-ester (165).

[0576]

[0577] To a solution of compound 164 (3.00 g, crude product, 3.03 mmol) in DMA (15.0 mL) were added NHS (0.38 g, 3.33 mmol) and EDC·HCl (0.87 g, 4.55 mmol). Stirring was continued at room temperature for 2 hours, then diluted with water (15 mL) and extracted with ethyl acetate (3×30 mL). The organic phases were combined and washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column (10-50% ethyl acetate / petroleum ether) to give a colorless oil (2.90 g, 90% yield). ESIMS m / z 1098.50 ([M+H] + ).

[0578] Example 65. Synthesis of Compound 166.

[0579]

[0580] To a solution of compound 42 (50 mg, 0.0565 mmol) and compound 165 (93 mg, 1.5 eq) in ethanol (10 mL) was added 0.1 M NaH2PO4 (10 mL) and stirred for 30 minutes. The reaction was concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (63 mg, 60% yield). ESIMS m / z 1868.80 ([M+H] + ).

[0581] Example 66. Synthesis of 14-(benzyloxy)-14-oxotetradecanoic acid (183).

[0582]

[0583] To a solution of tetradecanedioic acid (2.06 g, 8 mmol) in DMF (30 mL) was added K2CO3 (1.1 g, 8 mmol) and BnBr (1.36 g, 8 mmol). The mixture was stirred at room temperature overnight, then concentrated and purified by column chromatography (ethyl acetate / petroleum ether) to give the title compound 183 (1.2 g, 45% yield). ESIMS m / z 349.23 ([M+H] + ).

[0584] Example 67. Synthesis of tert-butyl 3-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (185).

[0585]

[0586] To a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq) in anhydrous THF (200 mL) was added sodium (0.1 g). The mixture was stirred until the sodium disappeared, and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq) was added dropwise. The mixture was stirred overnight and then quenched with hydrochloric acid solution (20.0 mL, 1 N) at 0°C. THF was removed by rotary evaporation, brine (300 mL) was added, and the resulting mixture was extracted with ethyl acetate (3×100 mL). The organic layer was washed with brine (3×300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a colorless oil (30.20 g, 79.0% yield) which was used without further purification. MS ESI m / z 278.17 ([M+H] + ).

[0587] Example 68. Synthesis of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (186).

[0588]

[0589] To tert-butyl 3-(2-(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq) and TsCl (41.37 g, 217.0 mmol, 2.0 eq) in anhydrous DCM (220 mL) at 0°C was added TEA (30.0 mL, 217.0 mmol, 2.0 eq). The mixture was stirred at room temperature overnight, then washed with water (3 x 300 mL) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated and purified on a silica gel column (3:1 hexanes / ethyl acetate) to give a colorless oil (39.4 g, 84.0% yield). MS ESI m / z 433.28 ([M+H] + ).

[0590] Example 69. Synthesis of tert-butyl 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate (187).

[0591]

[0592] To a solution of tert-butyl 3-(2-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq) in anhydrous DMF (100 mL) was added NaN3 (20.67 g, 316.6 mmol, 3.5 eq). The mixture was stirred at room temperature overnight. Water (500 mL) was added and extracted with ethyl acetate (3×300 mL). The combined organic layers were washed with water (3×900 mL) and brine (900 mL), dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column (5:1 hexanes / ethyl acetate) to give a yellow oil (23.8 g, 85.53% yield). MS ESI m / z 326.2 ([M+Na] + ).

[0593] Example 70. Synthesis of tert-butyl 3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (188).

[0594]

[0595] Raney-Ni (7.5 g, suspended in water) was washed with water (three times) and isopropanol (three times) and mixed with compound 187 (5.0 g, 16.5 mmol) in isopropanol. The mixture was stirred at room temperature for 16 hours under a balloon of H2, filtered, and then filtered over a pad of celite, washing the filter pad with isopropanol. The filtrate was concentrated and purified by column chromatography (5-25% methanol / dichloromethane) to give a light yellow oil (2.60 g, 57% yield). MS ESI m / z 279.19 ([M+H] + ).

[0596] Example 71. Synthesis of 27-benzyl-1-tert-butyl-14-oxo-4,7,10-trioxa-13-azaheptacosane-1,27-dioate (189).

[0597]

[0598] To a solution of compound 188 (2.60 g, 9.35 mmol) and compound 183 in dichloromethane (50 mL) was added EDC·HCl (2.15 g, 11.2 mmol) and DIPEA (3.6 mL, 20.6 mmol). The reaction mixture was stirred at room temperature for 1 hour, then diluted with 50 mL of dichloromethane and poured into a separatory funnel containing 50 mL of water. The organic phase was separated, washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (0-10% methanol / dichloromethane) to give the title compound 189 (4.94 g, 87% yield). MS ESI m / z 608.40 ([M+H] + ).

[0599] Example 72. Synthesis of 3,16-dioxo-1-phenyl-2,20,23,26-tetraoxa-17-azanonacosane-29-oic acid (190).

[0600]

[0601] To a solution of compound 189 (4.94 g, 8.14 mmol) in dichloromethane (20 mL) was added TFA (20 mL). The reaction was stirred at room temperature for 1 hour, then concentrated to dryness and concentrated twice with dichloromethane, and the residue was placed on a pump to give compound 190 (4.50 g, crude product). ESIMS m / z 552.35 ([M+H] + ).

[0602] Example 73. Synthesis of 40-benzyl 1-tert-butyl-14,27-dioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontane-1,40-dioic acid ester (191).

[0603]

[0604] To a solution of compound 190 (4.50 g, crude product, 8.14 mmol) and compound 188 (1.95 g, 7.00 mmol) in dichloromethane (50 mL) was added EDC·HCl (1.56 g, 8.14 mmol) and DIPEA (2.7 mL, 15.4 mmol). The reaction mixture was stirred at room temperature for 1 hour, then diluted with 50 mL of dichloromethane and poured into a separatory funnel filled with 50 mL of water. The organic phase was separated, washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (0-10% methanol / dichloromethane) to give the title compound 191 (5.22 g, 92% yield). MS ESI m / z 811.52 ([M+H] + ).

[0605] Example 74. Synthesis of 3,16,29-trioxo-1-phenyl-2,20,23,26,33,36,39-heptaoxa-17,30-diazatetradodecane-42-oic acid (192).

[0606]

[0607] To a solution of compound 191 (5.22 g, 6.44 mmol) in dichloromethane (15 mL) was added TFA (15 mL). The reaction was stirred at room temperature for 1 hour, then concentrated to dryness and co-concentrated twice with dichloromethane, and the residue was placed on the pump to give compound 192 (4.90 g, crude product). ESIMS m / z 755.46 ([M+H] + ).

[0608] Example 75. Synthesis of 40-benzyl 1-(2,5-dioxopyrrolidin-1-yl) 14,27-dioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontane-1,40-dioic acid ester (193).

[0609]

[0610] To a solution of compound 192 (4.90 g, crude product, 6.44 mmol) in dichloromethane (30 mL) were added NHS (0.81 g, 7.08 mmol), EDC·HCl (1.85 g, 9.66 mmol), followed by DIPEA (2.8 mL, 16.1 mmol). The reaction was stirred for 2 hours, then diluted with water (50 mL) and extracted with ethyl acetate (3×20 mL). The combined organic phases were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (10-50% ethyl acetate / petroleum ether) to give a colorless oil (4.90 g, 90% yield). ESIMS m / z 852.48 ([M+H] + ).

[0611] Example 76. Synthesis of 1-((2,5-dioxopyrrolidin-1-yl)oxy)-1,14,27-trioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontan-40-oic acid (194).

[0612]

[0613] In a hydrogenation bottle, Pd / C (10 wt%, 0.20 g) was added to a solution of compound 193 (4.90 g, 5.75 mmol) in methanol (20 mL), catalytic hydrogenation (1 atm H2) was stirred overnight, filtered through diatomaceous earth (filter aid), and the filtrate was concentrated to obtain compound 194 (4.50 g, yield> 100%). ESIMS m / z 762.44 ([M+H] + ).

[0614] Example 77. Synthesis of Compound 195.

[0615]

[0616] To a solution of compound 56 (40 mg, 1.5 eq) and compound 144 (50 mg, 1.5 eq) in ethanol (10 mL), 0.1 M NaH2PO4 (10 mL) was added and stirred for 30 minutes. The reaction was concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (31 mg, 40% yield). ESIMS m / z 1704.80 ([M+H] + ).

[0617] Example 78. Synthesis of Compound 196.

[0618]

[0619] To a mixture of compound 195 (31 mg, 0.0182 mmol) and compound 194 (17 mg, 0.0218 mmol) in DMA (10 mL) was added DIPEA (5 μL, 0.0273 mmol). The reaction was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (26 mg, 61% yield). ESIMS m / z 2351.60 ([M+H] + ).

[0620] Example 79. Synthesis of (6S,13S)-di-tert-butyl 9,10-bis(((benzyloxy)carbonyl)amino)-5,8,11,14-tetraoxo-6,13-bis(4-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioate (209).

[0621]

[0622] To a solution of (S)-tert-butyl 12-amino-2,2-dimethyl-6,13-dioxo-5-oxa-7,14-diaza-2-silaheptadecane-17-ol (6.02 g, 14.4 mmol) and 2,3-bis((((benzyloxy)carbonyl)amino)succinic acid (5.00 g, 12.0 mmol) in DMA (60 mL) was added EDC·HCl (2.76 g, 14.4 mmol) and DIPEA. (4.7 mL, 26.4 mmol). The reaction mixture was stirred at room temperature overnight, then diluted with 150 mL of dichloromethane and poured into a separatory funnel filled with 100 mL of water. The organic phase was separated, washed with brine (2×50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (10-80% ethyl acetate / petroleum ether) to give the title compound 209 (12.4 g, 85% yield). ESIMS m / z 1215.63 ([M+H] + ).

[0623] Example 80. Synthesis of (6S,13S)-di-tert-butyl 9,10-diamino-5,8,11,14-tetraoxo-6,13-bis(4-((((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioate (210).

[0624]

[0625] In a hydrogenation bottle, a mixture of a methanol (50 mL) solution of compound 209 (12.4 g, 10.2 mmol) and Pd / C (10 wt %, 0.10 g) was shaken for 2 hours, filtered through celite (filter aid), and the filtrate was concentrated to give compound 210 (9.47 g, 98% yield) as a colorless oil. ESIMS m / z 947.56 ([M+H] + ).

[0626] Example 81. Synthesis of (6S,13S)-di-tert-butyl 9,10-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-5,8,11,14-tetraoxo-6,13-bis(4-((((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioate (211).

[0627]

[0628] To a solution of compound 210 (9.47 g, 10.0 mmol) in dichloromethane (50 mL) were added NHS (1.39 g, 12.0 mmol), EDC·HCl (2.30 g, 12.0 mmol), and DIPEA (3.8 mL, 22.0 mmol). The reaction was stirred for 2 hours, then diluted with water (50 mL) and extracted with ethyl acetate (3×20 mL). The organic phases were combined and washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (10-80% ethyl acetate / petroleum ether) to give a colorless oil (9.49 g, 76% yield). ESIMS m / z 1249.72 ([M+H] + ).

[0629] Example 82. Synthesis of (6S,13S)-9,10-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propionamido)-5,8,11,14-tetraoxo-6,13-bis(4-((((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioic acid ester (212).

[0630]

[0631] A solution of compound 211 (9.49 g, 7.60 mmol) in THF (15 mL) and 4N hydrochloric acid (2 mL) was reacted at 0°C for 30 minutes, then concentrated, loaded onto a short silica gel column, and eluted with 0-15% methanol / dichloromethane to give a colorless oil (8.50 g, 90% yield). ESIMS m / z 1249.72 ([M+H] + ).

[0632] Example 83. Synthesis of Compound 213.

[0633]

[0634] To a solution of compound 212 (139.0 mg, 0.111 mmol) and compound 52 (50.0 mg, 0.0555 mmol) in DMF (10 mL) were added TBTU (35.6 mg, 0.111 mmol), DIPEA (20.0 μL, 0.111 mmol), and the mixture was stirred at room temperature for 2 hours. After removing DMF under high vacuum, the residue was purified by preparative HPLC (acetonitrile / water) to give a colorless oil (140.1 mg, 63% yield). ESIMS m / z 2002.84 ([M+H] + ).

[0635] Example 84. Synthesis of Compound 214.

[0636]

[0637] A solution of compound 213 (140 mg, 0.0699 mmol) in THF (10 mL) was reacted with TBAF (1.0 M in THF, 350 μL) at 0°C for 30 minutes, then concentrated and purified by a short silica gel column (0-10% methanol / dichloromethane) to give a colorless oil (100.3 mg, 88% yield). ESIMS m / z 1714.72 ([M+H] + ).

[0638] Example 85. Synthesis of Compound 215.

[0639]

[0640] A mixture of compound 214 (99.8 mg, 0.0583 mmol) and compound 194 (110.2 mg, 0.146 mmol) in THF (10 mL) and phosphate buffer solution (10 mL, 0.5 M, pH 7.7) was stirred overnight, then concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (79.2 mg, 45% yield). ESIMS m / z 3007.56 ([M+H] + ).

[0641] Example 86. Synthesis of 2-(1,3-dioxoisoindol-2-yl)acetyl chloride (223).

[0642]

[0643] To a solution of N-phthaloylglycine (10.0 g, 48.7 mmol) in dichloromethane (100 mL) was added oxalyl chloride (6.3 mL, 73.1 mmol) at room temperature, followed by dropwise addition of DMF. The reaction was stirred for 2 hours and then concentrated to afford compound 223 (10.8 g) as a yellow solid.

[0644] Example 87. Synthesis of tert-butyl 2-(2-(1,3-dioxoisoindolin-2-yl)acetyl)hydrazinecarboxylate (224).

[0645]

[0646] To a solution of tert-butyloxycarbonylhydrazine (7.08.g, 53.5mmol) in dichloromethane (200mL) was added Et3N (13.5mL, 97.4mmol), followed by compound 223 (10.8g, 48.7mmol) at 0°C. The reaction was stirred at room temperature for 30 minutes, poured into ice water (100mL), and extracted with dichloromethane (3×100mL). The combined organic phases were washed with water (100mL) and brine (100mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid (15.5g, 100% yield). ESIMS m / z 320.12 ([M+H] + ).

[0647] Example 88. Synthesis of 2-(1,3-dioxoisoindol-2-yl)acetohydrazide (225).

[0648]

[0649] Compound 224 (15.5 g, 48.7 mmol) was dissolved in dichloromethane (150 mL) and reacted with TFA (50 mL) at room temperature for 1 hour, then concentrated to give a white solid (10.6 g, 100% yield). ESIMS m / z 220.06 ([M+H] + ).

[0650] Example 89. Synthesis of 2-(1,3-dioxoisoindolin-2-yl)-N′-(2-(1,3-dioxoisoindolin-2-yl)acetyl)acetylhydrazine (226).

[0651]

[0652] To a solution of compound 225 (10.6 g, 48.7 mmol) in dichloromethane (200 mL) at 0°C, Et3N (13.5 mL, 97.4 mmol) and compound 223 (10.8 g, 48.7 mmol) were added. The reaction was warmed to room temperature and stirred overnight. The precipitate was collected by filtration and suspended in water (100 mL) and stirred for 20 minutes. Filtered again to give compound 218 (15.7 g, 80% yield). ESIMS m / z 407.09 ([M+H] + ).

[0653] Example 90. Synthesis of di-tert-butyl 2'-(1,2-bis(2-(1,3-dioxoisoindolin-2-yl)acetyl)hydrazine-1,2-diyl)diacetate (227).

[0654]

[0655] NaH (0.5 g, 12.3 mmol) was added in portions to a solution of compound 226 (2.0 g, 4.92 mmol) in DMF (40 mL) at 0°C. The mixture was heated to room temperature and stirred for 3 hours. After that, tert-butyl bromoacetate (2.0 g, 10.3 mmol) was added, the reaction was stirred overnight, then poured into ice water (100 mL) and extracted with dichloromethane (3×50 mL). The combined organic phase was washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column to give a white solid (1.5 g, 50% yield). ESIMS m / z 635.23 ([M+H] + ).

[0656] Example 91. Synthesis of di-tert-butyl 2'-(1,2-bis(2-aminoacetyl)hydrazine-1,2-diyl)diacetate (228).

[0657]

[0658] A mixture of compound 227 (1.5 g, 2.36 mmol) and hydrazine (442 mg, 7.08 mmol) in ethanol (30 mL) was refluxed for 1 hour, then cooled to room temperature and filtered. The filtrate was concentrated, diluted with ethyl acetate (20 mL), and filtered again. The filtrate was concentrated to give a white solid (750 mg, 85% yield). ESIMS m / z 375.22 ([M+H] + ).

[0659] Example 92. Synthesis of di-tert-butyl 2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)hydrazine-1,2-diyl)diacetate (229).

[0660]

[0661] A solution of compound 228 (750 mg, 2 mmol) in THF (2 mL) was added to an aqueous NaHCO3 solution (30 mL), cooled to 0°C, and then N-methoxycarbonylmaleimide (622 mg, 4 mmol) was added. The reaction was stirred at 0°C for 1 hour. The white solid (854 mg, 80% yield) was collected by filtration. ESIMS m / z 535.20 ([M+H] + ).

[0662] Example 93. Synthesis of 2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)hydrazine-1,2-diyl)diacetic acid (230).

[0663]

[0664] Compound 229 (854 mg, 1.6 mmol) was dissolved in dichloromethane (3 mL) and reacted with TFA (3 mL) at room temperature for 2 hours. The reaction was then concentrated to give compound 230 (675 mg, 100% yield). ESIMS m / z 423.07 ([M+H] + ).

[0665] Example 94. Synthesis of di-tert-butyl 4'-((2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)hydrazine-1,2-diyl)bis(acetyl))bis(azadiyl))dibutyrate (231).

[0666]

[0667] To a solution of compound 230 (200 mg, 0.47 mmol) in DMF (5 mL) at 0°C, tert-butyl 4-aminobutyrate (158 mg, 0.99 mmol) and EDC (189.7 mg, 0.99 mmol) were added. The reactants were warmed to room temperature, stirred overnight, poured into ice water, and extracted with dichloromethane (3×10 mL). The organic phases were combined, washed with 1N hydrochloric acid (5 mL), water (5 mL), and brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid (330 mg, 100% yield).

[0668] Example 95. Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 4,4′-((2,2′-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl)hydrazine-1,2-diyl))bis(azadiyl))dibutyrate (233).

[0669]

[0670] Compound 231 (330 mg, 0.47 mmol) was dissolved in dichloromethane (3 mL) and reacted with TFA (3 mL) at room temperature for 2 hours. The reactant was concentrated and redissolved in DMF (5 mL), cooled to 0°C, and NHS (113 mg, 0.98 mmol) and EDC (189 mg, 0.98 mmol) were added in sequence. The reactant was warmed to room temperature, stirred overnight, poured into ice water, and extracted with dichloromethane (3×20 mL). The combined organic phase was washed with water (5 mL), brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid (369 mg, 100% yield). ESIMS m / z 787.21 ([M+H] + ).

[0671] Example 96. Synthesis of (S)-48-((((benzyloxy)carbonyl)amino)-3,16,29,42-tetraoxo-1-phenyl-2,20,23,26,33,36,39-heptaoxa-17,30,43-triazatetranonadecan-49-oic acid (235).

[0672]

[0673] To a solution of compound 192 (1.00 g, 1.32 mmol) in dichloromethane (10 mL) at 0°C were added HATU (0.50 g, 1.32 mmol) and TEA (0.06 mL, 1.32 mmol). The reaction was stirred at 0°C for 30 minutes, then Z-Lys-OH (0.40 g, 1.43 mmol) was added. The reaction was stirred for 1 hour, then diluted with water (20 mL) and extracted with ethyl acetate (3×30 mL). The organic phases were combined and washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (0-10% methanol / dichloromethane) to give a colorless oil (1.28 g, 95% yield). ESIMS m / z 1017.60 ([M+H] + ).

[0674] Example 97. Synthesis of (S)-4,7-benzyl 1-(2,5-dioxopyrrolidin-1-yl) 2-((benzyloxy)carbonyl)amino)-8,21,34-trioxo-11,14,17,24,27,30-hexaoxa-7,20,33-triazatetratetradecane-1,47-dioic acid ester (236).

[0675]

[0676] To a solution of compound 235 (1.28 g, 1.26 mmol) in dichloromethane (10 mL) was added NHS (0.17 g, 1.51 mmol), EDC HCl (0.29 g, 1.51 mmol), followed by TEA (0.38 mL, 2.77 mmol). The reaction was stirred for 2 hours, then diluted with water (20 mL) and extracted with ethyl acetate (3×15 mL). The combined organic phases were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (0-10% methanol / dichloromethane) to give a colorless oil (1.28 g, 91% yield). ESIMS m / z 1114.62 ([M+H] + ).

[0677] Example 98. Synthesis of Compound 237.

[0678]

[0679] To a solution of compound 56 (50.2 mg, 0.0555 mmol) and compound 236 (136.1 mg, 0.122 mmol) in DMF (10 mL) was added DIPEA (20 μL, 0.122 mmol), and the mixture was stirred at room temperature for 2 hours. After removing DMF under high vacuum, the residue was purified by preparative HPLC (acetonitrile / water) to give a colorless oil (70.3 mg, 44% yield). ESIMS m / z 2899.80 ([M+H] + ).

[0680] Example 99. Synthesis of Compound 238.

[0681]

[0682] In a hydrogenation bottle, a solution of compound 237 (70.0 mg, 0.0241 mmol) in methanol (50 mL) was reacted with Pd / C (10 wt%, 145 mg) for 2 hours by catalytic hydrogenation (1 atm H2), filtered through diatomaceous earth (filter aid), and the filtrate was concentrated to obtain compound 238 (65 mg, yield> 100%). ESIMS m / z 2631.63 ([M+H] + ).

[0683] Example 100. Synthesis of Compound 239.

[0684]

[0685] A mixture of compound 238 (65 mg, 0.0247 mmol) and compound 233 (29 mg, 0.0371 mmol) in EtOH (10 mL) and phosphate buffer solution (10 mL, 0.5 M, pH 7.2) was stirred overnight, then concentrated and purified by preparative HPLC (acetonitrile / water) to give a white foam (28.3 mg, 36% yield). ESIMS m / z 3187.60 ([M+H] + ).

[0686] Example 101. Synthesis of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate.

[0687]

[0688] To a solution of di-tert-butyl hydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol) in DMF (150 ml) was added NaH (60%, 2.76 g, 68.8 mmol). After stirring at room temperature for 30 minutes, di-tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched by the addition of methanol (3 ml), concentrated, diluted with ethyl acetate (100 ml) and water (100 ml), separated, and the aqueous layer was extracted with ethyl acetate (2×50 ml). The organic layers were combined, dried over magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography (ethyl acetate / hexane 1:5 to 1:3) to give the title compound (12.98 g, 82% yield) as a colorless oil. MS ESI m / z C 22 H 41 N2O8[M+H] + Calculated value 461.28, measured value 461.40.

[0689] Example 102. Synthesis of 2'-(hydrazine-1,2-diyl)diacetic acid.

[0690]

[0691] To a solution of di-tert-butyl 1,2-di(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) in 1,4-dioxane (40 ml) was added hydrochloric acid (12 M, 10 ml). The mixture was stirred for 30 minutes, diluted with dioxane (20 ml) and toluene (40 ml), concentrated and concentrated to dryness with dioxane (20 ml) and toluene (40 ml) to give the crude title product which was used in the next step without further production (2.15 g, 103% yield, ~93% purity). MS ESI m / z C4H9N2O4[M+H] + Calculated value 149.05, measured value 149.40.

[0692] Example 103. Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid.

[0693]

[0694] To a mixed solution of 2,2'-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in THF (50 ml) and NaH2PO4 (0.1 M, 80 ml, pH 6.0) was added (E)-3-bromoacryloyl bromide (5.01 g, 23.60 mmol). The mixture was stirred for 6 hours, concentrated and purified on a silica gel column eluted with water / acetonitrile (1:9) containing 3% formic acid to give the title compound (2.35 g, 77% yield, ~93% purity). MS ESI m / zC 10 H 11 Br2N2O6[M+H] + Calculated value 412.89, measured value 413.50.

[0695] Example 104. Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetyl chloride.

[0696]

[0697] To a solution of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid (210 mg, 0.509 mmol) in dichloroethane (15 ml) was added (COCl)2 (505 mg, 4.01 mmol) followed by 0.040 ml of DMF. After stirring at room temperature for 2 hours, the mixture was concentrated and co-concentrated to dryness with dichloroethane (2 x 20 ml) and toluene (2 x 15 ml) to give the title crude product (unstable) which was used in the next step without further purification (245 mg, 107% yield). MS ESI m / zC 10 H9Br2Cl2N2O4[M+H] + Calculated values: 448.82, 450.82, 452.82, 454.82; measured values: 448.60, 450.60, 452.60, 454.60.

[0698] Example 105. Synthesis of tert-butyl 2,8-dioxo-1,5-oxazolidine-5-carboxylate.

[0699]

[0700] To a solution of 3,3'-azadiyldipropionic acid (10.00 g, 62.08 mmol) in 1.0 M NaOH (300 ml) was added a solution of di-tert-butyl dicarbonate (22.10 g, 101.3 mmol) in THF (200 ml) at 4°C over 1 hour, and after the addition was complete, the mixture was stirred at 4°C for 2 hours. The mixture was carefully acidified to pH 4 with 0.2 M H3PO4, concentrated in vacuo, extracted with dichloromethane, dried over Na2SO4, concentrated, and purified by flash silica gel chromatography eluting with acetic acid / methanol / dichloromethane (0.01:1:5) to give 3,3'-((tert-butyloxycarbonyl)azadiyl)dipropionic acid (13.62 g, 84% yield). ESIMS m / zC 11 H 19 NO6[M+H] + Calculated value: 262.27; measured value: 262.40.

[0701] Phosphorus pentoxide (8.70 g, 61.30 mmol) was added to a solution of 3,3'-((tert-butoxycarbonyl)azepinediyl)dipropionic acid (8.0 g, 30.6 mmol) in dichloromethane (500 ml) at 0°C. The mixture was stirred at 0°C for 2 hours, then continued to stir at room temperature for 1 hour, filtered through a short silica gel column, and the column was rinsed with ethyl acetate / dichloromethane (1:6). The filtrate was concentrated and slurried with ethyl acetate / n-hexane to give the title compound (5.64 g, 74% yield). ESIMS m / zC 11 H 17 NO5[M+H] + , calculated value 244.11; measured value 244.30.

[0702] Example 106. Synthesis of 2,5-dioxapyrrolidin-1-yl acrylate.

[0703]

[0704] A solution of acrylic acid (5.00 g, 71.4 mmol), NHS (9.01 g, 78.3 mmol) and EDC (20.0 g, 104.1 mmol) in dichloromethane (150 ml) and DIPEA (5 ml, 28.7 mmol) was stirred overnight, concentrated and purified by silica gel column chromatography (ethyl acetate / hexane 1:4) to give the title compound (9.30 g, 79% yield) as a colorless oil. 1 H NMR (500MHz, CDCl3) δ2.68 (s, 1H), 2.61 (s, 4H). MS ESI m / z C7H5NaNO4[M+Na] + Calculated value 190.02, measured value 190.20.

[0705] Example 107. Synthesis of tert-butyl 2-acryloylhydrazidecarboxylate.

[0706]

[0707] A solution of acrylic acid (5.00 g, 71.4 mmol), tert-butylhydrazinecarboxylate (9.45 g, 71.5 mmol) and EDC (20.0 g, 104.1 mmol) in dichloromethane (150 ml) and DIPEA (5 ml, 28.7 mmol) was stirred overnight, concentrated and purified by silica gel column chromatography (ethyl acetate / hexane 1:5) to give the title compound (7.92 g, 84% yield) as a colorless oil. 1 HNMR (500MHz, CDCl3) δ8.76 (m, 2H), 2.68 (s, 1H), 1.39 (s, 9H). MS ESI m / z C5H 12 NaN2O2[M+Na] + Calculated value 155.09, measured value 155.26.

[0708] Example 108. Synthesis of acrylhydrazide hydrochloride.

[0709]

[0710] At 4 ° C, tert-butyl 2-acryloylhydrazide carboxylate (4.01 g, 30.35 mmol) was dissolved in 1,4-dioxane (12 mL) and reacted with 4 ml of hydrochloric acid (concentrated). The mixture was stirred for 30 minutes, diluted with dioxane (30 ml) and toluene (30 ml) and concentrated under vacuum. The crude mixture was purified on silica gel, eluted with methanol (5% to 10%) in dichloromethane (containing 1% formic acid) to give the title compound (2.11 g, yield 83%), ESIMS m / z C3H5N2O[M+H] + , calculated value 85.03; measured value 85.30.

[0711] Example 109. Synthesis of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid.

[0712]

[0713] To a solution of maleic anhydride (268g, 2.73mol) in acetic acid (1L), 4-aminobutyric acid (285g, 2.76mol) was added. Stir at room temperature for 30 minutes, reflux the reactant for 1.5 hours, and cool to room temperature. Vacuum concentration gave a residue, which was dissolved in ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was crystallized from ethyl acetate and petroleum ether to give a white solid (400g, 80% yield). 1H NMR (500MHz, CDCl3) δ6.71 (s, 2H), 3.60 (t, J = 6.7Hz, 2H), 2.38 (t, J = 7.3Hz, 2H), 2.00–1.84 (m, 2H).

[0714] Example 110. Synthesis of 2,5-dioxopyrrolidin-1-yl-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate.

[0715]

[0716] At room temperature, 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (400 g, 2.18 mol, 1.0 eq) was dissolved in dichloromethane (1.5 L), N-hydroxysuccinimide (276 g, 2.40 mmol, 1.1 eq) and DIC (303 g, 2.40 mol, 1.1 eq) were added thereto, and stirred overnight. The reaction was concentrated and purified by column chromatography (1:2 petroleum ether / ethyl acetate) to give NHS ester (382 g, 63% yield) as a white solid. 1 H NMR (500MHz, CDCl3) δ6.74 (s, 2H), 3.67 (t, J=6.8Hz, 2H), 2.85 (s, 4H), 2.68 (t, J=7.5Hz, 2H), 2.13–2.03 (m, 2H).

[0717] Example 111. Synthesis of Compound S-1 (a control conjugable amatoxin compound).

[0718]

[0719] To a solution of 2,5-dioxopyrrolidin-1-yl 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate 125 (20.2 mg, 0.07 mmol) and compound 69 (15.1 mg, 0.0172 mmol) in DMF (10 mL) was added DIPEA (15 μL, 5 eq). The reaction was stirred at room temperature overnight, then concentrated and purified by preparative HPLC (acetonitrile / water) to give compound S-1 (15.0 mg, 82% yield) as a white foam. ESIMS m / z 1052.60 ([M+H] + ).

[0720] Example 112. Synthesis of tert-butyl 3-((2-aminoethyl)amino)propanoate.

[0721]

[0722] A solution of tert-butyl acrylate (12.81 g, 0.10 mmol) and ethyl-1,2-diamine (24.3 g, 0.40 mol) in THF (150 mL) was stirred at 45°C for 24 hours. The mixture was concentrated and purified on an Al2O3 column eluted with triethylamine / methanol / dichloromethane (5:15%:80%) to give the title compound (17.50 g, 92% yield). ESIMS m / z 189.20 ([M+H] + ).

[0723] Example 113. Synthesis of 3-(2-aminoethyl)amino)propanoic acid hydrochloride.

[0724]

[0725] A solution of tert-butyl 3-(((2-aminoethyl)amino)propanoate (17.00 g, 90.33 mmol) in 1,4-dioxane (50 mL) was reacted with concentrated hydrochloric acid (15 mL). The mixture was stirred at room temperature for 30 minutes, concentrated and diluted with purified water (150 mL) and ethyl acetate / n-hexane (40 mL, 1:5). The mixture was separated and the organic layer was extracted with water (2×10 mL). The aqueous layer was concentrated and dried on a vacuum pump to give the title compound (18.70 g, 100% yield, and 96% purity by LC-MS analysis). ESIMS m / z 133.20 ([M+H] + ).

[0726] Example 114. Synthesis of 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-propionic acid.

[0727]

[0728] To a solution of 3-((2-aminoethyl)amino)propanoic acid (18.70 g, 90.33 mmol) in THF (150 mL) at 0 °C was added maleic anhydride (8.85 g, 90.33 mmol). The mixture was stirred at 0-4°C for 4 hours and concentrated to give (Z)-4-((2-(((2-carboxyethyl)amino)ethyl)amino)-4-oxobutyl-2-enoic acid, and the product was confirmed by LC-MS. Toluene (150 ml) and DMA (50 ml) were then added thereto, and refluxed at 90°C using Dean-Stark. After 30 ml of solvent was collected in the collector, HMDS (hexamethyldisilazane, 9.0 mL, 43.15 mmol) and zinc chloride (16 mL, 1.0 M ether solution) were added. The mixture was heated to 115-125°C, and toluene was collected by Dean-Stark. The reaction mixture was refluxed at 120°C for 6 hours. During this period, 2×40 mL of anhydrous toluene was added to keep the mixture volume at around 50 mL. The mixture was then cooled, and 1 mL of 1:10 hydrochloric acid (conc.) / methanol. The mixture was concentrated and purified on a silica gel column, eluted with water / methanol (1:15), and dried on a vacuum pump to give 14.75 g of the title compound (77.0% yield). ESIMS m / z 213.10 ([M+H] + ).

[0729] Example 115. Synthesis of 2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl 4-methylbenzenesulfonate.

[0730]

[0731] To a solution of 2,5,8,11,14,17,20,23-octaoxapentacosan-25-ol (50.0 g, 0.130 mol) in DCM (200 ml) and pyridine (100 ml) was added TsCl (30.2 g, 0.159 mol). The mixture was stirred overnight, concentrated and purified on a silica gel column, eluted with acetone / dichloromethane (1:1 to 4:1) and dried on a vacuum pump to give the title compound 57.34 g (82.0% yield). ESIMS m / z 539.40 ([M+H] + ).

[0732] Example 116. Synthesis of S-2,5,8,11,14,17,20,23-octaoxapentacosan-25-ylthioacetate.

[0733]

[0734] To a mixture of 2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl 4-methylbenzenesulfonate (57.30 g, 0.106 mol) in THF (300 ml) and DIPEA (50 ml) was added HSAc (10.0 g, 0.131 mol). The mixture was stirred overnight, concentrated and purified on a silica gel column, eluted with ethyl acetate / dichloromethane (1:1 to 4:1) and dried on a vacuum pump to give the title compound 40.51 g (86% yield). ESIMS m / z 443.35 ([M+H] + ).

[0735] Example 117. Synthesis of 2,5,8,11,14,17,20,23-octaoxopentacosane-25-sulfonic acid

[0736]

[0737] S-2,5,8,11,14,17,20,23-octaoxopentacosane-25-ylthioacetate (40.40 g, 0.091 mol) was added to a mixture of acetic acid (200 mL) and 30% hydrogen peroxide (100 mL) at 35°C and stirred overnight. The mixture was concentrated, diluted with pure water (200 mL) and toluene (150 mL), separated, and the organic layer was extracted with water (2×25 mL). The aqueous solutions were combined, evaporated and dried on a vacuum pump to give the title compound 40.50 g (99% yield, 95% purity by LC-MS analysis). ESIMS m / z 449.30 ([M+H] + ).

[0738] Example 118. Synthesis of 3,3-N,N-(2"-maleimidoethyl)(2',5',8',11',14',17',20',23',26'-nonaoxaoctacosane-28'-sulfoxide)aminopropionic acid (70).

[0739]

[0740] To a mixture of 2,5,8,11,14,17,20,23-octaoxapentacosane-25-sulfonic acid (20.0 g, 44.62 mmol) in THF (100 ml) and DCM (100 ml) was added (COCl)2 (25.21 g, 200.19 mmol) and DMF (0.015 ml). The mixture was stirred at room temperature for 2 hours, concentrated, concentrated with dichloromethane / toluene (1:1, 2×50 ml), and then redissolved in THF (50 ml). To a solution of 3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-propionic acid (7.50 g, 35.36 mmol) in THF (100 mL) was added sulfonyl chloride. The mixture was stirred overnight, concentrated and purified on a silica gel column, eluted with methanol / dichloromethane (1:6 to 1:5), and dried on a vacuum pump to give the title compound 14.76 g (65% yield). ESIMS m / z 643.35 ([M+H] + ).

[0741] Example 119. Synthesis of N-N-succinimidyl 3,3-N,N-(2″-maleimidoethyl)(2′,5′,8′,11′,14′,17′,20′,23′,26′-nonaoxaoctacosane-28′-sulfoxide)aminopropanoate (70a).

[0742]

[0743] To a solution of 3,3-N,N-(2″-maleimidoethyl)(2′,5′,8′,11′,14′,17′,20′,23′,26′-nonaoxaoctacosane-28′-sulfoxide)aminopropionic acid (70) (7.50 g, 11.67 mmol) in THF (100 ml) was added N-hydroxysuccinimide (1.50 g, 13.04 mmol) and EDC (10.10 g, 52.60 mmol). The mixture was stirred overnight, concentrated and purified on a silica gel column eluted with methanol / dichloromethane (1:4 to 1:1) and dried on a vacuum pump to give the title compound 6.30 g (73% yield). ESIMS m / z 740.40 ([M+H] + ).

[0744] Example 120. General method for preparing conjugates 78a, 146, 154, 167, 197, 198, 216, 240 and S-2.

[0745] To 2.0 mL of 10 mg / ml Herceptin at pH 6.0-8.0, 0.70-2.0 mL of 100 mM NaH2PO4 in PBS buffer, pH 6.5-8.5 buffer, TCEP (14-35 μL, 20 mM aqueous solution), and compounds 71, 145, 153, 166, 195, 196, 215, 239, and S-1 (14-28 μL, 20 mM DMA solution) were added, followed by the addition of 4-(azidomethyl)benzoic acid (14-50 μL, 20 mM, pH 7.5 PBS buffer). The mixture was incubated at room temperature for 4-18 hours, and then DHAA (135 μL, 50 mM) was added. After continuous incubation overnight at room temperature, the mixture was purified on a G-25 column and eluted with 100 mM NaH2PO4, 50 mM NaCl pH 6.0-7.5 buffer to give 12.2-18.6 mg of conjugates 78a, 146, 154, 167, 197, 198, 216, 240 and S-2 (85%-94% yield) (in 13.4-15.8 ml of NaH2PO4 buffer). The drug / antibody ratio (DAR) of the conjugate was determined to be 3.5-4.2 by UPLC-QTOF mass spectrometry. The monomer content was 96-99% monomer by SEC HPLC analysis (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5 ml / min, 100 min).

[0746]

[0747] Example 121. In vitro cytotoxicity evaluation of conjugates 78a, 146, 154, 167, 197, 198, 216, 240 and S-2 compared to T-DM1:

[0748] The cell line used for the cytotoxicity assay was the human gastric cancer cell line NCI-N87, and the cells were grown in RPMI-1640 containing 10% FBS. For the assay, cells (180 μL, 6000 cells) were added to each well of a 96-well plate and incubated at 37°C, 5% CO2 for 24 hours. Then, the cells were treated with different concentrations of the test compound (20 μL) in the appropriate cell culture medium (total volume, 0.2 mL). The control wells contained cells and culture medium but no test compound. The well plate was incubated at 37°C and 5% CO2 for 120 hours, and then MTT (5 mg / mL) (20 μL) was added to the wells, and the well plate was incubated at 37°C for 1.5 hours. The culture medium was carefully removed, and DMSO (180 μL) was added, shaken for 15 minutes, and the absorbance was measured at 490nm and 570nm, with 620nm as the reference. The inhibition rate was calculated according to the following formula: inhibition rate % = [1-(analytical value-blank control value) / (control value-blank control value)] x 100. The results are listed in Table 1.

[0749] Table 1. Her2-amanitin homologue conjugates of the present patent application and their cytotoxicity (IC 50 ):

[0750]

[0751] Example 122. In vivo antitumor activity in the BALB / c nude mouse human gastric c...

Claims

1. A branched linker-connected conjugate, the structure of which is represented by formula (I): in represents a single bond; n is 1 to 30; T is a cell binding agent or molecule selected from an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody, a domain antibody fragment that binds to a target cell, an adnectin-like antibody, a DARPin protein, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule (transferrin) and / or a cell binding peptide, protein or small molecule attached to an albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle or a (viral) capsid; L1 and L2 are, the same or different, independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R 12 )、N(R 12 )N(R 12’ )、CH、CO、C(O)NH、C(O)O、NHC(O)NH、NHC(O)O、Polyethyleneoxy of the following structural formulas: (OCH2CH2) p OR 12 , or (OCH2CH-(CH3)) p OR 12 , or NH(CH2CH2O) p R 12 , or NH(CH2CH(CH3)O) p R 12 , or N[(CH2CH2O) p R 12 ]-[(CH2CH2O) p’ R 12’ ], or (OCH2CH2) p COOR 12 , or CH2CH2(OCH2CH2) p COOR 12 , wherein p and p' are independently selected from integers ranging from 0 to about 1000, or combinations thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or (Aa) r , r = 1-12 (1 to 12 amino acid units), including natural or unnatural amino acids, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units of the same or different sequences; W is a stretcher unit, typically a self-immolative spacer, a polypeptide unit, a hydrazone, a disulfide, a thioether, an ester or an amide bond; w is 1 or 2 or 3; V1 and V2 are independent spacer units selected from O, NH, S, C1-C8 alkyl; C2-C8 heteroalkyl, alkenyl or alkynyl; C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylalkyl, heteroalkylcycloalkyl, or alkylcarbonyl; or (Aa) r , r = 1-12 (1-12 amino acid units), including natural or unnatural amino acids, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units of the same or different sequences; or (CH2CH2O) p , p is 0-1000; v1 and v2 are independently 0, 1 or 2, but v1 and v2 are both 0. When v1 or v2 is 0, it means that the side chain Q1 or Q2 fragment is absent. Q1 and Q2 are independently represented by formula (I-q1): in is the position connected to L1 or L2, G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, or(Aa) q1 ; G3 is OH, SH, OR 12 , SR 12 、OC(O)R 12 、NHC(O)R 12 、C(O)R 12 、CH3、NH2、NR 12 , + NH(R 12 ), + N(R 12 )(R 12’ ), C(O)OH, C(O)N2, NHC(O)NH2, BH2, BR 12 R 12’ , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH、OC(O)NH(CH2) q1 C(O)OH、NHCO(CH2) q1 P(O)(OH)2、NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2、NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH、OS(O)2NH(CH2) q1 C(O)OH、NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR 12 、Ar、ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 , or (Aa) q1 ; (Aa) q1 is a peptide containing the same or different sequences of natural or unnatural amino acids; X1 and X2 are independently O, CH2, S, S(O), NHNH, NH, N(R 12 ), + NH(R 12 ), + N(R 12 )(R 12’ ), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y2 is O.NH, NR 12 、CH2.S、NHNH、Ar;R 12 , R 12’ , R 13 and R 13’ are independently H, C1-C8 alkyl; C2-C8 heteroalkyl, or heterocycle; C3-C8 aryl, aralkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocycle, or alkylhydroxyl; Y2 is O, NH, NR1, CH2, S, NHNH, Ar; p1, p2 and p3 are independently 0-100 but not all 0; q1 and q2 are independently 0-24; Preferably Q1 and Q2 are independently C2-C 100 Polycarboxylic acid or C-2-C 90 Polyalkylamine, C6-C 90 Oligosaccharides or polysaccharides, C6-C 100 Zwitterionic betaines or zwitterionic poly(sulfonyl betaines) (PSB) containing quaternary ammonium cations and sulfonate anions, C6-C 100 Biodegradable polymers such as polylactic acid / glycolic acid (PLGA), poly(acrylates), chitosan, copolymers of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethylphosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(ethylene glycol)-modified peptides, poly(ethylene glycol)-modified liposomes, poly(ethylene glycol)-modified alkyl carboxylic acids, poly(ethylene glycol)-modified alkylamines, hyaluronic acid (HA) (glycosaminoglycans), heparin or heparan sulfate (HSGAG), chondroitin sulfate or chondroitin sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfates, poly(ethylene glycol)-modified alkyl phosphates, or poly(ethylene glycol)-modified alkyl quaternary ammonium salts; Or any one or more of W, Q1, Q2, L1, L2, V1 or V2 may be independently defaulted, but Q1 and Q2 cannot be defaulted at the same time. D is an amatoxin homologue having the following formula (II): or one or more element isotope substitutions, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polycrystalline structures; or optical isomers, racemates, diastereomers or enantiomers thereof; in is the site of attachment to W; A single bond on an aromatic (indole) ring refers to its attachment to any carbon of the aromatic ring; Indicates an optional single key or optional default; R1 and R2 are independently selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH3)CH2OH, CH(OH)CH3, C1-C8 alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (ester), –OC(=O)OR 12 -(carbonate), –OC(=O)NHR 12 (Carbamate); C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl; R3 and R4 are independently selected from H, OH, –OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OCOOR 12 (carbonate), –OC(=O)NHR 12 (Carbamate), -OP(O)(OR 12 )(OR 12 ')(phosphate), OP(O)(NHR 12 )(NHR 12 ')(phosphoamine), O-SO3 - , or O-glycosides; R5 is selected from H, OH, NH2, NHOH, NHNH2, –OR 12、 –NHR 12 NHNHR 12 ,–NR 12 R 12 '、N(H)(R 12) R 13 CO(Aa) r , (amino acid or peptide, wherein Aa is an amino acid or a polypeptide, r = 0-6); R6 is selected from H, OH, CH2OH, CH(OH)CH2OH, CH(CH2OH)2, CH(CH3)OH, CH2CH2OH, PrOH, BuOH, C1~C8alkyl, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic; R7, R8 and R9 are independently selected from H, OH, CH3, CH(CH3)2, CH(CH3)CH2CH3, CH2OH, CH(OH)CH2OH, CH2CH(OH)CH2OH, CH(CH2OH)2, CH2C(OH)(CH2OH)2, CH2C(OH)(CH3)(CH2OH), CH2C(OH)(CH(CH3)2 )(CH2OH), CH2CH2OH, PrOH, BuOH, CH2COOH, CH2CH2COOH, CH(OH)COOH, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2CH2CH2NHC(=NH)NH2, C1~C8alkyl, CH2Ar, CH2SH, CH2SR 12 、CH2SSR 12 , CH2SSAr, CH2CH2SCH3, –OR 12 (ester), C2-C8 alkenyl, alkynyl, heteroalkyl, –OCOR 12 (Ester); C3-C8 aryl, heterocyclic or carbocyclic; R 10 and R 11 Independently selected from H, NH2, OH, SH, NO2, halogen, -NHOH, -N3 (azido); -CN (cyano); C1-C8 alkyl, C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic or carbocyclic; -OR 12 (ester), –OCOR 12 (ester), –OCOCH3(acetate), –OC(O)OR 12 (carbonate), -OC(O)CH(R 12 )NHAa (Aa is an amino acid group), -NR 12 R 12 '(amine), -NR 12 COR 12 '(amide), -R 12 NHCOR 12 '(alkylamide), -R 12 NHR 12 '(Amine), NHR 12 NHR 12 'NHR 12 ” (amine); –R 12 NCONR 12 '(urea); –R 12 NCOOR 12 '(Carbamate); –OCONR 12 R 12 '(Carbamate); –NR 12 (C=NH)NR 12 'R 12 ” (guanidine); –NR 12 CO(Aa) p , (amino acid or peptide, wherein Aa is an amino acid or peptide, and p is 0-6); –N(R 12 )CONR 12 'R 12 ” (urea); –OCSNHR 12 (Thiocarbamate); -R 12 SH (sulfhydryl); –R 12 SR 12 '(sulfide); –R 12 SSR 12 '(disulfide); –S(O)R 12 (sulfoxide); –S(O2)R 12 (sulfone); –SO3, HSO 3、 HSO2, or HSO3 - 、SO3 2- or -HSO2 - Salts (sulfites); –OSO3 - ;-N(R 12 )SOOR 12 '(Sulfonamide); H2S2O5 or S2O5 2- Salts (pyrosulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2 or PO3S 3- PO2S2 3- POS3 3- , PS4 3- Salts (mono-, di-, tri-, and tetra-thiophosphates); (R 12 O)2POSR 12 '(phosphorothioate); HS2O3 or S2O3 2- (Thiosulfate); HS2O4 or S2O4 2- Salts (dithionites); (P(=S)(OR 12 )(S)(OH) or salts formed with cations (dithiophosphates); -N(R 12 )OR 12 '(Hydroxyamine derivative); R 12 C(=O)NOH or salts formed with cations (hydroxamates); HOCH2SO2 - , or its salts (formaldehyde sulfoxylate); –N(R 12 )COR 12 '(amide); R 12 R 12 'R 12 "NPO3H (trialkylaminophosphoric acid or aminophosphoric acid); or ArAr'Ar"NPO3H (triarylphosphoric acid); OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1; O-glycoside (glucoside, galactoside, mannoside, glucuronide, alloside, fructoside, etc.), NH-glycoside, S-glycoside or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR1'R2'R3'; R1', R2' and R3 ’ are independently H, C1-C8 alkyl; Ar, Ar', and Ar" are C3-C8 aryl or heteroaryl; Where R 12 , R 12 ', and R 12 " is independently selected from H, C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or 1-8 carbon atom ester, ether or amide; or (OCH2CH2) p or (OCH2CH(CH3)) p of polyoxyethylene units, wherein p is an integer from 0 to 1000, or a combination thereof, or is omitted. X is S, O, NH, SO, SO2, or CH2; m' is 0 or 1.

2. The structural formula of the conjugate containing a branched linker is shown in (III): in, The definitions of D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, n and T are as described in claim 1.

3. The structure of the branched linker compound is shown in (IV), which can be conveniently reacted with the cell binding molecule T to form the conjugate in formula (I): in, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are defined as in claim 1; Lv1 can react with thiol, amine, carboxylic acid, selenol, phenol or hydroxyl on the cell binding molecule. Lv1 is independently selected from hydroxyl (OH), fluorine (F), chlorine (Cl), bromine (Br), iodine (I), nitrophenol, N-hydroxysuccinimide (NHS), phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, trifluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, trifluoromethanesulfonyl, imidazole, dichlorophenol, trichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, toluenesulfonyl, mesyl, 2-ethyl-5-phenylisoxazole-3'-sulfonyl, anhydride or anhydride formed by reaction with other anhydrides, such as acetic anhydride, formic anhydride, or an intermediate generated by reaction with a polypeptide condensation reagent or a Mitsunobu reaction reagent.Examples of condensing agents are as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide methyl p-toluenesulfonate (CMC or CME-CDI), 1,1'-carbonyldiimidazole (CDI), oxy-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-oxy-(1H-benzotriazol-1-yl)-ammonium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethylamino)-hexafluorophosphate (BTP), OP), (benzotriazol-1-yloxy)tripyrrolidinium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidine hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethyl-imidazolium hexafluorophosphate (CIP), chloropyrrolidinium hexafluorophosphate (PyCloP), fluoro-N,N,N',N' -bis(tetramethylene)formamidine hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxo-2-pyridyl)thiourea hexafluorophosphate, oxy-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxo-2-pyridyl)N,N,N',N'-tetramethylthiourea tetrafluoroborate, oxy-[(ethoxycarbonyl)-cyanomethylamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylaminooxy)dimethylamino-morpholino-hexafluorophosphate (COMU), oxy-(benzotriazol-1-yl)- N,N,N',N'-bis(tetramethylene)hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexyl-carbodiimide (with or without polymer binding), dipyrrolidinyl(N-succinimidyloxy)carbonium hexafluorophosphate (HSPyU), chlorodipyrrolidinylhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy)dipiperidinylcarbonium hexafluorophosphate (HBPipU), oxy-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromo(dimethylamino)-hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA,. ), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-oxy-(N-succinimidyl) hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyano-methyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N' , N'-tetramethyl-oxy-(N-succinimidyl) uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)-dipiperidinium (ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD). Lv1 can be an anhydride, or an anhydride formed by reacting with other C1-C8 anhydrides; Lv1 is preferably selected from: disulfide; Haloacetyl; Acyl halides; N-hydroxysuccinimide ester; Maleimide; Monosubstituted maleimide; Disubstituted maleimide; Monosubstituted succinimides; Disubstituted maleimide; Substituted maleic acid; -CHO aldehyde; Ethylenesulfonyl; Acryloyl; 2-(Tosyloxy)acetyl; 2-(methylsulfonyloxy)acetyl; 2-(Nitrophenol)acetyl; 2-(Dinitrophenyl)acetyl; 2-(Fluorophenol)-acetyl; 2-(Difluorophenol)-acetyl; 2-((trifluoromethylsulfonyl)oxy)acetyl; Ketone or aldehyde, 2-(Pentafluorophenol)acetyl; Methylsulfone phenyl oxadiazole (ODA); Anhydride, Alkoxyamine; Azide, Alkynyl, or Hydrazide; wherein X1′ is F, Cl, Br, I or Lv3; X2′ is O, NH, N(R1) or CH2; R3 is H, aryl or heteroaryl, wherein one or more H atoms may be independently substituted by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1 or -COOR1; Lv3 is a leaving group selected from F, Cl, Br, I, nitrophenyl, N-hydroxysuccinimide (NHS ), phenol group, dinitrophenol group, pentafluorophenol group, tetrafluorophenol group, difluorophenol group, monofluorophenol group, pentachlorophenol group, trifluoromethanesulfonyl group, imidazole group, dichlorophenol group, tetrachlorophenol group, 1-hydroxybenzotriazole group, toluenesulfonyl group, methanesulfonyl group, 2-ethyl-5-phenylisoxazole-3′-sulfonyl group, acid anhydride or acid anhydride formed by reaction with other acid anhydride, acetic anhydride, formic anhydride, or intermediates formed by reaction with polypeptide condensation reagents or Mitsunobu reaction reagents.

4. The structure of the branched linker compound is shown in (V), which can be conveniently reacted with the cell binding molecule T to form a conjugate in formula (III): in, The definitions of D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2 and n are as described in formula (I) in claim 1; wherein Lv1 and Lv2 are independently the same as the definition of Lv1 in formula (IV) in claim 3, and Lv1 and Lv2 may be the same as or different from formula (V).

5. The side chains Q1 and Q2 according to claim 1, 2, 3 or 4 are independently selected from Iq-01 to Iq-35: in, R 25 and R 25 'Independently selected from H, HC(O), CH3C(O), CH3C(NH), C1-C 18 Alkyl, C1-C 18 Alkyl-Y1-SO3H, C1-C 18 Alkyl-Y1-PO3H2, C1-C 18 Alkyl-Y1-CO2H, C1-C 18 Alkyl-Y1-N + R 12 'R 13 'R 13 'R 14 '、C1-C 18 Alkyl-Y1-CONH2, C2-C 18 Alkyl, C2-C 18 Esters, C2-C 18 Ether, C2-C 18 Amine, C2-C 18 Alkyl carboxamide, C3-C 18 Aryl, C3-C 18 Cycloalkyl, C3-C 18 Heterocycle, 1-24 amino acids, C2-C 18 Lipids, C2-C 18 Fatty acids or C2-C 18 Fatty ammonium lipid; X1 and X2 are independently selected from NH, N(R1'), O, CH2, S, C(O), S(O), S(O2), P(O)(OH), NHNH, CH=CH, Ar or (Aa) q1 , q1=0-24 (0-24 amino acids, q1=0 indicates default); X1, X2, X3, X4, Y1, Y2 and Y3 are independently selected from NH, N(R1'), O, C(O), CH2, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH, Ar or (Aa) q1 , X1, X2, X3, X4, Y1, Y2 and Y3 are independently optional; p1, p2 and p3 are independently 0-100, but cannot be 0 at the same time; q1, q2 and q3 are independently 0-24; R1', R2', R3' and R4' are independently selected from H and C1-C6 alkyl; Aa is a natural or non-natural amino acid; Ar or (Aa) q1 are the same or different peptide sequences; q1=0 means (Aa)q1 is absent.

6. D (amanitin) according to claims 1, 2, 3 and 4 can be selected from IIa, IIb, IIc, II-01, II-02, II-03, II-04, II-05, II-06, II-07, II-08, II-09, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23, II-24, II-25, II-26: or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; Where Z2 is oxygen or a lone pair of electrons; R 15 YesH、NHR 12 , OR 12 , C1-C8 straight chain or branched alkyl or heteroalkyl; C2-C8 straight chain or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 straight chain or branched aryl, arylalkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, carbonate (-R1C(O)OR 12 ), carbamate (-R 12 C(O)NR 12’ R 13 ); or a carboxylic acid ester, ester, ether or amide of 1 to 8 carbon atoms; or 1 to 8 amino acids; or such as (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is an integer from 0 to about 1000; Z1 is H, O, S, NH, NHNH, R 12 , or default; R 21 It is COR 12 、NHCOR 12 、COOR 12 ,CONHR 12 , R 12 , R 12 NH; R 22 YesR 12 , SR 12 、SCH(CH3)R 12 SC(CH3)2R 12 , X is O, S, NH, NHNH, or CH2. R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 12’ , R 13 The definitions of , and X1 are the same as above; 7. W, L1, L2, V1, and V2 according to claim 1, 2, 3, or 4 can be independently selected from one or more of the following linker components: 6-Maleimidocaproyl (MC), Maleimidopropionyl (MP), Valine-citrulline (val-cit), Alanine-phenylalanine (ala-phe), Lysine-phenylalanine (lys-phe), p-Aminobenzyloxy-carbonyl (PAB), 4-Thiopentanoyl (SPP), 4-thiobutyryl (SPDB), 4-(N-maleimidomethyl)cyclohexane-1-yl (MCC), Maleimidoethylamino (ME), 4-Sulfo-2-hydroxysulfonyl-butyryl (2-Sulfo-SPDB), Aryl sulfide group (PySS), (4-Acetyl)aminobenzoyl (SIAB), Oxybenzyl sulfide group, Aminobenzyl sulfide group, Dioxybenzyl sulfide group, Diaminobenzyl sulfide group, Aminooxybenzyl sulfide group, Alkoxyamino (AOA), Ethyleneoxy (EO), 4-Methyl-4-thio-pentanoyl (MPDP), Triazole, Disulfide, Alkylsulfonyl, Alkyl sulfonamides, Disulfonamide, Phosphodiamide, Alkylphosphonamides, Phosphonic acid, N-Methylalkylphosphonamide, N, N'-dimethylphosphoramide, Alkylphosphonic acid diamides, Hydrazine, Acetamidine; Oxime, Diacetylhydrazine, Aminoethylamine, Aminoethyl-aminoethylamine and L- or D-, natural or non-natural peptides containing 1-20 amino acids; wherein is a linking site; preferably, X2, X3, X4, X5 or X6 are independently selected from NH, NHNH, N(R 12 )、N(R 12 )N(R 12 '), O, S, C1-C6 alkyl, C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl, C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, CH2OR 12 、CH2SR 12 、CH2NHR 12 or 1 to 8 amino acids; 12 and R 12 ' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl, C3-C8 aryl, aralkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; an ester, ether or amide of 1-8 carbon atoms; or a polyoxyethylene unit of the formula (OCH2CH2)p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or a combination of the above.

8. W, L1, L2, V1, and V2 according to claim 1, 2, 3, or 4 may independently include: (A) Self-immolative or non-self-immolative components, peptide units, hydrazone bonds, disulfide, ester, oxime, amide or thioether bonds. The self-destruction units include aromatic compounds with electronic structures similar to p-aminobenzylcarbamoyl (PAB), derivatives of 2-aminoimidazole-5-methanol, heterocyclic PAB homologues, β-glucuronides, and o- or p-aminobenzyl acetals. Wherein, * marks the attachment point of additional spacers or releasable linkers, or cytotoxic agents, and / or binding molecules (CBA); X 1 , Y 1 , Z 2 and Z 3 are independently NH, O or S; Z 1 H, NHR1, OR1, SR 1、 COX1R1, wherein X1 and R1 are as defined above; v is 0 or 1; U 1 H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5') or OCH2PO(OR5(OR5'), wherein R5 and R5' are independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl or amino acid; C3-C8 aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroarylalkyl, alkylcarbonyl or glycoside; or a pharmaceutically cationic salt; (B): The non-self-immolative linker component comprises one of the following structures: Wherein, * marks the attachment point of additional spacers or releasable linkers, or cytotoxic agents, and / or binding molecules; X 1 , Y 1 , U 1 , R5, R5' are as defined above; r is 0-100; m and n are independently 0-20; (C): Releasable means that the linker contains at least one bond that is breakable under physiological conditions, pH, acid, base, oxidation, metabolism, biochemical or enzymatic labile, including one of the following structures: -(CR 15 R 16 ) m (Aa)r(CR 17 R 18 ) n (OCH2CH2) t -、-(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -、-(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -、-(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -、-(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -、-(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -、-(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m -(CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -、-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -、-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n- (OCH2CH2) r -、-(CR 15 R 16 ) m -phenyl-CO(Aa) t- (CR 17 R 18 ) n -、-(CR 15 R 16 ) m -Furan-CO(Aa) t (CR 17 R 18 ) n -、-(CR 15 R6) m -Oxazole-CO(Aa) t (CR 17 R 18 ) n -、-(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t (CCR 17 R 18 ) n -、-(CR 15 R 16 ) t -Thiophene-CO(CR 17 R 18 ) n -、-(CR 15 R 16 ) t -imidazole-CO-(CR 17 R 18 ) n -、-(CR 15 R 16 ) t -Morpholine-CO(Aa) t- (CR 17 R 18 ) n -、-(CR 15 R 16 ) t -Piperazine-CO(Aa) t (CR 17 R 18 ) n -、-(CR 15 R 16 ) t -N-Methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -、-(CR 15 R 16 ) m -(Aa) t Phenyl-,-(CR 15 R 16 ) m -(Aa) t Furan-, -(CR 15 R 16 ) m -Oxazole (Aa) t -、-(CR 15 R 16 ) m -Thiazolyl(Aa) t -、-(CR 15 R 16 ) m -Thienyl-(Aa) t -、-(CR 15 R 16 ) m -Imidazole (Aa) t -、-(CR 15 R 16 ) m -Morpholine-(Aa) t -、-(CR 15 R 16 ) m -Piperazine-(Aa) t -、-(CR 15 R 16 ) m -N-Methylpiperazine-(Aa) t -、-K(CR 15 R 16 ) m (Aa)r(CR 17 R 18 ) n (OCH2CH2) t -、-K(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -、-K(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -、-K(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -、-K(CR 15 R 16 ) m (CR 17 =CR 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r 、-K(CR 15 R 16 ) m (NR 11 CO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -、-K(CR5R6) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (O-CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (CO)(Aa) t- (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n -(OCH2CH2) r -、-K(CR 15 R 16 ) m- (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -、-K-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m -phenyl-CO(Aa) t (CR 17 R 18 ) n -、-K-(CR 15 R 16 ) m -Furan-CO(Aa) t (CR 17 R 18 ) n -、-K(CR 15 R 16 ) m -Oxazole-CO(Aa) t (CR 17 R 18 ) n -、-K(CR 15 R 16 ) m -Thiazolyl-CO(Aa) t- (CR 17 R 18 ) n -、-K(CR 15 R 16 ) t -Thiophene-CO(CR 17 R 18 ) n -、-K(CR 15 R 16 ) t Imidazole-CO-(CR 17 R 18 ) n -、-K(CR5R6) t Morpholine-CO(Aa) t -(CR 17 R 18 ) n -、-K(CR 15 R 16 ) t -Piperazine-CO(Aa) t- (CR 17 R 18 ) n -、-K(CR 15 R 16 ) t -N-Methylpiperazine-CO(Aa) t (CR 17 R 18 ) n -、-K(CR 15 R 16 ) m -(Aa) t Phenyl, -K-(CR 15 R 16 ) m- (Aa) t Furan-, -K(CR 15 R 16 ) m -Oxazole-(Aa) t -、-K(CR 15 R 16 ) m -Thiazolyl(Aa) t -、-K(CR 15 R 16 ) m -Thienyl-(Aa) t -、-K(CR 15 R 16 ) m -Imidazole (Aa) t -、-K(CR 15 R 16 ) m -Morpholine (Aa) t -、-K(CR 15 R 16 ) m Piperazine (Aa) t G, -K(CR5R6) m -N-Methylpiperazine (Aa) t -; where m, Aa, m, n, R 13 , R 14 and R 15 Definitions are as above; t and r are independently 0-100; R 16 , R 17 , R 18 , R 19 and R 20 independently selected from H, halogen, C1-C8 alkyl or heteroalkyl, C2-C8 aryl, alkenyl, alkynyl, ether, ester, amine or amide, C3-C8 aryl, optionally substituted with one or more halides, CN, NR 12 R 12’ , CF3, OR 12 , aryl, heterocyclic, S(O)R 12 、SO2R 12、 -CO2H, -SO3H, -OR 12 、-CO2R 12 、-CONR 12 ,-PO2R 12 R 13 、-PO3Hor P(O)R 12 R 12’ R 13 Replaced by; K is NR 12 , -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, Het (with C3-C 12 or a peptide containing 1-20 identical or different amino acids.

9. The conjugate of formula (I) according to claim 1, having the structures of a-01 to a-40, 78a-c, 91, 95, 97, 114, 117, 126, 132, 146, 154, 167, 179, 181, 197, 198, 206, 247, 250, 258, 260, 262: or an element isotope substitution, or a pharmaceutically acceptable salt, hydrate or hydrated salt; or a polycrystalline structure; or an optical isomer, racemate, diastereomer or enantiomer thereof; wherein X8 is O, S, NH, NHNH, NHR 12 , SR 12 、SSR 12 SSCH(CH3)R 12 、SSC(CH3)2R 12 , or R 12 ;X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 The definitions of mAb are as described above; Aa is a natural or unnatural amino acid; r is 0-100; (Aa)r is a peptide containing the same or different amino acid sequence, when r>2; when r=0, (Aa)r is omitted.

10. The conjugate of formula (III) according to claim 2, having structures of b-01 to b-22, 216, 221, 240: or one or more element isotope substitutions, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polycrystalline structures; or optical isomers, racemates, diastereomers or enantiomers thereof; Where X8 is O, S, NH, NHNH, NHR 12 , SR 12 、SSR 12 SSCH(CH3)R 12 、SSC(CH3)2R 12 , or R 12 ;X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 The definitions of , and mAb are as described above; Aa is a natural or unnatural amino acid; r is 0-12; (Aa)r is a peptide containing the same or different amino acid sequence, when r>2; r=0, (Aa)r is default.

11. The conjugate of formula (IV) according to claim 3, having the structure of c-01 to c-40, 71, 76, 77, 90, 94, 96, 113, 116, 126, 131, 145, 153, 166, 178, 180, 195, 196, 205, 246, 249, 257, 259, 261: or one or more element isotope substitutions, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polycrystalline structures; or optical isomers, racemates, diastereomers or enantiomers thereof; wherein X1, X2, X3, X4, X5, X8, Z2, Z3, p.p1, p2, p3, q1, q2, Lv3, m, n, R 12 , R 12’ , R 25 , R 25’ , (Aa)r and mA are as described above.

12. The conjugate of formula (V) according to claim 4, having structures of d-01 to d-25, 215, 220, 239: One or more element isotope substitutions, or pharmaceutically acceptable salts, hydrates or hydrated salts; or polycrystalline structures; or optical isomers, racemates, diastereomers or enantiomers thereof; wherein X1, X2, X3, X4, X5, X8, Z2, Z3, p.p1, p2, p3, q1, q2, Lv1, Lv2, Lv3, Lv 3’ , m, n, R 12 , R 12’ , R 15 , R 25 , R 25’ , (Aa)r and mAb were as described above.

13. The cell binding agent or cell binding molecule according to claim 1, 2, 9 or 10 is selected from the following structures: (A) antibodies, proteins, probodies, nanobodies, vitamins (including folic acid), peptides, polymer micelles, liposomes, lipoprotein drug carriers, nanoparticle drug carriers, dendrimers, and the above molecules or particles coated or linked to cell binding ligands, or combinations thereof; (B) Antibody proteins, complete antibodies (polyclonal antibodies, monoclonal antibodies, dimers, multimers), multispecific antibodies (e.g., bispecific, trispecific or tetraspecific antibodies), single-chain antibodies, antibody fragments that bind to target cells, monoclonal antibodies, single-chain monoclonal antibodies, monoclonal antibody fragments that bind to target cells, chimeric antibodies, chimeric antibody fragments that bind to target cells, domain antibodies, domain antibody fragments that bind to target cells, surface-modified antibodies, single-chain surface-modified antibodies, surface-modified antibodies that bind to target cells, humanized antibodies, single-chain humanized antibodies, single-chain humanized antibodies that bind to target cells, anti-idiotypic (anti-Id) antibodies, CDRs, bivalent antibodies, trivalent antibodies, tetravalent antibodies, minibodies, probody, probody fragments, small immune proteins (SIPs), lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules, large molecular weight proteins, fusion proteins, kinase inhibitors, gene targeting agents, nanoparticles or polymers modified with antibodies or large molecular weight proteins. (C) a ligand or receptor agonist selected from the following: folic acid derivatives; glutamate urea derivatives; somatostatin and its homologues, such as octreotide (Sandostatin) and lanreotide (Somatuline); aromatic sulfonamides; pituitary adenylate cyclase-activating peptide (PACAP) (PAC1); vasoactive intestinal polypeptide (VIP) (VPAC1, VPAC2); α-melanocyte stimulating hormone (α-MSH); cholecystokinin (CCK) or gastrin; bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp -Ala-Val-Gly-His-Leu-Met-NH2) or gastrin-releasing peptide (GRP); neurotensin receptors (NTR1, NTR2, NTR3); substance P; neuropeptide Y (Y1–Y6); homing peptides including RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (such as cRGDfV), TAASGVRSMH or LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands), and F3 peptides; cell penetrating peptides (CPPs);Peptide hormones, such as agonists and antagonists of luteinizing hormone-releasing hormone (LHRH), gonadotropin-releasing hormone (GnRH) agonists, act by targeting follicle-stimulating hormone (FSH) and luteinizing hormone (LH) as well as testosterone production, such as 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-AzGl y-NH2), histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-Bn)-Leu-Arg-Pro-NHEt), leuprolide (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), nafarelin, desloline, aberelin (Ac-D-2Nal-D-4- chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloro-Phe-D-3-(3-pyri) dyl)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(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2) and ganelli (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), such as Toll-like receptors (TLRs) ligands, C-type lectins and nodular receptors (NLRs) ligands; calcitonin receptor;Integrin receptors and their receptor subclasses (such as αVβ1, αVβ3, αVβ5, αVβ6, α6β4, α7β1, αLβ2, αIIbβ3, etc.) agonists (selected from GRGDSPK, cyclic (RGDfV) (L1) and its derivatives (cyclic (-N (Me) R-GDfV), cyclic (R-Sar-DfV), cyclic - (RG-N (Me) D-fV), cyclic (RGD-N (Me) fV), cyclic (RGDf-N (Me) V-) (cilengitide)); nanobodies (VHH derivatives (camelid Ig)); Domain antibodies (dAb, derivatives of VH or VL domains); bispecific T cell linkers (BiTE, bispecific diabodies); dual affinity retargeting antibodies (DART, bispecific bivalent antibodies); tetravalent tandem antibodies (TandAb, dimeric bispecific bivalent antibodies); Anticalin (derivatives of Lipocalins); Adnectin (10th FN3 (fibronectin)); designed ankyrin repeat proteins (DARPins); Avimer; agonists of EGF receptor or VEGF receptor. ; (D) The following small molecule cell binding molecules / ligands or cell receptor agonists: LB01 (folic acid), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, a somatostatin homolog), LB08 (lanreotide, a somatostatin homolog), LB09 (vapreotide (Sanvar), a somatostatin homolog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (gastrin-releasing peptide receptor (GRPr), MBA), LB LB13 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (cobalamin, vitamin B12 homolog), LB17 (cobalamin, vitamin B12 homolog), LB18 (cyclic RGD pentapeptide for αvβ3 integrin receptor), LB19 (heterobivalent peptide ligand for VEGF receptor), LB20 (neuromedullin B), LB21 (bombesin, acts on G protein-coupled receptor), LB22 (TLR2, acts on TLR-like Toll receptor), LB23 (acts on androgen receptor), LB24 (cilengitide or cyclic (-RGDfV-) conjugate, acts on αv integrin receptor) LB25 (rifabutin homologue), LB26 (rifabutin homologue), LB27 (rifabutin homologue), LB28 (fludrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclomethasone propionate), LB32 (triamcinolone acetate), LB33 (prednisolone), LB35 (methylprednisolone), LB36 (betamethasone), LB37 (irinotecan homologue), LB38 (crizotinib homologue), LB39 (bortezomib homologue), LB40 (carfilzomib homologue), LB41 (carfilzomib homologue), LB42 (leuprolide homologue), LB43 (triptorelin homologue), LB44 (clindamycin), LB45 (liraglutide homologue), LB46 (semivincristine homologue), LB47 (retapaline homologue), LB48 (dibulol homologue), LB49 (vinblastine homologue), LB50 (lixisenide homologue), LB51 (osimidine homologue), LB52 (nucleoside homologue), LB53 (erlotinib homologue) and LB54 (lapatinib homologue), whose structures are shown below: LB01 (Folic acid conjugate), LB02 (PMSA ligand conjugate), LB03 (PMSA ligand conjugate), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, a somatostatin homologue), LB08 (Lantide, somatostatin homologue), LB09 (aminopeptide (Sanvar), somatostatin homologue), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin-releasing peptide receptor (GRPr), MBA), LB13 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone (GnRH) ligand), LB14 (Luteinizing hormone-releasing hormone (LH-RH) and gonadotropin-releasing hormone (GnRH) ligand), LB15 (GnRH antagonist, abarelix), LB16 (cobalamin, a vitamin B12 homologue), LB17 (cobalamin, a vitamin B12 homologue), LB18 (cyclo-RGD pentapeptide, acting on α v β3 integrin receptor), LB19 (heterologous bivalent peptide ligand conjugate, acting on vascular endothelial growth factor VEGF receptor), LB20 (neuromedullin B), LB21 (bombesin conjugate, acting on G protein-coupled receptors), LB22 (TLR2 conjugate, acting on Toll-like receptors), LB23 (androgen receptor), LB24 (Cilengitide / cyclic (-RGDfV-) conjugate, acting on α v Integrin receptors LB25 (rifabutin homologue), LB26 (rifabutin homologue), LB27 (rifabutin homologue), LB28 (fludrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclomethasone propionate), LB32 (Anna Tate), LB33 (prednisone), LB34 (prednisolone), LB35 (methylprednisolone), LB36 (beflumethasone), LB37 (irinotecan homologue), LB38 (crizotinib homologue), LB39 (bortezomib homologue), wherein Y5 is N, CH, C(Cl), C(CH3) or C(COOR1); R1 is H, C1-C6 alkyl, C3-C8 aryl; LB40 (carfilzomib homologue), LB41 (carfilzomib homologue), LB42 (leuproreli homologue), LB43 (triptorelin homologue), LB44 (clindamycin), LB45 (liraglutide homologue), LB46 (semaglutide homologue), LB47 (retapalene homologue), LB48 (Dingbull homologue), LB49 (vinblastine homologue), LB50 (Lisisen peptide homologue), LB51 (oscitinib homologue), LB52 (nucleoside homologue), LB53 (erlotinib homologue), LB54 (lapatinib homologue), in is the connection position of the branch linker; X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH2, C(O)NHNHC(O) and C(O)NR1; X1 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1, NHR1, NR 1、 C(O)R1 or C(O)O; X5 is H, CH3, F or Cl; M1 and M2 are H, Na, K, Ca, Mg, NH4, N(R1R2R3 R4), respectively; R1, R2, R3 and R4 are as defined in claim 1.

14. The cell binding molecule T according to claim 1, 2 or 13, when connected to V1 and / or V2 in structural formula (I) and (III), or directly connected to L1 and / or L2, is characterized in that V1 and / or V2 are default and have one or more of the following structures: Where R 20 and R 21 are independently C1-C8 alkyl, C2-C8 heteroalkyl or heterocycle, C3-C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocycle or alkylcarbonyl, or C2-C 100 With the formula (CH2CH2O) p of polyalkylene glycol.

15. The cell-binding agent or cell-binding molecule of claim 1, 2, 9 or 10, characterized in that It can be used against the following cells: tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, melanocytes, or any cells expressing the following antigens or receptors: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD12w, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21a, CD21b, CD22c, CD23d, CD24, CD25 9. CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, C D57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD 67. CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD 85c, CD85d, CD85e, CD85f, CD85g, CD85g, CD85i, CD85j, CD85k, CD85m, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD 99. CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117,CD118、CD119、CD120、CD120a、CD120b、CD121、CD121a、CD121b、CD122、CD123、CD123a、CD124、CD125、CD126、CD127、CD128、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156、CD156a、CD156b、CD156c、CD156d、CD157、CD158、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f2、CD158g、CD158h、CD158i、CD158j、CD158k、CD159、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CDw198、CDw199、CD200、CD201、CD202、CD202(a、b)、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210a、CDw210b、CD211、CD212、CD213、CD213a1、CD213a2、CD214、CD215、CD216、CD217、CD218、CD218a、CD218、CD21b9、CD220、CD221、CD222、CD223、CD224、CD225、CD226、<h2 style=";text-align:left;direction:ltr">CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD235a, CD235b, CD236, CD237, CD238, CD239, CD240, CD240ce, CD240d, CD241, CD242 CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD26 3, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD281, CD282, CD283, CD284, CD2 85、CD286、CD287、CD288、CD289、CD290、CD291、CD292、CD293、CD294、CD295 、CD296、CD297、CD298、CD299、CD300、CD300a、CD300b、CD300c、CD301、CD302 、CD303、CD304、CD305、CD306、CD307、CD307a、CD307b、CD307c、CD307d、CD3 07e、CD307f、CD308、CD309、CD310、CD311、CD312、CD313、CD314、CD315、CD3 16、CD317、CD318、CD319、CD320、CD321、CD322、CD323、CD324、CD325、CD326 、CD327、CD328、CD329、CD330、CD331、CD332、CD333、CD334、CD335、CD336、CD 337、CD338、CD339、CD340、CD341、CD342、CD343、CD344、CD345、CD346、CD34 7、CD348、CD349、CD350、CD351、CD352、CD353、CD354、CD355、CD356、CD357、C D358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378,CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, WNT-activation inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, promoter receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha v beta 6, amino peptidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin protective antigen, antimetastatic protein receptor, AOC3 (VAP-1), B7-H3, anthrax Bacillus, BAFF (B cell activation factor), B lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, canine lupus erythematosus IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC fragment chemokine 11), CCR4 (CC chemokine receptor 4, CD194), CCR5, CD3E (ε), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), cluster factor A, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), CXC chemokine receptor 4, cyclic ADP ribonuclease, Cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, Dabigatran, DLL3 (Delta ligand-like 3), DLL4 (Delta ligand-like 4), DPP4 (Dipeptidyl-peptidase 4), DR5 (Death receptor 5), Escherichia coli shiga toxin type-1, Escherichia coli shiga toxin type-2, ED-B, EGFL7 (EGF domain-containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin (CD105), endothelin B receptor, endoglin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3,ERG (TMPRSS2 ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen 1, F protein of respiratory syncytial virus, frizzled receptor, fucose GM1, GD2 ganglioside, G-28 (cell surface antigen glycolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C), guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase C receptor, heat-stable enterotoxin receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (hepatocyte growth factor / scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28), IL31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, αIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αllβ3, α5β5, αvβ5), interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3,MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor, or glycosyl inhibitory factor (GIF)), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemoattractant protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1, MYCN, myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), N GF, neuronal apoptosis regulating proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, PAP, anti-(N-glycolylneuraminic acid) antibody binding site, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1), PDGF-Rα (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate co-transporter, PMEL 17. Polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutation, RGS5, ROBO4, respiratory syncytial virus, RON, ROR1, sarcoma translocation breakpoints, SART3, Sclerostin, SLAMF7 (SLAM member 7), Selectin P, SDC1 (syndecan 1), systemic lupus erythematosus (a), somatomedin C, SIP (sphingosine 1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial prostate antigen 1), STEAP2, STn, TAG-72 (tumor-associated glycoprotein), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B),TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (TNF-related necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signal sensor 2, tumor-specific glycosylated MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR-2, vimentin, WT1, XAGE 1, cells expressing insulin growth factor receptor, or cells expressing epidermal growth factor receptor.

16. The tumor cell of claim 15 is selected from lymphoma cells, myeloma cells, kidney cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell cancer cells, small cell lung cancer cells, non-small cell lung cancer cells, testicular cancer cells, malignant cells or any cell that causes cancer due to uncontrolled rapid growth and division.

17. A pharmaceutical composition comprising a therapeutically effective amount of the conjugate of claim 1, 2, 9 or 10 and a pharmaceutically acceptable salt thereof, a carrier, a diluent or an excipient, or a combination thereof, for treating or preventing cancer or an autoimmune disease or an infectious disease.

18. The pharmaceutical composition of claim 17 is a liquid formulation or a lyophilized solid, which, when reconstituted into a formulation for administration to a patient, comprises (by weight): 0.01%-99% of one or more conjugates of any one of claims 1, 2, 9 or 10; 0.0%-20.0% of one or more polyols; 0.0%-2.0% of one or more surfactants; 0.0%-5.0% of one or more preservatives; 0.0%-30% of one or more amino acids; 0.0%-5.0% of one or more antioxidants; 0.0%-0.3% of one or more metal chelators; 0.0%-30.0% of one or more buffer salts for adjusting the pH of the formulation to a pH of 4.5 to 7.5; and 0.0%-30.0% of one or more isotonic agents for adjusting the osmotic pressure to about 250-350 mOsm. in, The polyol is selected from fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbitol, melezitose, raffinose, mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, threitol, sorbitol, glycerol or L-gluconate and its metal salts. The surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81 or polysorbate 85, poloxamer, poly(ethylene oxide)-poly(propylene oxide), polyethylene-polypropylene, Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside, dodecyl, myristoyl, linoleyl or stearyl sulfobetaine, dodecyl, myristoyl, linoleyl or stearyl sarcosine, linoleic acid, myristoyl or hexadecyl betaine, Lauramidopropyl, cocamidopropyl, linoleamidopropyl, myristoylpropyl, palmitoylpropyl or isostearamido-propyl-betaine (e.g. lauramidopropyl), myristoylpropyl, palmitoylpropyl or isostearamidopropyl-dimethylamine, sodium methyl cocoyl or disodium methyl oleyl taurate, lauryl betaine, lauryl dimethylamine oxide, cocamidopropyl betaine and cocoamphoglycerate, isostearyl ethyliminium ethosulfate, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol. The preservative is selected from octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl and benzyl alcohol, alkyl parabens, such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol or m-cresol. The amino acid is selected from arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. The antioxidant is selected from ascorbic acid, glutathione, cystine or methionine. The metal chelator is selected from EDTA or EGTA. Buffer salts are selected from the sodium, potassium, ammonium or trishydroxyethylamine salts of citric, ascorbic, gluconic, carbonic, tartaric, succinic, acetic or phthalic acid, the hydrochloric, phosphate or sulfate salts of Tris or tromethamine, the acetate, chloride, phosphate, sulfate or succinate salts of arginine, glycine, glycylglycine or histidine. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate or sodium chloride. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate or sodium chloride.

19. The pharmaceutical composition according to claim 17 or 18, characterized in that Packaged as a liquid or lyophilized solid in a vial, bottle, prefilled syringe, or prefilled auto-injector.

20. The conjugate of claim 1, 2, 9 or 10, or the conjugate in the pharmaceutical composition of claim 17 or 18, having in vitro, in vivo or ex vivo cell killing activity.

21. The pharmaceutical composition according to claim 17 or 18, characterized in that It is administered simultaneously with chemotherapy drugs, radiotherapy, immunotherapy drugs, autoimmune disease drugs, anti-infective drugs or other conjugates for the synergistic treatment or prevention of cancer, autoimmune diseases or infectious diseases.

22. The chemotherapeutic drug as claimed in claim 21 is selected from: 1) Chemotherapy drugs: a) Alkylating agents: nitrogen mustards, chlorphenazone, chlorprenazine, cyclophosphamide, dacarbazine, estradiol nitrogen mustard, ifosfamide, nitrogen mustard, dimethoxamine hydrochloride, nitrogen oxide mustard, amlodipine hydrochloride, mycophenolic acid, dulcitol, guapobroman, new nitrogen mustard, phenylephrine, phenylephrine, thiotetracycline, trofosfamide, uracil; CC-1065, its adolesin, carzelesin, biszelesin and their synthetic homologues; duocarmycin and its synthetic homologues, KW-2189, CBI-TMI or CBI dimer; benzodiazepine dimer or pyrrolobenzodiazepine (PBD) dimer, tomemycin dimer, indole Dimers of benzodiazepines, imidazobenzothiadiazepines or oxazolidinones; nitrosoureas, including carmustine, lomustine, clomustine, fotemustine, nimustine, laromustine; alkyl sulfonates, including basufen, sufen, sulfamethoxazole and picofen; triazenes, dacarbazine; platinum-containing compounds, including carboplatin, cisplatin, oxaliplatin; aziridines, chromanone, calone, metodepa and uredopa; ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenetriamine, triethylphosphoramide, triethylenethiophosphoramide and trihydroxymethylmethylamine; b) Plant alkaloids: Vinca alkaloids, including vincristine, vinblastine, vindesine, vinorelbine, norvinblastine; Taxols, including paclitaxel, docetaxel and their homologues; Maytansines, including DM1, DM2, DM3, DM4, DM5, DM6, DM7, maytansine and ansamycin and their homologues; Cryptophycin (Cryptophycin 1 and Cryptophycin 8); Epothilone, Soft Coral Alcohol, Dimolide, Bronopolide, Dolastatin, Auristatin, Cephalostatin; Pancratistatin; Sarcodictyin; Sponge inhibitor; c) DNA topoisomerase inhibitors: etoposide, 9-aminocamptothecin, camptothecin, clenatol, doramycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, nosocomialin, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS2000); mitomycin and its homologues; d) Antimetabolites: antifolates, DHFR inhibitors (methotrexate, tramecil, dimethoate, pteropterin, aminopterin (4-aminobenzoic acid) or other folic acid analogs); IMP dehydrogenase inhibitors (mycophenolic acid, thiazofurine, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine); pyrimidine analogs, uracil analogs (ancitabine, azacitidine, 6-azauracil, capecitabine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, 5-fluorouracil, floxuridine, Ratitrexed (Tomudex); cytosine analogs (cytarabine, cytosine arabinoside, fludarabine); purine analogs (azathioprine, fludarabine, mercaptopurine, thiamine, thioguanine); folic acid supplements, florinic acid; e) Hormone therapy agents: receptor antagonists, antiestrogens (megestrol acetate, raloxifene, tamoxifen), LHRH stimulants (gostaline, leuprolide acetate); antiandrogens (bicalutamide, flutamide, calusone, betandron propionate, epiandrostenol, goserelin, leuprolide, metilidine, nilutamide, testolactone, trilostane and other androgen inhibitors); retinoids, vitamin D3 homologues (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); photodynamic therapy agents (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A); cytokines (interferon-α, interferon-γ, tumor necrosis factor (TNF), human protein containing TNF); f) Kinase inhibitors: BIBW 2992 (anti-EGFR / Erb2), Imatinib, Gefitinib, Guagaptanib, Sorafenib, Dasatinib, Sunitinib, Erlotinib, Nilotinib, Lapatinib, Axitinib, Pazopanib, Vandetanib, E7080 (anti-VEGFR2), Mubritinib, Ponatinib, Bafetinib, Bosutinib, Cabozantinib, Vismodegib, Iniparib, Ruxolitinib, CYT387, Axitinib, Tivozanib, Sorafenib, Bevacizumab, Cetuximab, Trastuzumab, Ranibizumab, Panitumumab, Ispins; g) Poly (ADP-ribose) polymerase (PARP) inhibitors: olaparib, niraparib, iniparib, talazoparib, veliparib, veliparib, CEP 9722 (Cephalon), E7016 (Eisai), BGB-290 (Beigene), 3-aminobenzamide; h) Antibiotics: Enediyne antibiotics (calicheamicin, calicheamicin gamma 1, delta 1, alpha 1 and beta 1, dynemycins, including dynemycin A and deoxyminomycin, esperamicins, cardamycins, C-1027, maduropeptin, neocarzacin and related chromoprotein enediyne antibiotics), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycins, carnosines, kanamycins, carminomicins, carmomycins, chromomycins, dazuromicin, daunorubicin , Desaurubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, Morpholine-doxorubicin, Cyanomorpholine-doxorubicin, 2-pyrroline-doxorubicin and Deoxydoxorubicin, Epirubicin, Aclarubicin, Idarubicin, Marcomycin, Nitomycin, Mycophenolic acid, Nogamycin, Olivomycin, Peplomycin, Potfiromycin, Puromycin, Quinamycin, Rodamycin, Streptozotocin, Streptozotocin, Tuberculin, Ubenimex, Netastatin, Zorubicin; i) Polyketides (Annona), Bullatacin and Bullatacinone; Gemcitabine, Cyclooxygenase Carfilzomib, Bortezomib, Thalidomide, Lenalidomide, Pomalidomide, Tosedostat, Zybrestat, PLX4032, STA-9090, Stimuvax, Allovectin-7, Xegeva, Provenge, Yervoy, Prenylation inhibitors, Lovastatin, Dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium ion, Cell cycle inhibitors (including staphylococcin), Actinomycins (such as actinomycin D, dactinomycin), Amatoxin, Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin), Anthracyclines (such as daunorubicin), Doxorubicin (adriamycin), Idarubicin, Epirubicin, Pirarubicin, Doxycycline, Mitoxantrone, MDR inhibitors or Verapamil, Ca 2+ 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; Antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane, acetoglucuronide, aldehyde phosphamide, aminolevulinic acid, amsacrine, arabinoside, Bestrabucil, bisantrene, Edatraxate, Defofamine, mecocin, diazocine, eflornithine (DFMO), elfomithine, elliptonium acetate, etoglu, gallium nitrate, cytosine, hydroxyurea, ibandronate, lentinan, lonidamine, mitoguanidine, mitoxantrone, moguadol, diamine nitracrine, pentostatin, mustard, pirarubicin, podophyllic acid, 2-ethylhydrazine, methylbenzalazine; Piperazinedione propane; rhizoxin; siroxicam; spirogermanium; tricrosporin; triimine quinone; trichlorotriethylamine; trichothecenes (including T-2 toxin, verrucosporin A, baculosporin A and anguiidine), polyurethane, siRNA, antisense drugs and nucleases. 2) Drugs for autoimmune diseases: cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, flucortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisone, sirolimus, tacrolimus. 3) Anti-infective drugs: a) Aminoglycosides: Amikacin, Astamicin, Gentomycin (Netilmicin, Sisomicin, Isopamicin), Hygromycin B, Kanamycin (Amikacin, Arbekacin, Aminodeoxykanamycin, Dibekacin, Tobramycin), Neomycin (Framycetin, Paromomycin, Ribomycin), Netilmicin, Spectinomycin, Streptomycin, Tobramycin, Methyl-Zisomicin; b) Amide alcohols: chloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) Ansamycins: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, panipenem; e) Cephem: carbacephem (loracabib), ceftriaxone, clomiphene cefuroxime, cephradine, cefoperazone, cefuroxime, cephalosporin, cephalothin or cephalothin, cephalexin, cefuroxime, cefadroxil, cefoperazone, cephalosporin, fluazifop, cephalosporin, cefadroxil, cephalosporin, cefoperazone, cefcaprine, cefuroxime, cefoperazone, cephalosporin, cefadroxil ... ceftriaxone, cefotaxime, cefotaxime, cefuroxime, cefoperazone, cefoperazone, cefoperazone, cefoperazone, cefoperazone, ceftriaxone, cephalosporin, cefazolin, cefalexin, ceftriaxone, cefoperazone, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefuroxime, cefoperazone, cefuroxime, cephalosporin (cefoxitin, cefotetan, cefuroxime), oxo(carbon) cephalosporins (floxacin, latamoxef); f) Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g) Glycylcyclines: Tigecycline; h) β-lactamase inhibitors: penicillins (sulbactam, tazobactam), oxopenicillins (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: daptomycin, A54145, calcium-dependent antibiotic (CDA); k) Macrolides: azithromycin, clotrimazole, clarithromycin, dirithromycin, erythromycin, fluramycin, josamycin, ketolides (telithromycin, cyromycin), midecamycin, micamycin, oleandomycin, rifamycins (isoniazid, rifampicin, rifabutin, rifapentine), ropimycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandomycin, telithromycin; l) Monocyclic amines: aztreonam, tigemonam; m) Oxazolidinones: linezolid; n) Penicillins: amoxicillin, ampicillin (pivacillin, helocillin, bacampicillin, ampicillin, doxorubicin), azlocillin, azlocillin, benzylpenicillin, benzathine penicillin, benzylpenicillin, benzathine penicillin phenoxymethylpenicillin, clocillin, procaine penicillin (meticillin), mezlocillin, methicillin, nafcillin, oxacillin, acetaminophen, penicillin, pheneticillin, phenoxymethylpenicillin, guaracillin, ampicillin, sulfenicillin, temocillin, ticarcillin; o) Peptides: bacitracin, colistin, polymyxin B; p) Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinas, danofloxacin, difloxacin, enoxacin, enrofloxacin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, levofloxacin, lomefloxacin, mabofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, toloxacin, trovafloxacin; q) Streptogramins: Primacycin, Quinupristin / Dalfopristin; r) Sulfonamides: sulfamethoxazole, sulfazosulfamide, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfapyridine, sulfisoxazole, trimethoprim, sulfamethoxazole (complex sulfamethoxazole); s) Steroid antibacterial drugs: such as fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, clomicycline, demeclocycline, ramuciline, mexicycline, methacycline, minocycline, oxytetracycline, panmecycline, pyrrolidinomethyltetracycline, tetracycline, glycylcycline (such as tigecycline); u) Other types of antibiotics: Annonaceous acetylcholine, arsphenamine, bacterial terpene alcohol inhibitors (bacitracin), DANAL / AR inhibitors (cycloserine), Dictyostatin, cyclosporin, cypermethrin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, NAM synthesis inhibitors (fosfomycin), nitrofurantoin, paclitaxel, pramoxetine, pyrazinamide, quinupristin / dalfopristin, rifampicin, tazobactam, tinidazole, and rutin. 4) Antiviral drugs: a) Invasion / fusion inhibitors: Apaviroc, Maraviroc, Vicriviroc, Gp41 (Enfuvirtide), PRO 140, CD4 (Ibalizumab); b) Integrase inhibitors: Raltegravir, Elvite-gravir, Globoidnan A; c) Maturation inhibitors: Bevirimat, Vivecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: Abacavir, Aciclovir, Adefovir, Amosivir, Abciximab, Brivudine, Cidofovir, Clavudine, Dexamethasone, Didanosine (ddI), Elvicitabine, Emtricitabine (FTC), Entecavir, Famciclovir, Fluracillin (5-FU), 3'-Fluoro substituted 2', 3'-deoxynucleoside homologues, such as 3'-Fluoro-2', 3'-dideoxythymidine (FLT) and 3'-Fluoro-2', 3'-dideoxyguanosine (FLG), fomivirsen, 9-guanine, iodoxuridine, lamivudine (3TC), 1-nucleosides (such as β-1-thymidine and β-1-2'-deoxycytidine), penciclovir, racivir, ribavirin, ditidine, stavudine (d4T), talibavirine (Viramidine), telbivudine, tenofovir, trifluridine valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleoside: Amantadine, Atipiridine, Capevirine, Diarylpyrimidine (Etravirine, Rilpivirine), Delavirdine, Docosanol, Emivirine, Efavirenz, Foscarnet (phosphocarboxylic acid), Imiquimod, Pegylated Interferon, Loviramide, Lodadenabinol, Methiazone, Nevirapine, NOV-205, Long-acting Interferon α, Podophyllotoxin, Rifampicin, Rimantadine, Resiquimod (R-848), Adamantane Acetamide; g) Protease inhibitors: Amprenavir, Atazanavir, Boceprevir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Nelfinavir, Pleconaril, Ritonavir, Saquinavir, Telaprevir (VX-950), Tipranavir; h) Other types of antiviral drugs: Abzyme, Arbidol, Aalanolide A, Ceragenin, Cyanavirin-n, Diarylpyrimidine, Epigallocatechin-3-gallate (EGCG), Foscarnet, Griffithsin, Taribavirin (viramidine), Hydroxyurea, KP-1461, Miltefosine, Pleconaril, Mixed inhibitors, Ribavirin, Seliciclib. 5) Pharmaceutically acceptable salts, acids, derivatives, hydrates or hydrated salts of the above drugs; or polymorphic crystals; or optical isomers, racemates, diastereomers or enantiomers thereof.

23. The synergistic therapeutic drug of claim 20 is selected from one or more of the following drugs: Abatacept, Abatamycin, Albicycline Acetate, Abalacin, Acetaminophen / Hydrocodone, Acalabrutinib, Aducanumab, Adalimumab, ADXS31-142, ADXS-HER2, Afatinib Maleic Acid, Aldesleukin, Alectinib, Alemtuzumab, Alitretinoin, ado-trastuzumab emtansine, Amphetamine / Dextroamphetamine, Anastrozole, Aripiprazole, Anthracycline, Aripiprazole, Atazanavir, Atezolizumab, Atorvastatin, Avelumab, Axicabtageneciloleucel, Axitinib, Belistat, BCG Live, Bevacizumab, Bexarotene, Brenatumumab, Bortezomib, Bosutinib, Brentuximab vedotin, budesonide, budesonide / formoterol, buprenorphine, cabazitaxel, cabozantinib, capmatinib, capecitabine, carfilzomib, chimeric antigen receptor engineered T cells (CAR-T), celecoxib, ceritinib, cetuximab, cedabenb, cyclosporine, cinacalcet, crizotinib, cobimetinib, Cosentyx, crizotinib, CTL019, dabigatran, dabrafenib, dacarbazine, daclizumab, dak Rotuzumab, daptomycin, daratumumab, darapotin alfa, darunavir, dasatinib, denileukin-diphtheria toxin, denosumab, sodium valproate, dexamethasone, denecap, dinutoximab, doxycycline, duloxetine, bivirinib, durvalumab, elotuzumab, emtricitabine / rilpivirine / tenofovir disoproxil, emtricitabine / tenofovir / efavirenz, enoxaparin, ensartinib, enzalutamide, Ep oetinα, erlotinib, esomeprazole, eszopiclone, etanercept, everolimus, exemestane, everolimus, exenatide, ezetimibe, ezetimibe / simvastatin, fenofibrate, filgrastim, fingolimod, fluticasone propionate, fluticasone / salmeterol, fulvestrant, gefitinib, glatiramer, goserelin acetate, icotinib, imatinib, ibritumomabtiuxetan, ibrutinib, idelalisib, Ifosfamide, Infliximab, Imiquimod, ImmuCyst, ImmunoBCG, Iniparib, Insulin Aspart, Insulin Detemir, Insulin Glargine, Insulin Lispro, Interferon α, Interferon α-1b, Interferon α-2a, Interferon α-2b, Interferon β, Interferon β1a, Interferon β1b, Interferon γ-1a, Lapatinib, Ipilimumab, Ipratropium / Salbuterol, Ixazomib, Kanuma,Lanreotide acetate, lenalidomide, lenalidomide, levatinib mesylate, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, lisdexamfetamine, LN-144, lorlatinib, memantine, methylguanidine, metoprolol, trametinib, mericitabine / rilpivirine / tenofovir, modafinil, mometasone, Mycidac-C, nexitovir Osimertinib, Neratinib, Nilotinib, Niraparib, Nivolumab, Ofatumumab, Obinutuzumab, Olaparib, Olmesartan Medoxomil, Olmesartan / Hydrochlorothiazide, Omalizumab, Omega-3 fatty acid ethyl esters, Encore, Oseltamivir, Osimertinib, Hydrocodone, Palbociclib, Palivizumab, Panitumumab, Panobinostat, Pazopanib, Pembr olizumab, PD-1 antibody, PD-L1 antibody, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, ProscaVax, propranolol, quetiapine, rabeprazole, radium 223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, regliafil, ribociclib, rituximab, rivaroxaban, romidepsin, rosuvastatin, ruxolitinib phosphate, salbutamol, savolitinib, sumaglutide, sevelamer, sildenafil, sutuximab, sipuleucel-T, sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonigib, sorafenib, sunitinib, tacrolimus, tacrimus, tadalafil, tamoxifen, dabrafenib, talimogene laherparepvec, Talazoparib, Telaprevir, Talazoparib, Temozolomide, Temsirolimus, Tenofovir / Emtricitabine, Tenofovir Disoproxil, Testosterone Gel, Thalidomide, TICE BCG, Tiotropium, Tisagenlecleucel, Toremifene, Trastuzumab, Trabectedin (Ecteinascidin 743), Trametinib, Tremelimumab, Trifluorouracil / Tipiracil, Retinoic Acid, Uro-BCG, Utekinumab, Valsartan, Veliparib, Vandetanib, Vemurafenib, Venetoclax, Vorinostat, Aflibercept, Zostavax and its homologues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof.

Images