Cell-binding molecule-tubulysin derivative conjugate and preparation method therefor

Abstract

The present invention relates to the conjugates of Tubulysin derivatives (anologs) and cell-binding molecules using branched (side-chain) linkers, and the resulting conjugates have better pharmacokinetic properties, and thus can more accurately target and kill abnormal cells. The invention also relates to the conjugation methods of the Tubulysin derivatives (anologs) to cell-binding molecules, and methods for synthesizing the small molecules, and methods of using the conjugates for targeted therapy for cancers, infections and autoimmune diseases. The conjugates of Tubulysin derivatives with long branched linkers demonstrated increased half-life, minimal exposure to non-targeted cells, tissues or organs in system circulation, leading to reduced off-target toxicity.

Description

Cell binding molecule-tubulysin derivative conjugate and method of making same TECHNICAL FIELD

[0001] The present invention relates to conjugating Tubulysin derivatives (homologues) with cell binding molecules with branched (pendent) linkers, resulting in conjugates with superior pharmacokinetic properties, thereby enabling more precise targeting of abnormal cells for killing. The invention also relates to methods of synthesizing Tubulysin homologues and cell binding agents and molecules contained therein, as well as methods of using the conjugates for targeted therapy of cancer, infections, and autoimmune diseases. BACKGROUND

[0002] The clinical success of Adcetris in treating relapsed or refractory Hodgkin lymphoma (Okeley, N. et al. Hematol Oncol. Clin. North. Am, 2014, 28, 13-25; Gopal, A., et al., Blood 2015, 125, 1236-43) and Kadcyla for the treatment of relapsed HER2-positive breast cancer (Peddi, P., Hurvitz, S., Ther. Adv. Med. Oncol. 2014, 6(5), 202-9; Lambert, J. and Chari, R., J. Med. Chem. 2014, 57, 6949-64) demonstrate that antibody drug conjugates (ADCs) are a very promising approach for targeted cancer therapy. Important factors for the success of ADCs include the three important components therein, namely the monoclonal antibody, the cytotoxic molecule, and the linker, as well as the site of attachment of the linker-cytotoxic molecule (L. Ducry and B Stump, Bioconjugate Chem., 2010, 21, 5-13; GS Hamilton, Biologicals 2015, 43, 318-32). Research on the components of ADCs has been ongoing for three decades. The linker needs to satisfy the ability to react with a specific reactive functional group on the drug, stability in the human blood circulation system, and easy release of the drug after binding to the antigen and endocytosis into the cell, and importantly, the linker-cytotoxic molecule should not harm normal tissues once off-target in the blood circulation, and existing linkage technologies are still limited (Ponte, J. et al., Bioconj. Chem., 2016, 27(7), 1588-98; Dovgan, I. et al. Sci. Rep. 2016, 6, 30835; Ross, P. L. and Wolfe, J. L., J Pharm. Sci. 105(2), 391-7; Chen, T. et al. J. Pharm. Biomed. Anal., 2016, 117, 304-10).

[0003] Early ADCs were mainly used for targeting liquid tumors, using very labile linkers, free drug was released in the blood circulation and off-target toxicity occurred (Bander N.H. et al., Clin. Adv. Hematol. Oncol., 2012, 10, 1-16). Current generation ADCs have more stable linkers and the cytotoxic agent is more active (Behrens, C.R. and Liu, B., mAbs, 2014. 6, 46-53). However, off-target toxicity is still one of the main challenges in ADC drug development (Roberts, S.A. et al., Regul. Toxicol. Pharmacol. 2013, 67, 382-91). For example, in clinical practice T-DM1 A stable (non-cleavable) MCC linker was used, which had a great benefit for patients with HER2-positive metastatic breast cancer (mBC) or who had received the corresponding treatment or had a recurrence of HER2 tumors 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, T-DM1 has failed in clinical trials as a first-line therapy for HER2-positive unresectable locally advanced or metastatic breast cancer patients and as a second-line therapy for HER2-positive advanced gastric cancer, the benefit for patients was not significant compared to the toxic side effects it produced (Ellis, PA, et al., J. Clin. Oncol. 2015, 33 (2015 ASCO Meeting Abstract 507); Shen, K. et al., Sci Rep. 2016, 6, 23262; de Goeij, B.E. and Lambert, J.M. Curr Opin Immunol 2016, 40, 14-23; Barrios, C.H. et al., J Clin Oncol 2016, 34, (2016 ASCO Meeting Abstract 593).

[0004] To address the issue of off-target toxicity, one direction of ADC chemistry development is to expand the linker-cytotoxic agent component and coupling chemistry beyond the use of a single cytotoxic agent, while also addressing the issue of the linker-cytotoxic agent pair's activity against the target disease (Lambert, JM Ther Deliv 2016, 7, 279-82; Zhao, RY et al. 2011, J. Med. Chem. 54, 3606-23). Many pharmaceutical developers and academic institutions have focused efforts on developing new and reliable specific coupling linkers, site-directed ADC coupling methods, which appear to have longer circulating half-lives, higher efficacy, reduced off-target toxicity, ADCs with narrow in vivo pharmacokinetic (PK) profiles, and better batch-to-batch consistency in production processes (Hamblett, K. J. et al. Clin. Cancer Res. 2004, 10, 7063-70; Adem, Y. T. et al. Bioconjugate Chem. 2014, 25, 656-664; Boylan, N. J. Bioconjugate Chem. 2013, 24, 1008-1016; Strop, P. et al. Chem. Biol. 2013, 20, 161-67; Wakankar, A. mAbs, 2011, 3, 161-172). These site-directed coupling methods that have been reported include: the introduction of engineered cysteines in the antibody (Junutula, J. R. et al. Nat. Biotechnol. 2008, 26, 925-32; Junutula, JR, et al. 2010 Clin. Cancer Res. 16, 4769; US patents 8,309,300; 7,855,275; 7,521,541; 7,723,485, WO2008 / 141044), selenocysteines (Hofer, T. et al. Biochemistry 2009, 48, 12047-57; Li, X. et al. Methods 2014, 65, 133-8; US patent 8,916,159), cysteines tagged with perfluorinated aromatic reagents (Zhang, C. et al. Nat. Chem. 2015, 8, 1-9), thiofurfuranes (Okeley, N. M. et al. Bioconjugate Chem. 2013, 24, 1650), unnatural amino acids (Axup, J. Y. et al. Proc. Nat. Acad. Sci. USA. 2012, 109, 16101-6; Zimmerman, E. S. 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; US Patent 8,778,631 and US Patent Application No. 20100184135, WO2010 / 081110; WO2006 / 069246, 2007 / 059312; US Patents 7,332,571, 7,696,312 and 7,638,299; WO2007 / 130453, US Patents 7,632,492 and 7,829,659); intermolecular disulfide bonds are rebridged after reduction by dibromomaleimide (Jones, MW et al. J. Am. Chem. Soc. 2012, 134, 1847-52), bisulfone reagents (Badescu, G. et al. Bioconjug. Chem. 2014, 25, 1124-36; WO2013 / 190272, WO2014 / 064424) and bisbromopyridazinedione (Maruani, A. et al. Nat. Commun. 2015, 6, 6645); galactose and sialyltransferases (Zhou, Q. et al. Bioconjug. Chem. 2014, 25, 510-520; US Patent Application No. 20140294867 for Sanofi-Genzyme), formylglycine-generating enzymes (FGEs) (Drake, PM et al. Bioconj. Chem. 2014, 25, 1331-41; Carrico, IS et al. US Patents 7,985,783; 8,097,701; 8,349,910, and US Patent Application Nos. 20140141025, 20100210543), phosphopantetheinyl transferases (PPTases) (Grünewald, J. et al. Bioconjug. Chem. 2015, 26, 2554-62), sortase A (Beerli, RR, et al. PLoS One 2015, 10, e0131177), glutamine tags introduced with the use of the moapa chain wheel filamentous fungus transglutaminase (mTG) (Strop, P., Bioconj. Chem., 2014, 25, 855-62; Strop, P., et al., Chem. Biol. 2013, 20, 161-7; US Patent 8,871,908), or glutamine tags introduced with the use of microbial transglutaminase (MTGase) (Dennler, P., et al. 2014, Bioconjug. Chem. 25, 569-78; Siegmund, V. et al. Angew. Chem.-Int. Ed. 2014, 53, 11092-6; US Patent 8,486,491), or the use of the enzyme phosphopanthetheinyl transferase (PPTase) (Grünewald, J. et al. Bioconjug. Chem. 2015, 26, 2554-62).2015, 54, 13420-4; US Patent Application No. 20130189287; US Patent 7,893,019), by enzymes or bacteria to form isopeptide 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, M J Am. Chem. Soc. 2010, 132, 4526-7).

[0005] We have disclosed several conjugation methods to re-bridge a pair of thiols generated by reduction of the interchain disulfide of native antibodies, such as using bromomaleimide and dibromomaleimide linkers (WO2014 / 009774), 2,3-disubstituted succinic acid / 2-monosubstituted / 2,3-disubstituted fumaric or maleic acid linkers (WO2015 / 155753, WO20160596228), acetylenedicarboxylic acid linkers (WO2015 / 151080, WO20160596228) or hydrazine linkers (WO2015 / 151081). Compared to traditional non-selective methods of conjugation to cysteine or lysine residues on antibodies, ADCs prepared with these linkers and conjugation methods have a larger therapeutic window. Here we disclose a Tubulysin conjugate containing a long branched linker. The long branched linker can prevent the antibody drug conjugate from being hydrolyzed by hydrolytic enzymes such as proteases or esterases, making the conjugate more stable in the circulatory system.

[0006] Tubulysins are a class of potent cytotoxic agents that are well known in the art and can be isolated from natural products based on known methods or prepared by methods of organic synthesis (e.g., Balasubramanian, R., et al. J. Med. Chem., 2009, 52, 238-40; Wipf, P., et al. Org. Lett., 2004, 6, 4057-60; Pando, O., et al. J. Am. Chem. Soc, 2011, 133, 7692-5; Reddy, J. A., et al. Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33; Patterson, A. W., et al. J. Org. Chem., 2008, 73, 4362-9; Pando, O., et al. Org. Lett., 2009, 11(24), 5567-9; Wipf, P., et al. Org. Lett., 2007, 9(8), 1605-7; Friestad, G. K., Org. Lett., 2004, 6, 3249-52; Peltier, H. M., et al. J. Am. Chem. Soc, 2006, 128, 16018-9; Chanrasekhar, S., et al. J. Org. Chem., 2009, 74, 9531-4; Liu, Y., et al. Mol. Pharmaceutics, 2012, 9, 168-75; Friestad, G. K., et al. Org. Lett., 2009, 11, 1095-8; Kubicek, K., et al., Angew Chem Int Ed Engl, 2010. 49:4809-12; Chai, Y., et al., Chem Biol, 2010, 17:296-309; Ullrich, A., et al., Angew Chem Int Ed Engl, 2009, 48, 4422-5; Sani, M., et al. Angew Chem Int Ed Engl, 2007, 46, 3526-9; Domling, A., et al., Angew Chem Int Ed Engl, 2006, 45, 7235-9; Zanda, M., et al, Can. Pat. Appl. CA 2710693 (2011); Chai, Y., et al. Eur. Pat. Appl. 2174947 (2010), WO 2010034724; Leamon, C. et al, WO2010033733, WO 2009002993; Ellman, J., et al., PCT WO2009134279; WO 2009012958; US Patent Applications 20110263650, 20110021568; Matschiner, G., et al., WO2009095447; Vlahov, I., et al., WO2009055562, WO 2008112873; Low, P., et al., WO2009026177; Richter, W., WO2008138561; Kjems, J., et al., WO 2008125116; Davis, M.; et al., WO2008076333; Diener, J.; et al., US Patent Application 20070041901, WO2006096754; Matschiner, G., et al., WO2006056464; Vaghefi, F., et al., WO2006033913; Doemling, A., Ger. Offen. DE102004030227, WO2004005327, WO2004005326, WO2004005269; Stanton, M., et al., US Patent Application 20040249130; Hoefle, G., et al., Ger. Offen. DE10254439, DE10241152, DE10008089; Leung, D., et al., WO2002077036; Reichenbach, H., et al., Ger. Offen. DE19638870; Wolfgang, R., US20120129779; Chen, H., US Patent Application 20110027274. We previously disclosed the construction of Tubulysin conjugates for targeted therapy of cancer, infections and autoimmune diseases (PCT / IB2012 / 053554). The Tubulysin conjugates with long branched linkers in the present invention have increased half-life during targeted delivery, minimal exposure to non-target cells, tissues or organs in the blood circulation, resulting in reduced off-target toxicity.

[0007] SUMMARY

[0008] The present invention relates to conjugation of Tubulysin homologs with cell binding molecules with branched (pendant) linkers, resulting in conjugates with better pharmacokinetic properties, thus enabling more precise targeted killing of abnormal cells. The present invention also relates to the conjugation of Tubulysin homologs with cell binding agents and methods of synthesis of the molecules contained therein, as well as methods of using the conjugates for targeted therapy of cancer, infections and autoimmune diseases.

[0009] In one aspect, the present application relates to an antibody-Tubulysin B derivative conjugate, characterized in that said conjugate has the structure of formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt, hydrate or hydrated salt of a structure of formula I, a polymorph of a structure of formula I, an optical isomer of a structure of formula I, one or more deuterium ( 2 H) atoms in place of hydrogen ( 1 H) atoms of a structure of formula I, or one or more 13 C atoms in place of 12 C atoms of a structure of formula I;

[0012] wherein P 1 is H, COCH3, COH, PO(OH)2, CH2OPO(OH)2, SO2CH3, C6H 11 O5 (glycoside), CONHCH3, CON(CH3)2, CON(CH2CH2)2NCH3, CON(CH2CH3)2, or CON(CH2CH2)2CHN(CH2CH2)2CH2;

[0013] R1, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkoxyl (R1OR2), C1-C6 alkylcarbonyl (R1COR2), C1-C6 alkylester (R1COOR2), C1-C6 alkylcarboxyl ((R1COOH), or C1-C6 alkylamide ((R1CONHR2);

[0014] Alternatively, R1and R2together, R1and R3together, R2and R3together, or R3and R4together form a C2-C7 heterocyclic ring or a C2-C7 cycloalkyl structure;

[0015] R5 is H, O-C1-C6 alkyl, C(O)-H, C(O)-C1-C6 (straight or branched) alkyl, C(O)-NH-C1-C6 (straight or branched) alkyl, or C(O)-N(C1-C6 (straight or branched) alkyl)2;

[0016] R6, R7, and R8 are each independently H, C1-C6 alkyl, C1-C6 alkoxyl (R1OR2), C1-C6 alkylcarbonyl (R1COR2), C1-C6 alkylester (R1COOR2), C1-C6 alkylcarboxyl ((R1COOH), or C1-C6 alkylamide ((R1CONHR2); preferably R6, R7, and R8 are each independently H or CH3;

[0017] mAb is an antibody, antibody fragment, monoclonal antibody, polyclonal antibody, nanobody, probody, or an antibody and antibody fragment modified with synthetic molecules or proteins;

[0018] L is a linker containing hydrophilic branches, whose main structure is C2-C 100 peptide units (1-12 natural or unnatural amino acids), hydrazone groups, disulfide groups, ester groups, oxime groups, amide groups or thioether groups.

[0019] In one aspect, in the conjugate of the application, L has the structure:

[0020]

[0021] wherein Aa is a L- or D- natural or unnatural amino acid;

[0022] r is an integer between 0 and 12; when r is not 0, (Aa) r is a peptide unit composed of the same or different amino acids;

[0023] m1 = an integer between 1 and 18; m2 = an integer between 1 and 100; m3 = an integer between 1 and 8; m4 = an integer between 0 and 8; m5 = an integer between 1 and 8;

[0024] Y is NHC(=0), NHS(02), NH(SO), NHS(02)NH, NHP(0)(OH)NH or C(0)NH;

[0025] R9 is H, (0=)CR1, (0=)CNHR1, R1COOH, R1(COCH2NH) m2 H, R1(Aa) r or R1(COCH2NCH3) m2 H, and

[0026] R1, m2 and (Aa) r as defined in claim 1 and in the above definitions.

[0027] In addition, the cell surface receptor binding molecule mAb can be any form of cell binding body, including peptides or peptide-like structures: antibodies, single chain antibodies, antibody fragments that can bind to target cells, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments that can bind to target cells, chimeric antibodies, chimeric antibody fragments that can bind to target cells, functional region antibodies, functional region antibody fragments that can bind to target cells, antibody-like genetically engineered proteins, fibronectin binders adnectins, pre-designed ankyrin repeat proteins (DARPin), lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules, transferrin, cell surface small molecule ligands, or albumin, high molecular weight, or dendrimer-linked cell binding bodies, high molecular weight materials, proteins, liposomes, nanoparticles, vesicles, or (viral) microcapsules containing cell binding molecules (binding peptides, proteins, antibodies, or cell surface small molecule ligands) on their surface.

[0028] Preparation of cell binding molecule-Tubulysin B derivative conjugates

[0029] In a particular embodiment, synthesis of the cell binding molecule-Tubulysin B derivative conjugate

[0030] comprising one or more of the following steps:

[0031]

[0032] wherein P in structural formula (II) 1 、 R1, R2, R3, R4, R5, R6, R7, and R8, and mAb are as described in structural formula (I);

[0033] L' has the structure (II-0) and (II-00):

[0034]

[0035] wherein m1, m2, m3, m4, m5, Aa, r, Y, and R9 are as described in structural formula (I).

[0036] A particular desirable structure for L' is:

[0037]

[0038]

[0039] wherein m1, m2, m3, m4, m5, Aa, r, and R9 are as described in any one of claims 1-2.

[0040] In another embodiment for preparing the conjugate as described above, the preparation method of the mAb-SH subjected to conjugation comprises any one of the following a) to c):

[0041] a). reducing the disulfide bond between the heavy and light chains, between the heavy chains, or between the chains of the antibody, antibody fragment, monoclonal antibody, polyclonal antibody, nanobody, probody, or antibody and antibody fragment modified with synthetic molecules or proteins by a reducing agent (preferably, tris (2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT), dithiothreitol (DTE), L-glutathione (GSH), 2-mercaptoethylamine (β-MEA), or / and β-mercaptoethanol (β-ME, 2-ME));

[0042] b). preparing a thiol by reacting a Traut reagent or a thiolactone with the amine of the antibody molecule:

[0043]

[0044] c). introducing a more easily reduced disulfide bond group on the antibody by a biochemical reaction under the condition of a buffer system, and then reducing by TCEP, DTT, GSH, β-MEA, β-ME:

[0045]

[0046] In another embodiment for preparing the conjugate as described above, the buffer system used in the synthesis of the conjugate is a buffer solution of phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (tris-hydroxymethyl aminomethane), benzoic acid or triethanolamine, or a mixture thereof, with a pH of 5.0 to 9.5 and a concentration of 1 mM to 1000 mM, and containing 0% to 35% of a water-soluble organic solvent: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA or DMSO, and the conjugation reaction temperature is controlled at 0°C to 45°C, and the conjugation reaction time is 5 minutes to 96 hours.

[0047] In another embodiment for preparing the conjugate as described above, after the completion of the conjugation reaction, the conjugate of formula (I) is obtained by purification using ultrafiltration or column chromatography. Commonly used column chromatography includes molecular sieve column, cation column, anion column, hydrophobic (HIC) column, reverse phase column, or protein A or G affinity column.

[0048] In another embodiment for preparing the conjugate as described above, the synthesis of the conjugate of formula (II) is obtained by condensation reaction of a Tubulysin B derivative of formula (III) and a compound of formula (L’):

[0049]

[0050] wherein X is OH, halogen (F, Cl, Br, or I), phenol, pentachlorophenol, trifluoromethylsulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3'-sulfonic acid, a self acid anhydride or an acid anhydride formed with other acid anhydrides such as acetic anhydride, formic anhydride; or a polypeptide condensation reaction intermediate or a Mitsunobu reaction intermediate;

[0051] wherein, the condensation reaction is completed in 5 minutes to 120 hours under the condition of temperature control at -20°C to 150°C in an organic solvent dichloromethane, dichloroethane, DMF, DMA, tetrahydrofuran (THF), DMSO, acetone, isopropanol, n-butanol or acetonitrile containing pyridine, triethylamine or diisopropylethylamine at a volume ratio of 1% to 100%, or a mixed solvent of two or more of the above solvents, or with or without inert gas (nitrogen, argon, helium) protection;

[0052] Alternatively, the condensation reaction is carried out in a buffer system of pH 5.0 to 9.5, 1 mM to 1000 mM of phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (tris-hydroxymethyl aminomethane), benzoic acid or triethanolamine, or a mixture thereof, and containing 0% to 35% of water-soluble organic solvent: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA or DMSO, and the coupling reaction temperature is controlled at 0°C to 45°C, and the coupling reaction is 5 minutes to 96 hours.

[0053] In addition, the NH2 group in structural formula (III) is desirably in the form of trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate, sulfonate for condensation reaction.

[0054] When X is OH, the above condensation reaction requires a condensation reagent. Commonly used condensation reagents include: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC or CME-CDI), carbonyl diimidazole (CDI), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate. Salts (BOP), benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (PyBOP), diethyl pyrocarbonate (DEPC), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, 2-(7-benzotriazolium oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 1-[(dimethylamine)(morpholinyl)methylene]-1[1,2,3]triazolo[4,5-b]1-pyridine-3-oxohexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), chlorotripyrrolylphosphonium hexafluorophosphate (PyCloP), bis(tetramethylene)fluoroformamide (BTFFH), N,N,N',N'-Tetramethyl-thio-(1-oxo-2-pyridyl)thiourea hexafluorophosphate, 2-(2-pyridone-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TPTU), thio-(1-oxo-2-pyridyl)-N,N,N',N'-tetramethylthiourea hexafluorophosphate, O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiourea hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholine-carbomony hexafluorophosphate (COMU), (benzotriazol-1-yloxy)dipyrrolidine Carbofluorophosphate (HBPyU), N-benzyl-N′-cyclohexylcarbodiimide (or loaded on a polymer), dipyrrolidinyl(N-succinyliminooxy)carbomonyhexafluorophosphate (HSPyU), 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinylhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy)piperidine carbofluorophosphate (HBPipU), 6-chlorobenzotriazolium-1,1,3,3-tetramethylurea tetrafluoroborate (TCTU), tris(dimethylamino)phosphine hexafluorophosphate bromide (BrOP), 1-n-propylphosphonic anhydride (PPACA), ), 2-isocyanatoethylmorpholine (MEI), N,N,N',N'-tetramethyluronium- oxy-(N-succinimidyl) hexafluorophosphate (HSTU), 2-bromo-l- ethylpyridinium tetrafluoroborate (BEP), oxy-[(ethoxycarbonyl) cyanomethylamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), 2-succinimidyl-l, 1, 3, 3-tetramethyluronium tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(3, 4-dihydro-4-oxo-l, 2, 3-benzotriazin-3- yl)uronium tetrafluoroborate (TDBTU), azodicarbonyl dipiperidine (ADD), bis(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD) or diethyl azodicarboxylate (DEAD).

[0055] In another embodiment, the process for preparing the conjugate described above is characterized in that the synthesis of the Tubulysin B derivative of structural formula (III) comprises one or more of the following steps:

[0056]

[0057]

[0058] wherein R5' is H, C1-C6alkyl, C1-C6alkyl, or C1-C6(linear or branched) aminoalkyl; and the other groups are as previously defined.

[0059] In another embodiment, the process for preparing the conjugate described above is characterized in that the synthesis of the Tubulysin B derivative of structural formula (III) comprises one or more of the following steps:

[0060] Step 1. Stiring diethoxyacetonitrile with aqueous ammonium sulfide at room temperature to obtain compound 1, 2,2-diethoxythioacetamide;

[0061]

[0062] Step 2. Condensing compound 1 with bromopyruvate in an anhydrous solvent (such as anhydrous tetrahydrofuran, dichloromethane, acetonitrile, N,N- dimethylformamide, methanol, isopropanol) by heating to obtain compound 2;

[0063]

[0064] Step 3. Compound 2 is dissolved in a solvent (such as tetrahydrofuran, dichloromethane, ethyl acetate, n-heptane, dioxane, acetonitrile) and hydrolyzed with a Lewis acid or a protonic acid (including hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, p-toluenesulfonic acid pyridine, AICI3, FeCI3, ZnCI2, BF3, BCI3, BBr3, TiCI4, ZnBr2, LiBF4) to give compound 3;

[0065]

[0066] Step 4. The unsaturated sulfonamide is dehydrogenated with a base such as n-butyllithium at low temperature (such as -45°C to -78°C), and then undergoes an addition reaction with compound 3 in the presence of a Lewis acid to give compound 4;

[0067]

[0068] The Lewis acid is selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, p-toluenesulfonic acid pyridine, AICI3, FeCI3, ZnCI2, BF3, BCI3, BBr3, TiCI4, ZnBr2, LiBF4;

[0069] Step 5. Compound 4 is selectively reduced with a reducing agent (such as NaBH4, LiBH4, Na(OAc)3BH, Na(CN)BH3, etc.) at low temperature (such as -45°C to -78°C), and its stereochemistry is controlled by adding a Lewis acid (such as Ti(Oet)4) to give compound 5;

[0070]

[0071] Step 6. Compound 5 is dissolved in a solvent (such as methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile) and is removed from the tert-butylsulfinyl group with an acid such as hydrochloric acid, sulfuric acid, and phosphoric acid to give compound 6;

[0072]

[0073] Step 7. Compound 6 and azotic acid are dissolved in a solvent (such as n-heptane, tetrahydrofuran, dichloromethane, N,N-dimethylformamide) in the presence of a condensing reagent (such as DIC / HOBt, DCC / HOBt, EDC / HOBt, HATU, BOP, T3P, BrOP) or by a condensation reaction route to undergo a condensation reaction to give compound 7;

[0074] Alternatively, azide acid is reacted with isobutyl chloroformate in the presence of an organic base (such as triethylamine, diisopropylethylamine, N-methylmorpholine, etc.) in THF to give a mixed anhydride, which is then condensed with the hydrochloride salt of compound 6 to give compound 7;

[0075] Alternatively, azide acid is reacted with oxalyl chloride, triethylamine and a catalytic amount of DMF in a solvent (such as n-heptane, n-hexane, dichloromethane, tetrahydrofuran) to convert it to an acid chloride, which is then condensed with the hydrochloride salt of compound 6 to give compound 7;

[0076]

[0077] Step 8. The hydroxyl group on compound 7 is reacted with a hydroxyl protecting reagent (such as TESCl) in the presence of an organic base (such as imidazole, triethylamine, pyridine) in a solvent (such as dichloromethane, tetrahydrofuran, acetonitrile) to give compound 8;

[0078]

[0079] Step 9. Compound 8 is dissolved in a solvent (such as tetrahydrofuran, dichloromethane, acetonitrile) and deprotonated with a base (such as KHMDS, LiHMDS, NaHMDS, KOtBu, NaH, KH) and then alkylated with iodomethane, bromomethane, dimethyl sulfate, methyl triflate or iodoethane, etc. to give compound 9;

[0080]

[0081] Step 10. Compound 9 is dissolved in a solvent (such as tetrahydrofuran, dichloromethane, ethyl acetate) and the azido group is reduced to an amino group under conditions such as in the presence of hydrogen gas and a palladium on carbon catalyst, triphenylphosphine and water (Staudinger reaction), and then condensed with an acid or a reactive acid derivative to give compound 10;

[0082]

[0083] Step 11. The hydroxyl protecting group PG1 on compound 10 is removed under appropriate conditions (such as the TES protecting group can be removed in the presence of hydrochloric acid, THF / MeOH / AcOH, nBu4NF or pyridine hydrofluoride in THF) to give compound 11;

[0084]

[0085] Step 12. The ester group in compound 11 is converted to the acid compound 12 under the action of a base (such as LiOH, NaOH, KOH), or other appropriate conditions (such as methyl ester can be converted to carboxylic acid under the action of reagents such as LiCl, LiI, Me3SiOK, etc.);

[0086]

[0087] Step 13. Compound 12 reacts with acid anhydride (such as acetic anhydride, propionic anhydride, isopropionic anhydride, etc.), acid halide (such as acetyl halide, propionic halide, propionic halide, formic halide, isomide halide, dimethylamide halide, etc.) in the presence of a base (such as triethylamine, N,N-diisopropylethylamine, pyridine) and a catalyst (such as DMAP) under certain temperature conditions (such as 0°C to 23°C) to obtain compound 13. The reaction can also be carried out without using a base or a catalyst;

[0088]

[0089] Step 14. Compound 13 condenses with an appropriate hydroxyl-containing compound such as pentafluorophenol or N-hydroxysuccinimide in the presence of a condensation reagent (such as EDC, DIC, DCC, HATU, HBTU) to obtain a reactive ester compound 14;

[0090]

[0091] Step 15. Compound 15 condenses with compound 14 in an aqueous phase under certain pH conditions (such as pH = 5.0-8.0), or in an organic phase in the presence of an organic base (such as TEA, DBU, DIPEA) or an inorganic base (such as Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain compound 16. The reaction can also be carried out without using any base, provided that the reaction temperature (such as 0°C to 23°C) and the reaction time (such as 30 minutes to 18 hours) are controlled;

[0092]

[0093] Step 16. The nitro group in compound 16 is reduced to an amino group under reducing conditions, such as in the presence of hydrogen and palladium-carbon catalyst, hydrazine hydrate and FeCl3, iron powder and acetic acid, etc., to obtain compound III;

[0094]

[0095] In another embodiment for preparing the conjugate described above, the synthesis of the compound of structural formula (L') comprises one or more of the following steps:

[0096]

[0097]

[0098] Under ideal conditions, the synthesis of a compound of formula (L') comprises one or more of the following steps:

[0099] Step 1. Compound 1-1 and compound 1-2 are condensed under the action of a condensing agent (such as EDC, HATU, DIC, DCC), or undergo condensation reaction through a condensation reaction route (such as compound 1-2 undergoes condensation reaction with pentafluorophenol, nitrophenol or N-hydroxysuccinimide under the action of condensing reagents such as DIC and EDC to produce the corresponding active ester, and then reacts with compound 1-1), to obtain compound 1a;

[0100] Alternatively, compound 1-3 and compound 1-4 are condensed under the action of a condensing agent (such as EDC, HATU, DIC, DCC), or undergo condensation reaction through a condensation reaction route, to obtain compound 1b;

[0101]

[0102] Step 2. The carboxyl protecting group PG2 in compound 1 is removed under the action of a deprotecting agent (such as t-butyl ester under the action of acid), to obtain compound 2;

[0103]

[0104] Step 3. Carboxyl-containing compound 2 and amino-containing compound 3 are condensed under the action of a condensing agent (such as EDC, HATU, DIC, DCC), or undergo condensation reaction through a condensation reaction route, to obtain compound 4;

[0105]

[0106] Step 4. The amino protecting group PG1 on compound 4 is removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions, to obtain compound 5;

[0107]

[0108] Step 5. Carboxyl-containing compound 6 and amino-containing compound 5 are condensed under the action of a condensing agent (such as EDC, HATU, DIC, DCC), or undergo condensation reaction through a condensation reaction route, to obtain compound 7;

[0109]

[0110] Step 6. The carboxyl protecting group PG3 on compound 7 is removed under deprotection conditions (such as t-butyl ester protecting group on carboxyl group can be cleaved under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.), to obtain compound 8;

[0111]

[0112] Step 7. Compound 8 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, DCC) to obtain an ester compound with reactivity, or reacts with other acid-activated groups to obtain a compound L' with condensation reactivity;

[0113]

[0114] 16. The conjugate of any one of claims 9-13, wherein the synthesis of the compound of formula (L') comprises one or more of the following steps:

[0115]

[0116] Ideally, the synthesis of the compound of formula (L') comprises one or more of the following steps:

[0117] Step 1. The amino protecting group PG1 on compound 1 is removed under deprotection conditions, such as the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be cleaved under acidic conditions, to obtain compound 2;

[0118]

[0119] Step 2. Amino-containing compound 2 and carboxyl-containing compound 3 are condensed under the action of a condensation reagent (such as EDC, HATU, DIC, DCC), or through a condensation reaction route, to obtain compound 4;

[0120]

[0121] Step 3. The carboxyl protecting group PG2 on compound 4 is removed under deprotection conditions, such as the t-butyl ester protecting group on the carboxyl group can be cleaved under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc., to obtain compound 5;

[0122]

[0123] Step 4. Carboxyl-containing compound 5 and amino-containing compound 6 are condensed in the presence of a condensing agent (e.g., EDC, HATU, DIC, DCC) or by a condensation reaction scheme to provide compound 7;

[0124]

[0125] Step 5. The protecting group PG3on the carboxyl group of compound 7 is removed under deprotection conditions, e.g., a t-butyl ester protecting group on a carboxyl group can be cleaved in the presence of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc., to provide compound 8;

[0126]

[0127] Step 6. Compound 8 is condensed with a hydroxyl-containing compound (e.g., pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent to provide a reactive ester compound or other acid-activated group to provide compound 9;

[0128]

[0129] In another embodiment of the process for preparing the conjugate described above, the synthesis of the compound of formula (II) is obtained by condensation of a compound of formula (IV) and a compound of formula (V):

[0130]

[0131] wherein X is as defined above and the condensation conditions are as described above;

[0132] The NH2group in the compound of formula (V) is preferably in the form of a trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate salt for condensation.

[0133] In another embodiment of the process for preparing the conjugate described above, the synthesis of the compound of formula (IV) comprises one or more of the following steps:

[0134]

[0135] In an ideal embodiment, the synthesis of the compound of formula (IV) comprises any of the following steps:

[0136] The carboxylic acid compound 1 is condensed with a hydroxyl-containing compound (e.g., pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent (e.g., EDC, DIC, DCC, HATU, HBTU) to provide a reactive ester;

[0137] Alternatively, carboxylic acid compound 1 is reacted with ethyl chloroformate, isobutyl chloroformate, or the like, in the presence of an organic base (such as N-methylmorpholine, triethylamine, diisopropylethylamine, or the like) to provide a reactive mixed anhydride;

[0138] Alternatively, carboxylic acid compound 1 is reacted with oxalyl chloride in the presence of an organic base (such as triethylamine) and a catalytic amount (such as 0.01 equivalents to 0.5 equivalents) of DMF to provide the acid chloride.

[0139] In another embodiment of the process for preparing the conjugate described above, the synthesis of compound 2 comprises one or more of the following steps:

[0140]

[0141] In another embodiment of the process for preparing the conjugate described above, the synthesis of compound 2 comprises one or more of the following steps:

[0142] Step 1. Compound 1 is condensed with compound 2 in an aqueous phase at a certain pH (such as pH = 5.0-8.0) or in an organic phase in the presence of an organic base (such as TEA, DBU, DIPEA) or an inorganic base (such as Na2CO3, Cs2CO3, K2CO3, NaHCO3) to provide compound 3; the reaction can also optionally be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled;

[0143]

[0144] Step 2. The amino protecting group PG4 on compound 3 is removed under deprotection conditions (such as the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be cleaved under acidic conditions) to provide compound V;

[0145]

[0146] In another embodiment of the process for preparing the conjugate described above, the synthesis of compound 2 comprises one or more of the following steps:

[0147]

[0148] wherein compound 8 (compound XIVa) obtained in the synthesis step is the target compound 2; PG4 is an amino protecting group.

[0149] In another embodiment of the process for preparing the conjugate described above, the synthesis of compound 2 comprises one or more of the following steps:

[0150] Step 1. Dissolve L-tyrosine ester derivative 1 in a suitable solvent, such as acetone, tetrahydrofuran, acetonitrile, dichloromethane, or a mixture of these solvents and water, and react it with benzyl chloride, benzyl bromide, or other benzyl compounds at 0 to 60°C. Suitable organic or inorganic bases, such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, DBU, sodium hydride, etc., can be added to the reaction system. Suitable additives, such as sodium iodide or phase transfer catalysts, such as benzyltriethylammonium chloride (TEBA), tetrabutylammonium bromide (TBAB), tetrabutylammonium chloride, tetrabutylammonium bisulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, etc., can also be added to the system to obtain compound 2.

[0151] Step 2. Dissolve compound 2 in organic solvents such as dichloromethane, tetrahydrofuran, methanol, ethanol, diethyl ether, etc., and reduce it with reducing agents such as lithium aluminum hydride, DIBAL, sodium borohydride, lithium borohydride, sodium dihydrobis(2-methoxyethoxy)aluminate (Red-Al), diborane, etc. Additives such as I2, ferric chloride, zinc chloride, magnesium chloride, lithium chloride, calcium chloride, etc. can be added to the reaction system to control the activity of the reducing agent to obtain compound 3;

[0152] Step 3. Alcohols 3 are oxidized to aldehydes 4 under appropriate oxidation conditions, such as Swern oxidation (oxalyl chloride, DMSO, triethylamine), Parikh-Doering oxidation (sulfur trioxide pyridine oxidation), Dess-Martin oxidation, etc.

[0153] Step 4. Aldehyde 4 reacts with a phosphate ester (Horner-Wadsworth-Emmons reaction) or a phosphorus ylide reaction (Wittig reaction) to elongate the carbon chain and give compound 5;

[0154] Step 5. The double bond in compound 5 is hydrogenated and reduced under the action of a homogeneous or two-phase catalyst, and the benzyl group is also removed at the same time to obtain a chiral compound with a single stereostructure, or a mixture of two diastereomers; the catalysts include two-phase catalysts such as Pd / C, Pd(OH)2 / C, Pd / BaSO4, PtO2, Pt / Al2O3, Ru / C, Raney nickel, etc., and homogeneous asymmetric hydrogenation catalysts such as Crabtree catalysts, [Ru(II)-(BINAP)] catalysts, [(Ph3P)CuH]6 catalysts, etc.

[0155] Step 6. Dissolve compound 6 in an organic solvent, such as tetrahydrofuran, acetonitrile, or dichloromethane, and nitrate it under nitration conditions. Nitrating agents include nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, tert-butyl nitrosamine, nitric acid / trifluoroacetic anhydride, NO2BF4, and nitropyridinium salts.

[0156] Step 7. The nitro group in compound 7 is reduced to amino group under the following conditions including H2 / Pd / C, Fe or Zn / HOAc, SnCl2 / HCl.

[0157] In another embodiment for preparing the conjugate described above, it is characterized in that the synthesis of compound 2 comprises one or more of the following steps:

[0158]

[0159] wherein the compound 8 (compound XIVb) obtained in the synthesis step is the target compound 2.

[0160] Ideally, the synthesis of compound 2 comprises one or more of the following steps:

[0161] Step 1. Aldol reaction of compound 1 with Evans chiral N-acyloxazolidinone or thiothione 2, wherein X = O or S, R 16 = H, methyl, phenyl, R 17 = H, methyl, isopropyl, phenyl, benzyl, etc., to obtain stereospecific compound 3;

[0162] Step 2. The hydroxyl group on compound 3 is removed under the following conditions including Barton-McCombie deoxygenation, i.e. the alcohol is first converted into a thioacyl derivative such as alkyl xanthate, phenyl thiochloroformate, thioimidazole ester, and then treated with Bu3SnH to undergo radical cleavage to obtain the dehydroxylated product; the conditions for radical cleavage include: n-Bu3SnH / AIBN, n-Bu3SnH / AIBN / n-BuOH / PMHS, (Bu4N)2S2O8 / HCO2Na;

[0163] Step 3. Compound 4 is dissolved in tetrahydrofuran, and the Evans chiral auxiliary is cleaved under the condition of LiOH / H2O2 to obtain the corresponding acid 5;

[0164] Step 4. Compound 5 is dissolved in an organic solvent such as ethyl acetate, methanol, dichloromethane, ethanol or acetic acid, and the benzyl group is catalytically hydrogenated in the presence of palladium-carbon catalyst to obtain compound 6;

[0165] Step 5. Compound 6 is dissolved in an organic solvent such as tetrahydrofuran, acetonitrile, dichloromethane, and is nitrated under nitration conditions, and the nitrating agent includes nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, nitroso tert-butyl ester, nitric acid / trifluoroacetic anhydride, NO2BF4, nitropyridinium salt, etc.

[0166] Step 6. The nitro group in compound 7 is reduced to an amino group under conditions including H2 / Pd / C, Fe or Zn / HOAc, SnCl2 / HCl, etc. to give chiral compound 8 with a single stereochemistry.

[0167] In another embodiment, the process for preparing the conjugate described above is characterized in that the synthesis of the structure (II) is obtained by condensation of the structure (VI) and the structure (VII):

[0168]

[0169] wherein X is as defined and the condensation conditions are as described in any one of claims 9-11.

[0170] In one embodiment, the synthesis of the structure (VI) comprises one or more of the following steps:

[0171]

[0172]

[0173] In an ideal embodiment, the synthesis of the structure (VI) comprises one or more of the following steps:

[0174] Step 1. Compound 1 is condensed with an appropriate hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent to give a derivative of the reactive acid compound 2;

[0175]

[0176] Step 2. Compound 2 is condensed with compound 3 in an aqueous phase at a certain pH (such as pH = 5.0-8.0) or in an organic phase in the presence of an organic base (such as TEA, DBU, DIPEA) or an inorganic base (such as Na2CO3, Cs2CO3, K2CO3, NaHCO3) to give compound 4; the reaction can also optionally be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled;

[0177]

[0178] Step 3. The amino protecting group PG4 on compound 4 is selectively removed under deprotection conditions (such as the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be cleaved under acidic conditions) to give compound 5;

[0179]

[0180] Step 4. Compound 5 is condensed with compound of formula (IV) in an aqueous phase at a certain pH (e.g. pH = 5.0-8.0) or in an organic phase in the presence of an organic base (e.g. TEA, DBU, DIPEA) or an inorganic base (e.g. Na2CO3, Cs2CO3, K2CO3, NaHCO3) to give compound 6; the reaction can also be optionally carried out without any base, but the reaction temperature and reaction time need to be controlled;

[0181]

[0182] Step 5. The amino protecting group PG1 on compound 6 is removed under deprotection conditions (e.g. the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium on carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions) to give compound VI;

[0183]

[0184] In another embodiment for preparing the conjugate described above, the synthesis of compound of formula (VII) comprises one or more of the following steps:

[0185]

[0186] In another embodiment for preparing the conjugate described above, the synthesis of compound of formula (VII) comprises one or more of the following steps:

[0187] Step 1. Carboxylic acid compound 1 is condensed with a hydroxyl-containing compound (e.g. pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent (e.g. EDC, HATU, DIC, DCC) to give a reactive ester;

[0188] Alternatively, carboxylic acid compound 1 is reacted with ethyl chloroformate, isobutyl chloroformate, etc. in the presence of an organic base (e.g. N-methylmorpholine, triethylamine, diisopropylethylamine, etc.) to give a reactive mixed anhydride;

[0189] Alternatively, carboxylic acid compound 1 is reacted with oxalyl chloride in the presence of an organic base (e.g. triethylamine) and a catalytic amount (e.g. 0.01 equivalent to 0.5 equivalent) of DMF to give an acid chloride;

[0190]

[0191] In another embodiment for preparing the conjugate described above, the synthesis of compound of formula (II) is obtained by condensation of compound of formula (VIII) and compound of formula (IX):

[0192]

[0193] wherein X is defined as above and the condensation reaction conditions are as described above. The NH2group in structure (VIII) is desirably in the form of a trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate salt for the condensation reaction.

[0194] In particular embodiments, the synthesis of structure (VIII) comprises one or more of the following steps:

[0195]

[0196]

[0197] In particular embodiments, the synthesis of structure (VIII) comprises one or more of the following steps:

[0198] Step 1. The carboxyl protecting group PG3on compound 1 is removed under deprotection conditions (such as the t-butyl ester protecting group on the carboxyl group can be cleaved off in the presence of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.) to give compound 2;

[0199]

[0200] Step 2. Compound 2 is condensed with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent (such as EDC, HATU, DIC, DCC) to give a reactive ester compound 3;

[0201]

[0202] Step 3. Compound 3 is condensed with compound 4 in an aqueous phase at a certain pH value (such as pH = 5.0-8.0) or in an organic phase in the presence of an organic base (such as TEA, DBU, DIPEA) or an inorganic base (such as Na2CO3, Cs2CO3, K2CO3, NaHCO3) to give compound 5; the reaction can also optionally be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled;

[0203]

[0204] Step 4. The amino protecting group PG3on compound 5 is removed under deprotection conditions (such as the Cbz protecting group on the amino group can be cleaved off in the presence of hydrogen gas and a palladium on carbon catalyst, and the Boc protecting group on the amino group can be cleaved off under acidic conditions) to give compound 6;

[0205]

[0206] Step 5. Compound 6 and structural formula (IV) undergo a condensation reaction in an aqueous phase under specific pH conditions (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to yield compound 7. Alternatively, the reaction may be carried out without any base, but appropriate reaction temperature and reaction time must be controlled.

[0207]

[0208] Step 6. The amino protecting group PG1 on compound 7 is removed under deprotection conditions. For example, the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium on carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound VIII.

[0209]

[0210] In another specific embodiment of the preparation of the above-described conjugate, the synthesis of structural formula (IX) is characterized by comprising one or more of the following steps:

[0211] Carboxylic acid compound 1 undergoes a condensation reaction with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing agent to give a reactive ester IX;

[0212] Alternatively, carboxylic acid compound 1 reacts with ethyl chloroformate, isobutyl chloroformate, etc., in the presence of organic bases (such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc.) to obtain a reactive mixed anhydride IX;

[0213] Alternatively, carboxylic acid compound 1 reacts with oxalyl chloride in the presence of an organic base such as triethylamine and a catalyst amount of DMF to give acyl chloride IX;

[0214]

[0215] In another specific embodiment of the preparation of the above-described coupling, the characteristic feature is that the synthesis of structural formula (II) is obtained by a condensation reaction of structural formula (X) and structural formula (XI):

[0216]

[0217] Where Y 1 and Y 2 Group condensation forms Y group; Y 1 and Y 2 They are NH2 and - +NH3, COOH, COX, SO2CI, P(O)CI2, NHCOX, NHSO2CI, NHP(O)CI2, NHP(O)(OH)CI,

[0218]

[0219] In another embodiment for preparing the conjugate described above, the synthesis of the compound of formula (X) comprises one or more of the following steps:

[0220]

[0221] In another embodiment for preparing the conjugate described above, the synthesis of the compound of formula (X) comprises one or more of the following steps:

[0222] Step 1. The carboxyl-containing compound 1 and the compound VI undergo condensation reaction under the action of condensing agent (such as EDC, HATU, DIC, DCC), or through condensation reaction route, to obtain compound 2; wherein Z 1 is the precursor of Y 1 , such as the appropriately protected amino group, carboxyl group, amide group, phosphoramide group and sulfonamide group, carboxylic acid ester, phosphate ester, phosphonate, etc.

[0223]

[0224] Step 2. The amino protecting group PG1 on compound 2 is removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions, to obtain compound 3;

[0225]

[0226] Step 3. The carboxyl-containing compound 4 and the amino-containing compound 3 undergo condensation reaction under the action of condensing agent, or through condensation reaction route, to obtain compound 5;

[0227]

[0228] Step 4. The functional group Z 1 in compound 5 is converted into the functional group Y 1 through appropriate chemical conversion, such as deprotection of carboxyl and amino groups, to obtain compound X;

[0229]

[0230] In another embodiment for preparing the conjugate described above, characterized in that the synthesis of structural formula (XI) comprises one or more of the following steps:

[0231]

[0232] In an ideal condition, the synthesis of structural formula (XI) comprises one or more of the following steps:

[0233] Step 1. Compound 1 is dissolved in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and deprotonated with a base such as sodium hydride, sodium, sodium hydroxide, etc., then stirred with compound 2 (wherein X is halogen such as chlorine, bromine, iodine or other leaving group) at a certain temperature, and the reaction gives compound 3;

[0234] Step 2. The carboxyl protecting group PG1 on compound 3 is removed under deprotection conditions, such as the t-butyl ester protecting group can be cleaved under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc., to give compound XIa-1;

[0235] Step 3. Compound 1 is dissolved in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and deprotonated with a base such as sodium hydride, sodium, sodium hydroxide, etc., then stirred with compound 4 at a certain temperature, and the reaction gives compound 5;

[0236] Step 4. The carboxyl protecting group PG1 on compound 5 is removed under deprotection conditions, such as the t-butyl ester protecting group can be cleaved under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc., to give compound XIa-2;

[0237] Step 5. Compound 6 is dissolved in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc., and an appropriate organic base such as triethylamine, N,N-diisopropylethylamine, pyridine, etc. is added, and methyl sulfonyl chloride, 4-toluenesulfonyl chloride, etc. is reacted at 0-5°C to give compound 7;

[0238] Step 6. Compound 7 is reacted with ammonia water in an aqueous phase or an organic solvent such as methanol, ethanol, acetonitrile, tetrahydrofuran, epoxide hexacyclic, etc., and the reaction can be heated appropriately to give compound XIb.

[0239] Step 7. Compound 7 is reacted with sodium azide in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc. to give compound 8;

[0240] Step 8. The azide compound 8 is hydrogenated in the presence of a palladium-carbon catalyst or reduced by the action of triphenylphosphine and water to give compound XIb;

[0241] Step 9. Compound 7 is reacted with dibenzylamine in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethylsulfoxide, and the like, preferably N,N-dimethylformamide, at 100 °C to yield compound 9;

[0242] Step 10. Compound 9 is dissolved in a solvent such as ethyl acetate, methanol, ethanol, acetic acid, tetrahydrofuran, and the like, and reduced over palladium on carbon catalyst under a hydrogen atmosphere, with optional heating to 45 °C, to yield compound XIb.

[0243] In another embodiment of the process for preparing the conjugates described above, the synthesis of Formula (II) is obtained by condensation of Formula (XII) and Formula (XIII):

[0244]

[0245]

[0246] wherein X is as defined above and the condensation conditions are as described above.

[0247] Desirably, the NH2group of Formula (XII) is in the form of a trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate salt for condensation.

[0248] In another embodiment of the process for preparing the conjugates described above, the synthesis of Formula (XII) comprises one or more of the following steps:

[0249]

[0250] Desirably, the synthesis of Formula (XII) comprises one or more of the following steps:

[0251] Step 1. Compound 1 is condensed with an appropriate hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent to yield a reactive acid derivative compound 2;

[0252]

[0253] Step 2. Compound 2 is condensed with compound 3 in an aqueous phase at a certain pH (e.g., pH = 5.0-8.0) or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to give compound 4; the reaction can also optionally be carried out without any base, but the appropriate reaction temperature and reaction time need to be controlled;

[0254]

[0255] Step 3. The amino protecting group PG4 on compound 4 is selectively removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium on charcoal catalyst, and the Boc protecting group on the amino group can be cleaved under acidic conditions) to give compound 5;

[0256]

[0257] Step 4. Compound 5 is condensed with structural formula (IV) (i.e., structural formula (IV) of any one of claims 18-21) in an aqueous phase at a certain pH (e.g., pH = 5.0-8.0) or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to give compound 6; the reaction can also optionally be carried out without any base, but the appropriate reaction temperature and reaction time need to be controlled;

[0258]

[0259] Step 5. The amino protecting group PG1 on compound 6 is removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium on charcoal catalyst, and the Boc protecting group on the amino group can be cleaved under acidic conditions) to give compound XII;

[0260]

[0261] In another specific embodiment for preparing the conjugate described above, the synthesis of structural formula (XIII) comprises one or more of the following steps:

[0262]

[0263] Preferably, the synthesis of structural formula (XIII) comprises one or more of the following steps:

[0264] Step 1. Carboxyl-containing compound 1 and amino-containing compound 2 are condensed in the presence of a condensing agent (such as EDC, HATU, DIC, DCC) or via a condensation reaction pathway to give compound 3;

[0265]

[0266] Step 2. The protecting group PG1on the carboxyl group of compound 3 is removed under deprotection conditions (such as the t-butyl ester protecting group on the carboxyl group can be cleaved in the presence of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.) to give compound 4;

[0267]

[0268] Step 3. Carboxylic acid compound 4 is condensed with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing reagent to give a reactive ester structure (XIII);

[0269] Alternatively, carboxylic acid compound 4 is reacted with ethyl chloroformate, isobutyl chloroformate, etc. in the presence of an organic base such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc. to give a reactive mixed anhydride structure (XIII);

[0270] Alternatively, carboxylic acid compound 4 is reacted with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount of DMF to give an acid chloride structure (XIII),

[0271]

[0272] Preferred structures of the compound of formula (II) are as follows:

[0273]

[0274]

[0275] In another embodiment, a pharmaceutical composition comprises a conjugate as described in any of the above embodiments or a conjugate formed by reacting a compound comprising a linker as described above with a cell binding molecule, and a pharmaceutically acceptable excipient. The conjugate of any of the above embodiments for use in the manufacture of a medicament for the treatment of cancer, infection or autoimmune disease. BRIEF DESCRIPTION OF DRAWINGS

[0276] Figure 1 shows the synthesis of Tubulysin derivative fragments 13 and 18.

[0277] Figure 2 shows the synthesis of Tubulysin derivative fragment 34.

[0278] Figure 3. Shows synthesis of Tubulysin derivative fragments 37, 38 and 45.

[0279] Figure 4. Shows synthesis of Tubulysin derivative fragment 57.

[0280] Figure 5. Shows synthesis of Tubulysin derivative fragment 71.

[0281] Figure 6. Shows synthesis of conjugatable Tubulysin derivative 72.

[0282] Figure 7. Shows in vivo anti-tumor activity of conjugates in NCI-N87 xenograft tumor bearing BALB / c nude mice.

[0283] Figure 8. Shows toxicity study of Her2 antibody conjugates of Tubulysin derivatives (compared to T-DM1)

[0284] Glossary of Related Terms:

[0285] Alkyl refers to a linear or cyclic straight chain or branched chain aliphatic hydrocarbon containing 1 to 8 carbon atoms. Branched refers to one or more lower alkyl groups, such as methyl, ethyl or propyl, attached to the linear alkyl group. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-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-methylhexyl, 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 iso-octyl. C1-C8alkyl can be unsubstituted or substituted with one or more groups including but not limited to C1-C8alkyl, C1-C8alkoxy, aryl, acyl, acyloxy, ester, -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen (-F, -Cl, -Br, -I), -N 3 2 , -NHR', -N(R')2and -CN; wherein R' refers to C1-C8alkyl or aryl.

[0286] ​C3-C8carbocycle refers to a saturated or unsaturated non-aromatic ring compound containing 3, 4, 5, 6, 7, or 8 carbon atoms. Typical C3-C8carbocycles 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. C3-C8carbocycles can be unsubstituted or substituted with one or more groups including, but not limited to, C1-C8alkyl, C1-C8alkoxy, aryl, acyl, acyloxy, ester, -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -S(O)2R', -S(O)R', -OH, halogen (-F, -Cl, -Br, -I), -N3, -NH2, -NHR', -N(R')2, and -CN; wherein R' is C1-C8alkyl or aryl.

[0287] C3-C8carbocyclyl refers to a group in which one hydrogen atom of the above C3-C8carbocycle is replaced by a bond.

[0288] Alkenyl refers to a straight or branched chain aliphatic hydrocarbon containing a carbon-carbon double bond and having from 2 to 8 carbon atoms. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl, and octenyl.

[0289] Alkynyl refers to a straight or branched chain aliphatic hydrocarbon containing a carbon-carbon triple bond and having from 2 to 8 carbon atoms. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, hexynyl, heptynyl, and octynyl.

[0290] Heteroalkyl refers to an alkyl group having from 2 to 8 carbon atoms wherein one to four of the carbon atoms are independently replaced with O, S, or N.

[0291] Aryl or aromatic group refers to an aromatic or heteroaromatic group having one or more rings composed of from 3 to 14 carbon atoms (most often 6 to 10 carbon atoms). Heteroaromatic group refers to an aromatic group having one or more carbon atoms (most often 1, 2, 3, or 4 carbon atoms) replaced with O, N, Si, Se, P, or S (most often O, S, or N). Aryl or aromatic group also refers to an aromatic group having one or more hydrogen atoms replaced with R 13 , F, Cl, Br, I, OR 13 , SR 13 , NR 13 , R 14 , N=NR 13 , N=R13 NR 13 R 14 NO2, SOR 13 R 14 SO2R 13 SO3R 13 OSO3R 13 PR 13 R 14 POR 13 R 14 PO2R 13 R 14 OPO3R 13 R 14 or PO3R 13 R 14 wherein R 13 and R 14 are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl, or a pharmaceutically acceptable salt.

[0292] Halogen atom means a fluorine, chlorine, bromine, or iodine atom, preferably fluorine and chlorine.

[0293] Heterocycle means an aromatic, non-aromatic ring or a heterocyclic ring having 2 to 8 carbon atoms, and 1 to 4 carbon atoms of the ring are replaced by heteroatoms. The heteroatoms are O, N, S, Se, and P, preferably O, N, and S. Useful heterocycles can also be found in The Handbook of Chemistry and Physics, 78th edition, CRC Press, 1997-1998, pages 225-226. Suitable non-heteroaryl groups include, but are not limited to, epoxide, epinitrane, episulfide, pyrrolidine, pyrazolidine, imidazolidine, oxirane, tetrahydrofuran, dioxolane, tetrahydropyran, dioxane, piperidine, piperazine, morpholine, pyran, imidazoline, pyrrolizine, pyrazoline, thiazolidine, tetrahydrothiopyran, dithiane, thiomorpholine, dihydropyran, tetrahydropyran, tetrahydropyridine, dihydropyridine, tetrahydropyrimidine, dihydrothiopyran, azepane, and fused rings thereof with phenyl.

[0294] Heteroaryl means an aromatic heteromonocyclic, bicyclic, or polycyclic ring structure having 3 to 14 (preferably 5 to 10) atoms. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thiophenyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolinyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, N-oxopyridyl, and fused rings thereof with phenyl.

[0295] The terms alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl also refer to the corresponding alkylidene, cycloalkylidene, alkenylidene, alkynylidene, arylidene, heteroarylidenes and heterocyclylidenes formed by the loss of two hydrogen atoms from the corresponding hydrocarbon.

[0296] "Arylalkyl" refers to a non-cyclic alkyl radical having one hydrogen atom removed from an alkyl radical, typically a terminal or sp3hybridized carbon atom, which is replaced with an aryl radical. Typical arylalkyl groups include benzyl, 2-phenylethyl, 2-phenylethenyl, naphthylmethyl, 2-naphthylethyl, 2-naphthylethenyl, naphthylbenzyl, 2-naphthylphenylethyl, and the like.

[0297] "Heteroarylalkyl" refers to a non-cyclic alkyl radical having one hydrogen atom removed from an alkyl radical, typically a terminal or sp3hybridized carbon atom, which is replaced with a heteroaryl radical. Heteroarylalkyl groups are represented by 2-benzimidazolylmethyl, 2-furylethyl, and the like.

[0298] "Hydroxyl protecting group" refers to methyl oxymethyl ether (MOM), 2-methyloxymethyloxymethyl ether (2-MOEOM), tetrahydropyranyl ether, benzyl ether, p-methyloxylbenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, ethyl acetate, substituted ethyl acetate, benzoate, benzyl formate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate, adamantane acetate, mesitoate, methylsulfonate, and tosylate.

[0299] The amino acids can be natural and / or unnatural amino acids, typically in the L or D form, and preferably alpha amino acids. Natural amino acids are those encoded by the genetic code, which 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 derivations of the proteinogenic amino acids. For example, hydroxyproline, lanthionine; 2-aminobutyric acid, dehydroalanine, gamma-aminobutyric acid (a neurotransmitter), ornithine, citrulline, beta-alanine (3-aminopropanoic acid), gamma-carboxyglutamate, selenocysteine (now in many non-eukaryotes as well as most eukaryotes, but not directly encoded by DNA), pyrrolysine (found only in some archaea and bacteria), N-formylmethionine (which is the first amino acid of bacterial, mitochondrial and chloroplast proteins), 5-hydroxytryptophan, L-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimetics. An analog has the same structure of H2N(R)CHCO2H of a natural amino acid, but the R group is not found in a natural amino acid. For example, analogs include seryl, hexanoic acid, methionine sulfoxide, and methionine methylsulfonium salts. Preferably, an analog amino acid has a structure different from the conventional chemical structure of an alpha amino acid, but functions similarly. The term "unnatural amino acid" is in the "D" stereoform, as opposed to the "L" stereoform of a natural amino acid. When 1-12 amino acids are used in the present application, the amino acid sequence is preferably recognized and cleaved by a protease.Many recognition and cleavage amino acid sequences are known (e.g., 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 contain citations to other references), and, in particular, the amino acid sequences selected from the group consisting of Val-Cit, Ala-Val, Ala-Lys, Gly-Lys, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Asp-Lys, Glu-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.

[0300] A "peptide" is a compound formed by the linkage of two or more amino acids through the amino group of one amino acid and the carboxyl group of another amino acid by a peptide bond (i.e., an amide bond). A compound in which two amino acids are linked by a peptide bond is referred to as a dipeptide; a compound in which three amino acids are linked by peptide bonds is referred to as a tripeptide, and so on, up to a compound in which thirty amino acids are linked by peptide bonds is referred to as a thirty-peptide. A peptide formed entirely of natural alpha amino acids is a natural peptide (natural protein). A peptide containing one or more unnatural amino acids or amino acid analogs is an unnatural peptide (peptidomimetic compound). A peptide of two or more amino acids is a peptide unit.

[0301] A "glycoside" is a molecule in which a sugar group is linked to another group through its anomeric carbon by a glycosidic bond. Glycosides can be linked by O-(O-glycoside), N-(glycosylamine), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds. Its core is the empirical formula C m (H2O) n(wherein m can be different from n, m and n are < 36), the glycosides herein include glucose (dextrose), fructose (levulose), allose, altrose, mannose, gulose, idose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfonoquinolone (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbose, mannose, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide), and stachyose. The glycosides can be in the D form or the L form, in the 5-atom cyclic furanose form, the 6-atom cyclic pyranose form, or the acyclic form, in the alpha-anomer (the -OH of the anomeric carbon below the plane of the Haworth projection) or the beta-anomer (the -OH of the anomeric carbon above the plane of the Haworth projection). The glycosides of most common use herein are monosaccharides, disaccharides, polyols, or oligosaccharides (containing 3-6 saccharide units).

[0302] "Antibody", as used herein, is used in the broadest sense of the term, specifically encompassing intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and antibody fragments), and antigen binding fragments of antibodies which retain the ability to specifically bind to an antigen. The native form of an antibody is a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chains are linked together via disulfide bonds. In each pair, the light and heavy chains are also linked to each other by disulfide bonds. The variable regions of the light and heavy chains (VL and VH) are responsible for binding to an antigen. The variable regions of the light and heavy chains are composed of a framework region interrupted by three high variable regions, also known as "complementarity determining regions" or "CDRs". The CDR regions are the regions most likely to be recognized as foreign by the immune system. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antibody can be obtained from any suitable species. In certain aspects, the antibody is of human or murine origin. The antibody can be human, humanized, or chimeric.

[0303] The terms "specific binding" and "bind specifically" mean that the antibody or antibody derivative will bind to its corresponding target antigen in a highly selective manner, and not to many other antigens. Typically, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -7 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -8 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -9 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -10 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -11 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10 -12 M. Preferably, the antibody or antibody derivative will bind to its target antigen with a dissociation constant (Kd) of less than about 1 x 10

[0304] "Pharmaceutically acceptable" or "pharmacologically acceptable" means that the corresponding compound or composition of compounds is not biologically or otherwise undesirable, i.e., the compound or composition of compounds is capable of being used in a pharmaceutical composition administered to an animal or human without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0305] Pharmaceutically acceptable excipients include all carriers, diluents, adjuvants or formers such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like. In the medical field, it is a very common practice to add such excipients to the active pharmaceutical ingredients. It can be said that the addition of excipients to the pharmaceutical ingredients is not unreasonable unless the excipients are not compatible with the active ingredients. To achieve good results, active excipients can also be added to the pharmaceutical ingredients.

[0306] In the present application, the pharmaceutically acceptable salts refer to the salt derivatives of the compounds of the present application. The compounds of the present application can form corresponding acid or base salts by appropriate modification. The pharmaceutically acceptable salts include common non-toxic salts or quaternary ammonium salts which can be formed from the compounds of the present application and corresponding non-toxic inorganic or organic acids. For example, the pharmaceutically acceptable salts can be formed from inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and organic acids including 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 and lactic acid. Other salts include aminobutanol, aminotriethanol, meglumine, pyrrolidinoethanol and the like ammonium salts and metal salts such as sodium, potassium, calcium, zinc, magnesium and the like.

[0307] The pharmaceutically acceptable salts of the present application can be prepared by conventional chemical methods. In general, such salts can be prepared by the addition of an appropriate acid or base to the free acid or base of the compounds of the present application in a water or organic solvent or a mixture thereof. Non-aqueous solvents are generally ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of applicable salts can be found in Remington's Pharmaceutical Sciences, 17th Ed. Mack Publishing Company, Easton, PA, 1985, p. 1418.

[0308] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a Ligand Drug Conjugate or Linker Drug Conjugate. The Conjugate can contain at least one amino group and thus can form an acid addition salt with the amino group. Nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts can include additional molecules such as acetate, succinate, or other counter ions. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Embodiments of pharmaceutically acceptable salts that are part of a multiple charged atom can have multiple counter ions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ions.

[0309] The phrase "pharmaceutically acceptable solvate" or "solvate" refers to an association of one or more solvent molecules and a Ligand Drug Conjugate or Linker Drug Conjugate. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, butanol, t-butanol, acetone, glycerol, DMSO, ethyl acetate, formic acid, acetic acid, triethanolamine, and ethanolamine.

[0310] Hydrate refers to a compound containing water. The water can be associated with other moieties, such as a metal ion hydration ligand to form a complex, or it can be covalently bound, such as in the hydrate trichloroacetaldehyde. It can also refer to a crystal or liquid molecule formed by some compounds with water under certain temperature and pressure conditions. The water in a hydrate is present in a defined amount, for example, the hydrate of anhydrous sodium sulfate, Na2S04, has the composition Na2S04-10H20. The water in a hydrate can be associated in several different ways: one is as a ligand, coordinated to a metal ion, called a coordination water of crystallization; another is associated with an anion, called an anion water of crystallization. Water can also be present in a crystal in a certain proportion without being directly associated with a cation or anion, occupying a certain position in the crystal lattice. This form of water is called lattice water, which generally contains 12 water molecules. Some crystalline compounds also contain water, but without a certain proportion. The salt of a hydrate refers to a pharmaceutically acceptable salt formed on the basis of this hydrate.

[0311] Optical isomers, also known as enantiomers, are molecules that cannot be superimposed on their stereoisomer mirror image. A substance containing one chiral carbon atom has two optical isomers, which are related to each other as real objects and mirror images. Enantiomers have equal optical power but opposite directions of rotation, and their physical and chemical properties can be similar. A molecule containing two identical chiral carbon atoms has three optical isomers. When a molecule contains several different chiral atoms, the number of optical isomers is 2n, where n is the number of different chiral atoms. n When two substances of equal amounts of optical isomers are mixed uniformly, the optical properties cancel each other out, forming a racemate.

[0312] Examples of "patients" or "subjects" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. In exemplary embodiments, the patient or subject is a human.

[0313] "Administering" refers to any means of transferring, delivering, introducing or conveying a drug or other agent to a subject, including oral, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, or intrathecal administration. The use of devices or instruments to administer the agent is also contemplated, such devices can utilize active or passive transport, and can be slow-release or rapid-release delivery devices.

[0314] The following abbreviations can be used herein, and have the indicated definitions: Boc, tert-butyloxycarbonyl; BroP, bromotripyrrolidinophosphonium hexafluorophosphate; CDI, 1,1'- carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, dichloroethane; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethyl cyanophosphonate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high pressure liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine. DETAILED DESCRIPTION

[0315] Specific embodiments of the present application will now be described in greater detail below. While the application is susceptible to various modifications and alternative forms, specific embodiments of the application have been shown by way of example in the drawings and will be described in detail below. It should be understood that the application is not intended to be limited to the particular embodiments described herein, but on the contrary, is intended to cover all modifications and alternatives falling within the scope of the application.

[0316] The disclosed subject matter can take form in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope thereof to those skilled in the art. Accordingly, the disclosed subject matter is not limited to the specific embodiments described herein, but can be practiced with modifications and changes within the scope of the subject matter.

[0317] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs.

[0318] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0319] The term "a" does not denote a limitation of the number of items to one, but rather denotes that at least one of an item is present. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it were individually recited herein. The endpoints of all ranges are included in the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of "example" or "exemplary" is intended to present examples or exemplary embodiments, and is not intended to be limiting unless otherwise indicated.

[0320] This invention includes compounds of formula (I) having at least one desired atomic isotopic substitution in an amount exceeding the natural abundance (i.e., enrichment) of the isotope, and the use of the compounds. An isotope is an atom having the same atomic number but different mass numbers, i.e., the same number of protons but different numbers of neutrons. Isotopic substitution, such as deuterium substitution, can be partial or complete. Partial deuterium substitution refers to at least one hydrogen atom being substituted with deuterium. In some embodiments, the isotope is enriched at 90%, 95%, or 99% or more at any site of interest. In one embodiment, deuterium is enriched at 90%, 95%, or 99% at the desired site.

[0321] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0322] The term "C1-C6" refers to groups containing 1 to 6 carbon atoms.

[0323] The term "linker containing hydrophilic branches" indicates that the main structure is C2-C. 100 It is composed of peptide units (1 to 12 natural or non-natural amino acids), hydrazone groups, disulfide groups, ester groups, oxime groups, amide groups, or thioether groups.

[0324] The term "pharmaceutically acceptable salt" means a salt of a compound suitable for use in pharmaceutical preparations. When the compound has one or more basic groups, the salt can be an acid addition salt, such as sulfates, hydrobroms, tartrates, methanesulfonates, maleates, citrates, phosphates, acetates, pyrates, hydroiodates, nitrates, hydrochlorides, lactates, methyl sulfates, fumarates, benzoates, succinates, methanesulfonates, lactobionates, octanoates, toluenesulfonates, etc. When the compound has one or more acidic groups, the salt can be a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, aminobutanetriol salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, etc. Polycrystalline forms and solvates are also included within the scope of this invention.

[0325] The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by adding to the free acid or base, as appropriate, the stoichiometrically equivalent amount of the desired salt former. Non-aqueous media are employed which can be ethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts are found in "Remington's Pharmaceutical Sciences", 17th Ed. Mack Publishing Company, Easton, PA, 1985, p. 1418.

[0326] Pharmaceutically acceptable excipients include all carriers, diluents, adjuvants or formers such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. In the medical field, it is very common practice to add such excipients to the active pharmaceutical ingredients. It can be said that the addition of excipients to the pharmaceutical ingredients is not without reason, unless the excipients are not compatible with the pharmaceutical active components. To achieve good results, active auxiliary components can also be added to the pharmaceutical ingredients.

[0327] In this context, the chiral carbon atom site in formula (I) is selected to be pure R, pure S or a mixture of R / S in different proportions.

[0328] Unless specifically indicated otherwise, all stereoisomers of the compounds described herein, including but not limited to, other enantiomers, diastereomers or geometric isomers, as well as mixtures of stereoisomers, are included in the scope of the present application. Individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof, are included within the scope of the present application unless otherwise specified.

[0329] It will be appreciated by those skilled in the art that compounds can have tautomeric forms (e.g., keto and enol forms), resonance forms, and zwitterionic forms, which are equivalent to those depicted in the structural formulae used herein, and that the structural formulae include such tautomeric, resonance or zwitterionic forms.

[0330] Another aspect of the present application is the production of antibodies. This includes the in vivo, in vitro production or combination thereof. The production of polyclonal antibodies to a peptide segment of a receptor is well known in the art, as exemplified by U.S. Patent No. 4,493,795 (Nestor et al.). The classic method for producing monoclonal antibodies is to immunize a mouse with the antigen of interest, and to fuse the resulting mouse spleen cells with myeloma cells (Kohler, G; Milstein, C. 1975. Nature 256:495-497). Detailed procedures are described in antibodies - A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988), which is incorporated herein by reference. In particular, a specific monoclonal antibody can be obtained by immunizing a mouse, rat, hamster or other mammal with the antigen of interest. The antigen of interest includes whole cells, antigens isolated from cells, whole viruses, attenuated whole viruses and viral proteins. The spleen cells are fused with myeloma cells using PEG 6000. The resulting hybridomas are screened for their sensitivity to HAT. The monoclonal antibodies produced by the hybridoma cells are useful in the practice of the present application by virtue of their immunoreactivity with the specific target cell receptor or their ability to inhibit receptor activity.

[0331] The resulting monoclonal hybridoma cells secrete monoclonal antibodies to the specific antigen of interest. The monoclonal antibodies used in the practice of the present application are produced by growing the monoclonal hybridoma cells in a nutrient medium under conditions permitting the production and secretion of the antibodies into the medium. The antibody-containing medium is then collected and the antibodies are purified by well-known techniques. The purification methods include protein A affinity chromatography; anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography and gel filtration chromatography (particularly the use of antigen- coupled protein A affinity chromatography and gel filtration chromatography are widely used); centrifugation; sedimentation or other standard purification methods

[0332] The appropriate culture medium and artificial culture medium for the hybridoma cells can be obtained commercially or synthesized using known techniques. A typical artificial culture medium is DMEM (Dulbecco et al. Virol 8:396 (1959)) supplemented with 4.5 mg / L glucose, 20 mM glutamine, 20% fetal bovine serum and an antifoaming agent such as polyoxyethylene polyoxypropylene copolymer.

[0333] In addition to cell fusion techniques, cell lines producing antibodies can be constructed by other methods, such as by transfecting B lymphocytes with oncogenic DNA or by introducing oncogenic viral genes (e.g., EBV, also known as HHV-4 or KSHV) into B lymphocytes. See, e.g., U.S. Pat. Nos. 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 against receptor polypeptides or polypeptide analogs containing carboxy-terminal sequences. See, e.g., Niman et al. Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983); Geysen et al. Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Lei et al. Biochemistry 34(20):6675-6688 (1995). Typically, the receptor polypeptides or polypeptide analogs are used as immunogens, either alone or conjugated to an immunogenic carrier, to prepare monoclonal antibodies against the receptor polypeptides.

[0334] There are other well-established methods for producing antibodies as binding molecules in the invention. Of particular interest is the production of fully human antibodies. Phage display technology is used to obtain fully human antibodies that specifically bind to a known antigen from a library of fully human antibodies by affinity selection. Phage display technology, vector construction and library screening are well documented in the literature. See, e.g., Dente et al. Gene. 148(1):7-13 (1994); Little et al. Biotechnol Adv. 12(3):539-55 (1994); Clackson et al. Nature 352:264-628 (1991); Huse et al. Science 246:1275-1281 (1989).

[0335] Monoclonal antibodies obtained from other species (e.g., mouse) using hybridoma technology need to be humanized. The modified antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Among them, the common method of humanization of antibodies is the transplantation and remodeling of complementarity determining regions. See: US Patent Nos. 5859205 and 6797492; Liu et al., Immunol Rev. 222:9-27 (2008); Almagro et al., Front Biosci. 1; 13: 1619-33 (2008); Lazar et al. Mol Immunol. 44(8): 1986-98 (2007); Li et al. Proc. Natl. Acad. Sci. USA. 103(10):3557-62 (2006) incorporated herein by reference. Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys and other mammals carrying a large number of human immunoglobulin light chains and heavy chains with antigens. Take mice as an example: Xenomouse (Abgenix, Inc.), HuMab-Mouse (Medarex / BMS), VelociMouse (Regeneron), see: US Patent Nos: 6596541, 6207418, 6150584, 6111166, 6075181, 5922545, 5661016, 5545806, 5436149 and 5569825. In human therapy, the immunogenicity of chimeric antibodies constructed by integrating mouse variable region genes with human constant region genes in the human body is much lower than that of mouse antibodies (Kipriyanov et al., Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002) incorporated herein by reference. In addition, site-directed mutagenesis of some sites in the variable region of the antibody can effectively improve the affinity and specificity of the antibody (Brannigan et al., Nat Rev Mol Cell Biol. 3:964-70 (2002); Adams et al., J Immunol Methods. 231:249-60 (1999)). Partial replacement of the constant region of the antibody can also effectively improve its affinity to immune effector factors and enhance cytotoxicity.

[0336] Antibodies with immune specificity to malignant cell antigens can be obtained by commercial channels or some mature technical methods, such as chemical synthesis or recombinant expression technology. The genes encoding such antibodies can also be obtained by some commercial channels, such as GenBank database or other similar databases, published literature, or conventional cloning and sequencing methods.

[0337] In addition to antibodies, polypeptides or proteins can also be used as binding molecules to bind, block, attack or otherwise interact with the corresponding receptors or epitopes on the surface of target cells. As long as these polypeptides or proteins can specifically bind to a particular epitope or its corresponding receptor, they do not necessarily have to belong to the immunoglobulin family. These polypeptides can also be isolated by techniques similar to those used for phage display of antibodies (Szardenings, J Recept Signal Transduct Res. 2003; 23(4):307-49). Peptides obtained from random polypeptide libraries can be used similarly to antibodies and antibody fragments. The specificity of the antigen binding of the polypeptide or protein molecules can be maintained by linking them to some macromolecules or agents through their binding molecules. These macromolecules and agents include albumin, polymers, liposomes, nanoparticles or dendrimers.

[0338] In the treatment of cancer, autoimmune diseases and infectious diseases, antibodies for conjugation are, for example, the following (but not limited to): 3F8 (anti-GD2 antibody), Abagovomab (anti-CA-125 antibody), Abciximab (anti-CD41 antibody (integrin alpha-IIB), Adalimumab (anti-TNF-alpha antibody), Ado-trastuzumab (anti-EpCAM antibody, CD326), Afelimab (anti-TNF-alpha); Afutuzumab (anti-CD20 antibody), Alacizumab pegol (anti-VEGFR2 antibody), ALD518 (anti-IL-6 antibody), Alemtuzumab (alias: Campath, MabCampath, Campath, anti-CD52 antibody), Altumomab (anti-CEA antibody), Anatumomab (anti-TAG-72 antibody), Anrukinzumab (alias: IMA-638, anti-IL-13 antibody), Apolizumab (anti-HLA-DR antibody), Arcitumomab (anti-CEA antibody), Aselizumab (anti-L-selectin (CD62L) antibody, Atlizumab (alias: Tocilizumab, Actemra, RoActemra, anti-IL-6 receptor antibody), Atorolimumab (anti-rhesus factor antibody), Bapineuzumab (anti-beta-amyloid antibody), Basiliximab (Simulect, anti-CD25 (alpha chain of the IL-2 receptor) antibody, Batumix (anti-phosphatidylserine antibody), Bebtuximab (alias: LymphoScan, anti-CD22 antibody), Belimumab (alias: Benlysta, LymphoStat-B, anti-BAFF antibody), Benralizumab (anti-CD125 antibody), Beractant (anti-CCL11 (eotaxin-1) antibody), Berumask (alias: Scintimun, anti-CEA-related antigen antibody), Bevacizumab (alias: Avastin, anti-VEGF-A antibody) Biciromab (alias: FibriScint, anti-fibrin II beta chain antibody), Bivatuzumab (anti-CD44v6 antibody), Blinatumomab (alias: BiTE, anti-CD19 antibody), Brentuximab (CAC10, anti-CD30 TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), Canakinumab (alias: Ilaris, anti-IL-1 antibody), Cantuzumab (alias: C242, anti-CanAg antibody), Caplacizumab, Catumaxomab (alias: removab, anti-EpCAM, anti-CD3 antibody), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD4 antibody), Cimzia (alias: Cimzia anti-TNF-alpha antibody),Cetuximab (aka Erbitux, IMC-C225, anti-EGFR antibody), Cetuximab (anti-EpCAM antibody), Cixutumumab (anti-IGF-1 antibody), Clenoliximab (anti-CD4 antibody), Clivatuzumab (anti-MUC1 antibody), Conatumumab (anti-TRAIL-R2 antibody), CR6261 (anti-influenza A hemagglutinin antibody), Dacetuzumab (anti-CD40 antibody), Daclizumab (aka Zenapax, anti-CD25 (alpha chain of IL-2 receptor) antibody), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase) antibody), Denosumab (aka Prolia, anti-RANKL antibody), Denosumab (anti-B-lymphoma cell antibody), Atorolimumab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside antibody), Eculizumab (aka Soliris, anti-C5 antibody), Ebertuximab (anti-endotoxin antibody), Edrecolomab (aka Panorex, MAb 17-1A, anti-EpCAM antibody), Efalizumab (aka Raptiva, anti-LFA-1 (CD11a) antibody), Efungumab (aka Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody), Ertliumab (anti-IL-6 antibody), Enlimomab (anti-ICAM-1 (CD54) antibody), Epitumomab (anti-episialin antibody), Epratuzumab (anti-CD22 antibody), Erlizumab (anti-ITGB2 (CD18) antibody), Ertumaxomab (aka Rexomun, anti-HER2 / neu, CD3 antibody), Etarneptat (aka Abegrin, anti-integrin αvβ3 antibody), Ibalizumab (anti-hepatitis B surface antigen antibody), Fanolesomab (aka NeutroSpec, anti-CD15 antibody), Faralimomab (anti-interferon receptor antibody), Farletuzumab (anti-folate receptor 1 antibody), Felvizumab (anti-respiratory syncytial virus antibody), Fezakinumab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor antibody), Fontolizumab (anti-IFN-γ antibody), Fendriove (anti-rabies glycoprotein antibody), Fresolimumab (anti-TGF-β antibody), Galiximab (anti-CD80 antibody), Gantenerumab (anti-β-amyloid antibody), Gavilimomab (anti-CD147 (basigin) antibody), Gemtuzumab (anti-CD33 antibody), Girentuximab (anti-carbonic anhydrase 9 antibody),Glembatumumab (aka: CR011, anti-GPNMB antibody), Golimumab (aka: SIMPONI, anti-TNF-alpha antibody), Gomiliximab (anti-CD23 (IgE receptor) antibody), Ibalizumab (anti-CD4 antibody), Ibritumomab (anti-CD20 antibody), Igovomab (aka: Indimacis-125, anti-CA-125 antibody), Ineximab (aka: Myoscint, anti-cardiac myosin antibody), Infliximab (aka: Remicade, anti-TNF-alpha antibody), Intetumumab (anti-CD51 antibody), Inolimomab (anti-CD25 (IL-2 receptor alpha chain) antibody), Inotuzumab (anti-CD22 antibody), Ipilimumab (anti-CD152 antibody), Iratumumab (anti-CD30 (TNFRSF8) antibody), Keliximab (anti-CD4 antibody), Labetuzumab (aka: CEA-Cide, anti-CEA antibody), Lebrikizumab (anti-IL-13 antibody), Lemelizumab (anti-NCA-90 (granulocyte antigen) antibody), Lecatumumab (anti-TGF beta-2 antibody), Lexatumumab (anti-TRAIL-R2 antibody), Libilomo (anti-hepatitis B surface antigen antibody), Linratuzumab (anti-CD33 antibody), Lucatumumab (anti-CD40 antibody), Lumiliximab (anti-CD23 (IgE receptor) antibody), Mapatumumab (anti-TRAIL-R1 antibody), Maslimomab (anti-T-cell receptor antibody), Matuzumab (anti-EGFR antibody), Mepolizumab (aka: Bosatria, anti-IL-5 antibody), Metelimumab (anti-TGF beta-1 antibody), Milatuzumab (anti-CD74 antibody), Minretumomab (anti-TAG-72 antibody), Muclotuzumab (aka BEC-2, anti-GD3 ganglioside antibody), Morolimumab (anti-rhesus factor antibody), Motavizumab (aka: NUMAX, anti-respiratory syncytial virus antibody), Muromonab-CD3 (aka: OKT3 ORTHOCLONE, anti-CD3 antibody), Nacolomab (anti-C242 antibody), Narnatumab (anti-5T4 antibody), Natalizumab (aka: Tysabri, anti-integrin alpha 4 antibody), Nebacumab (anti-endotoxin antibody), Necitumumab (anti-EGFR antibody), Nerelimomab (anti-TNF-alpha antibody), Nimotuzumab (aka: Theracim, Theraloc, anti-EGFR antibody), Nofetumomab, Ocrelizumab (anti-CD20 antibody),Odalasvir (anti-HCV antibody), Odomzo (anti-CD20 antibody), Olaratumab (anti-PDGF-Ra antibody), Omalizumab (anti-IgE Fc region antibody), Oportuzumab (anti-EpCAM antibody), Oregovomab (anti-CA-125 antibody), Otelixizumab (anti-CD3 antibody), Pagibaximab (anti-lipoteichoic acid antibody), Palivizumab (anti-respiratory syncytial virus antibody), Panitumumab (anti-EGFR antibody), Panobacumab (anti-Pseudomonas aeruginosa antibody), Pascolizumab (anti-IL-4 antibody), Pemtumomab (anti-MUC1 antibody), Pertuzumab (anti-HER2 / neu antibody), Pexelizumab (anti-C5 antibody), Pintumomab (anti-adenocarcinoma antigen antibody), Priliximab (anti-CD4 antibody), Protumumab (anti-vimentin antibody), PRO 140 (anti-CCR5 antibody), Racotumomab (anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-ganglioside GM3) antibody), Ravulizumab (anti-canine distemper glycoprotein antibody), Ramucirumab (anti-VEGFR2 antibody), Ranibizumab (anti-VEGF-A antibody), Resibaciulin (anti-anthrax toxin, protective antigen antibody), Regavirumab (anti-cytomegalovirus glycoprotein B antibody), Reslizumab (anti-IL-5 antibody), Rilotumumab (anti-HGF antibody), Rituximab (anti-CD20 antibody), Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-IFN-a antibody), Rovelizumab (anti-CD11, CD18 antibody), Ruplizumab (anti-CD154 (CD40L) antibody), Siltuximab (anti-IL-6 antibody), Siplizumab (anti-CD2 antibody), (Smart) MI95 (anti-CD33 antibody), Solanezumab (anti-beta-amyloid antibody),Sonepcizumab (anti-sphingosine-1 -phosphate antibody), Solitomab (anti-episialin antibody), Stamulumab (anti-myostatin antibody), Sulfuramod (aka: LeukoScan, (anti-NCA-90 (granulocyte antigen) antibody))), Tacatuzumab (anti-alpha-fetoprotein antibody), Tedezolid (anti-integrin alphaIIb beta3 antibody), Talizumab (anti-IgE antibody), Tanezumab (anti-NGF antibody), Taplitumomab (anti-CD19 antibody), Tefibazumab (aka: Aurexis, anti-agrin antibody), Atinumab, Tenatumomab (anti-fibroblast growth protein C antibody), Tenocticumab (anti-CD40 antibody), Teplizumab (anti-CD3 antibody), TGN1412 (anti-CD28 antibody), Ticilimumab (aka: Tremelimumab (anti-CTLA-4 antibody), Tigatuzumab (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody), Tocilizumab (aka: Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor antibody), Toralizumab (anti-CD154 (CD40L) antibody), Tositumomab (anti-CD20 antibody), Trastuzumab (Herceptin, (anti-HER2 / neu protein antibody), Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab celmoleukin (anti-EpCAM antibody), Tovorimab (anti-B hepatitis virus antibody), Urtoxazumab (anti- E. coli antibody), Ustekinumab (aka: Stelara, anti-IL-12, IL-23 antibody), Vapaliximab (anti-AOC3 (VAP-1) antibody), Vedolizumab, (anti-integrin alpha4 beta7 antibody), Veltuzumab (anti-CD20 antibody), Vepalimomab (anti-AOC3 (VAP-1) antibody, Visilizumab (aka: Nuvion, anti-CD3 antibody), Vitaxin (anti-vascular integrin avb3 antibody), Volociximab (anti-integrin alpha5 beta1), Volocithumab (aka: HumaSPECT, anti-tumor antigen CTAA16.88 antibody), Zolimumab (aka: HUMAX-EGFR, (anti-EGFR antibody), Zanolimumab (aka: HUMAX-CD4, anti-CD4 antibody), Ziralimumab (anti-CD147 (basal immunoglobulin) antibody), Almoztumab (anti-CD5 antibody), Etanercept, Aflibercept Abatilcept Linacidep (ARCALYST), 14F7 [anti-IRP-2 (Iron Regulatory Protein 2) antibody], 14G2a (anti-GD2 ganglioside antibody, treats melanoma and solid tumors, from nat. cancer inst.), J591 (anti-PSMA antibody, treats prostate cancer, Weill Medical College), 225.28S [anti-HMW-MAA (high molecular weight melanoma-associated antigen) antibody, Soling Radiofarmaci SRL (Milan, Italy) treats melanoma], COL-1 (anti-CEACAM3 antibody, CGM1, nat. cancer inst. Treats colorectal and gastric cancer), CYT-356 Lym-1 (anti-HLA-DR10 antibody, Biogen Idee, for cancer), MAK-195F [anti-TNF antibody (also known as: tumor necrosis factor; TNFA, tumor necrosis factor-alpha; TNFSF2), Abbott / Northern, for treating septic shock in sepsis], MEDI-500 [alias: T10B9, anti-CD3 antibody, TR alpha / beta (T cell receptor alpha / beta), complex, MedImmune, for treating graft versus host disease], RING SCAN [anti-TAG72 (tumor-associated glycoprotein 72 antibody), Neoprobe Group, for treating breast, colon, and rectal cancer. Avicidin (anti-EpCAM antibody (epithelial cell adhesion molecule), anti-TACSTD1 antibody (tumor-associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2 antibody (epithelial glycoprotein 2); anti-KSA antibody; KS1 / 4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx, for treating colon cancer, ovarian cancer, prostate cancer, and non-Hodgkin's lymphoma; LymphoCide (IMMUNOMEDICS, NJ), smart ID10 (Protein Design Labs), Oncolym (Techniclone, CA), Allomune (BioTransplant, CA), anti-VEGF antibody (Genentech, CA); CEAcide (IMMUNOMEDICS, NJ), IMC-1C11 (ImClone, NJ), and cetuximab (ImClone, New Jersey).

[0339] Other antibodies for binding antigens include (but are not limited to): aminopeptidase N (CD13), Annexin Al, B7-H3 (CD276, various cancers), CA125, CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA242, placental alkaline phosphatase (cancer), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, lymphoma of non-Hodgkin's lymphoma, multiple myeloma), epsilon of CD3 (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30, CD33, CD37, CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia), CD51 (metastatic melanoma, sarcoma), CD52, CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumors, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD79,CD80 (lymphoma), CD98 (cancer), Mucin (cancer), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colon cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer, and lung cancer), DLL4 (Delta-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hematopoietic tumors, solid tumors), Endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, prostate non-Hodgkin's lymphoma, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2; lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (a cell surface antigen glyvolipid, melanoma), Idiotypic GD3 (cancer), Heat shock proteins (cancer), HER1 (lung, gastric cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (non-Hodgkin's lymphoma, B-cell leukemia), Human chorionic gonadotropin (cancer), IGF1R (insulin-like growth factor 1 receptor, solid tumors, hematological cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman disease, IL6-dependent tumors), Integrins (integrin ανβ3, α5β1, α6β4, αllβ3, α5β5, ανβ5 cell adhesion factors, various cancers), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE4 (cancer), Anti-transferrin receptor (cancer), P97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (large intestine), GP100 (melanoma), MART1 (melanoma) MPG (melanoma), MS4A1 (membrane-spanning 4 protein A, small cell lung cancer, non-Hodgkin's lymphoma), Nucleolin, Neuro-oncological gene product (cancer),P21 (cancer), anti (N-glycolylneuraminic acid, breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG 72 (tumor-associated glycoprotein 72, leukemia, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, non-Hodgkin's lymphoma, and other cancers, rheumatoid arthritis and systemic lupus erythematosus), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1, lymphoma, non-Hodgkin's lymphoma, colon cancer, lung cancer), VCAM-1 (CD106, melanoma), vascular endothelial growth factor, vascular endothelial growth factor-A, VEGF-2 (CD309) (various cancers). Some other tumor-associated antigens recognized by antibodies have been reviewed (Gerber et al., mAbs 1 :3, 247-253 (2009); Novellino et al., cancer immunol immunother. 54(3), 187-207 (2005) Franke et al., cancer biother radiopharm. 2000, 15, 459-76). There are many other antigens: other different clusters (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, CDw17, CD18, CD19, 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, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E,CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85g, CD85i, CD85j, CD85k, CD85m, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, 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, 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, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD281, CD282, CD283, CD284, CD285, 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, CD307e, CD307f, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, 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, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC,5T4), APO2, ASLG659, BMPR1B (bone morphogenetic protein receptor), CRIPTO, Annexin A1, Nucleostemin, Endoglin (CD105), ROBO4, Aminopeptidase N, Delta-like 3 (DLL3), Delta-like 4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, Individual genotype, MAGE A3, P53 nonmutant, NY-ESO-1, GD2, CEA, MelanA / MART1, Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34, member 2, Type II sodium-dependent phosphate transporter 3b), Ras mutations, gp100, p53 mutant, Proteinase3 (PR1), BCR-abl, Tetratocarcinoma-derived growth factor), EphA receptor, EphB receptor, EGFR, EGFRvIII, ETBR (endothelin), HER2 / neu, HER3, HLA-DOB (MHC class II molecule IA antigen), Integrin, IRTA2, MPF (MPF, MSLN, SMR, megakaryocyte-potentiating factor, mesothelin), CRIPTO, Sema 5b (FLJ10372, KIAA1445, Mm42015, SEMA5B, 5EMAG, semaphoring 5 bHlog, sdema domain, seven transmembrane epithelial prostatic antigen, and STEAP2 (HGNC 8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate), tyrosinase, survivin, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSMA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Trop2,Page 4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, Fos protein-related antigen 1.

[0340] The conjugates of the present application are useful for the treatment of cancer. These cancers include, but are not limited to, adrenocortical carcinoma, rectal cancer, bladder cancer, brain tumors (adult: brain stem glioma, childhood, cerebellar astrocytoma, astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, pineal, visual pathway and hypothalamic glioma), breast cancer, carcinoid tumor, gastrointestinal, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family tumor (PNET), extracranial malignant germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumor, gonadal, 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 cavity cancer, liver cancer, lung cancer (non-small cell, small cell, lymphoma (AIDS-related, central nervous system, cutaneous T-cell, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, primary occult squamous cell carcinoma of the neck, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial cell, germ cell tumor, low malignant potential tumor), pancreatic cancer (exocrine, islet cell carcinoma), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary tumor, plasma cell neoplasm, prostate cancer rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sezary syndrome, skin cancer, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small bowel cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), unusual tumors of childhood, vaginal cancer, vulvar cancer, Wilms' tumor.

[0341] The conjugate of the present application is suitable for the prevention and treatment of autoimmune diseases. The autoimmune diseases include, but are not limited to, achlorhydria autoimmune chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic encephalomyelitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-glomerular basement membrane / anti-tubular basement membrane nephritis, antiphospholipid syndrome, anti-synthetase 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 neuropathies, autoimmune pancreatitis autoimmune polyendocrinopathies type I, II, III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo disease / Balo concentric sclerosis, Behcet's syndrome, Berger's disease, Bickerstaff's brainstem encephalitis, Blau's syndrome, bullous pemphigoid, Castleman's disease, Chagas' disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic relapsing Lyme disease, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, coeliac disease, cochleai vestibular syndrome, cold agglutinin disease, complement C2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease (an idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Darier's disease, dermatomyositis, diabetes mellitus type 1, diffuse cutaneous scleroderma, Dressier's syndrome, discoid lupus erythematosus, eczema, endometriosis, enthesitis, eosinophilic fasciitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evan's syndrome, fibrosing alveolitis, fibromyalgia, fibromyositis, fibrosing mediastinitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hidradenitis suppurativa, Hughes' syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating disease, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease, lupoid hepatitis, lupus erythematosus, Maghedi's syndrome, Meniere's disease, microscopic polyangiitis,Miller Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neurogenic muscle stiffness, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, panda syndrome (autoimmune neuropsychiatric disorder of childhood complicated by streptococcal infection), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, progressive hemifacial atrophy, Behcet's syndrome, pars planitis, pemphigus, pemphigus vulgaris, pernicious anemia, periarteritis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthropathies, sticky blood syndrome, Still's disease, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, acute febrile neutrophilic dermatosis, Sydenham chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), thalidomide-induced painful ophthalmoplegia syndrome, transverse myelitis, ulcerative colitis (an idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondylarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, Wiskott-Aldrich syndrome.

[0342] In another embodiment, antigen binding molecules for conjugation for the treatment or prevention of autoimmune diseases 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-double stranded DNA antibodies; anti-single stranded DNA antibodies, anti-cardiolipin antibodies IgM, IgG; anti-celiac antibodies; anti-phospholipid antibodies IgK, IgG; anti-ribosomal protein antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies, T-cell antibodies; thyroid globulin antibodies, anti-scleroderma-70 antibodies (anti-SCL-70); human anti-Jo antibodies (anti-jo); anti-self antibodies of systemic lupus erythematosus patients; anti-Sjogren's syndrome antibodies (Anti-La / SSB); anti-systemic lupus erythematosus antibodies; anti-parietal cell antibodies; anti-histone antibodies; anti-ribonucleoprotein antibodies (anti-RNP); cytoplasmic antibodies of neutrophils (C-ANCA); perinuclear antibodies of neutrophils (P-ANCA); anti-centromere antibodies; anti-nuclear fibrin antibodies, and anti-glomerular basement membrane antibodies (GBM) antibodies, anti-ganglioside antibodies; anti-Desmogein 3 antibodies; anti-human P62 antibodies; anti-human sp 100 antibodies; anti-mitochondrial M2 antibodies; rheumatoid factor antibodies; anti-mutant citrullinated vimentin antibodies (anti-MCV); anti-topoisomerase antibodies; anti-neutrophil cytoplasmic (CANCA) antibodies.

[0343] In certain preferred embodiments, the binding molecules for conjugation in the present application can bind to receptors or receptor complexes expressed by activated lymphocytes associated with autoimmune diseases. These include members of the immunoglobulin gene superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD70, CD79, CD79b, CD90, CD123, CD125, CD138, CD152 / CTLA-4, PD-1, or ICOS), members of the TNF receptor superfamily (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, Trop2 and 30 APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (C-type, S-type or I-type) or complement regulatory proteins.

[0344] In another embodiment, useful binders that are immunospecific for viral or bacterial antigens are human or human monoclonal antibodies. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide segment, polypeptide protein that elicits an immune response (e.g., HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, human T lymphotropic virus infection modulator tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen). As used herein, the term "bacterial antigen" includes, but is not limited to, any microbial peptide segment, polypeptide protein, saccharide, polysaccharide, lipid molecule that elicits an immune response (e.g., bacterial, fungal, pathogenic protozoan, yeast polypeptides including, for example, lipopolysaccharide and capsular polysaccharide 5 / 8). Useful type I antibodies for treating viral or bacterial infections include, but are not limited to, palivizumab, a humanized anti-respiratory syncytial virus monoclonal antibody for treating RSV infection; PRO 542, a CD4 fusion antibody for treating HIV infection; Ostavir, a humanized antibody for treating hepatitis B virus; PROTVIR, a humanized antibody IgG.sub.1 for treating cytomegalovirus, and anti-lipopolysaccharide (anti-LPS) antibodies.

[0345] The conjugates of the present application 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 immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis 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, Candida infection (candidiasis, thrush), Cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Chlamydia pneumoniae infection, Cholera, Chromomycosis, Clonorchiasis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever, Common cold (acute viral rhinopharyngitis; acute coryza), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothrium infection, Dracunculosis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (fifth disease), Exanthem subitum, Fascioliasis, Familial fatal insomnia, Filariasis, Food poisoning by Clostridium perfringens, Non-parasitic amoebic infection, Fusobacterium infection, Gas gangrene (clostridial myonecrosis), Geotrichosis, Gistman-Strauss syndrome, Giardiasis, Glanders, Glossopharyngeal candidiasis, Gonorrhea, Granuloma inguinale, 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 renal syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ehrlichiosis, Human granulocytic anaplasmosis, Human metapneumovirus infection, Human monocytotropic ehrlichiosis, Human papillomavirus infection, Human parainfluenza virus infection, Hymenolepidosis, Epstein-Barr virus infectious mononucleosis (single), Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionnaires' disease (Veterans' Association disease), Legionnaires' disease (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Legionnaires' disease (Veterans' Association disease), Li's disease, Lyme disease (Lyme borreliosis),Postparum schistosomiasis, microsporidiosis, molluscum contagiosum, mumps, spotted fever (endemic typhus), mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis (ophthalmia neonatorum), Creutzfeldt-Jakob syndrome (vCJD, nvCJD), nocardiosis, onchocerciasis (river blindness), paracoccidiomycosis (South American blastomycosis), paragonimiasis, pasteurellosis, pediculosis capitis (head lice), pediculosis corporis (body lice), pediculosis pubis (pubic lice), pelvic inflammatory disease, pertussis, plague, pneumococcal infection, pneumocystis pneumonia, pneumonia, poliomyelitis, prion disease, primary amoebic meningoencephalitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat-bite fever, respiratory syncytial virus infection, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsia, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, shigellosis, shingles (herpes zoster), smallpox (variola), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, nematode, syphilis, taeniasis, tetanus (lockjaw), tinea capitis, tinea corporis, tinea cruris, tinea manuum, tinea nigra, tinea pedis, tinea unguium, tinea versicolor, toxocariasis (ocular larva migrans), toxocariasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomoniasis, trichuriasis (whipworm infection), tuberculosis, tularemia, ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white piedra (white piedra), pseudotuberculosis infection, yersiniosis, yellow fever, zygomycosis.

[0346] The binding molecules of the previously described antibodies of this patent can be used against pathogenic strains including, but not limited to: Acinetobacter baumannii, Actinomyces israelii, Actinomyces georgiae and Actinomyces naeslundii, Brucia brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma, Bacillus anthracis, Helcococcus catus, Junin virus, Ascaris, Aspergillus, Astrovirus family, Babesia, Bacillus cereus, Bacteroides, Bacteroides, Balantidium coli, Baylisascaris, BK virus, Boides, Blastocystis hominis, Blastomyces dermatitidis, Arenavirus, Borrelia, Clostridium botulinum, Cinnamomum burmanni, Brucella, Burkholderia cenocepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter, Candida albicans and other Candida, Bartonella, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Phialophora pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immitis and Coccidioides posadasii, Colorado tick fever virus, Rhinovirus, Coronavirus, Prion Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium, Ancylostoma ceylanicum; multiple parasites, Cyclospora, Taenia solium, Cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) arbovirus, Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium latum, Dracunculus medinensis, Ebola virus, Echinococcus, Ehrlichia, Enterobius vermicularis, Enterococcus, Enterovirus, Rickettsia prowazekii, Parvovirus B19, Human herpesvirus 6 and Human herpesvirus 7, Clonorchis sinensis, Fasciola hepatica and Fasciola magna, FFI prion, Filariodea superfamily, Neisseria gonorrhoeae, Fusobacterium, Clostridium perfringens, other Clostridium, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma and Angiostrongylus cantonensis, Neisseria gonorrhoeae, Mycobacterium granulomatis, Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, Enterovirus, most Coxsackie A viruses and Enterovirus 71, Sindbis virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma, Ancylostoma duodenale and Necator americanus, Haemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasmataceae, Human metapneumovirus, Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenza virus, Haplorchis pumilus and Nanophyetus salmincola, Epstein-Barr virus, Orthomyxoviridae, Balantidium coli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenas, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila,Leishmania, Mycobacterium leprae and M. lepromatosis, Leptospira, Listeria, Borrelia burgdorferi and other Borrelia species, Brugia malayi and B. timori, Lymphocytic choriomeningitis virus (LCMV), Plasmodium, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitidis, Metagonimus yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia rickettsii, Mycoplasma pneumoniae, multiple bacteria (Madura foot mycosis) and fungi (Madura foot mycosis), parasitic dipteran fly larvae, Chlamydia trachomatis and gonococcus, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Blastomyces brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella, Pediculosis capitis, Pediculosis corporis, Pediculosis pubis, Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis carinii, Poliovirus, Prevotella, Naegleria fowleri, JC virus, Chlamydophila psittaci, Rickettsia, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory syncytial virus, Cibicides siberi, Rhinovirus, Rickettsia, Rickettsia akari, Rift Valley fever virus, Rickettsia conorii, Rotavirus, Rubella virus, Salmonella, SARS coronavirus, Sarcoptes scabiei, Schistosoma, Shigella, Varicella-zoster virus, Variola major or smallpox, Sporothrix schenckii, Staphylococcus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia, Clostridium tetani, Trichophyton, Trichophyton tonsurans, Trichophyton, Trichophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, Exophiala werneckii, Trichophyton, Malassezia, Toxocara canis or cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, White piedra, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales (Mucormycosis) and Entomophthorales (Entomophthoromycosis), Pseudomonas aeruginosa, Campylobacter fetus (Vibrio), Aeromonas hydrophila, Edwardsiella tarda, Yersinia, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Spirochaeta yaws, Treponema carateneum, Borrelia afzelii, Borrelia burgdorferi, Leptospira icterohaemorrhagiae, Pneumocystis carinii, Brucella, Brucella suis, Brucella melitensis, Mycoplasma, Rickettsia tsutsugumushi, Clostridium tetani; pathogenic fungi (Aspergillus, Candida albicans, Histoplasma capsulatum); protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum,Malaria (Plasmodium falciparum) ; or helminths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms),

[0347] Other antibodies as binding ligands of the present application for the treatment of viral diseases include, but are not limited to: antibodies acting on antigens of pathogenic viruses including the following examples but not limited to: Variola virus, Herpes virus, Adenovirus, Papovaviridae, Enterovirus, Picornaviridae, Parvoviridae, Reovirus, Retroviridae, Influenza virus, Parainfluenza virus, Mumps, Measles, Respiratory syncytial virus, Rubella, Arbovirus, Rhabdovirus, Arenaviridae, Non-A / Non-B Hepatitis virus, Rhinovirus, Coronavirus, Rotavirus, Oncovirus [e.g., Hepatitis B virus (hepatocellular carcinoma), Human papillomavirus (cervical cancer, anal cancer), Kaposi sarcoma-associated herpesvirus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (simian virus 40), Hepatitis C virus (liver cancer), Human T-lymphotropic virus type 1 (adult T-cell leukemia / lymphoma), Immune disorder-causing viruses: [e.g., Human immunodeficiency virus (AIDS)]; Central nervous system viruses: [e.g., JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), Arboviral encephalitis, Orthomyxoviridae (possible) (sleeping sickness), RV (rabies), Vesicular stomatitis-India virus, Rhabdoviral meningitis, Ramsay Hunt syndrome type II; Poliomyelitis (infantile paralysis, post-polio syndrome), Human T-lymphotropic virus type 1 (tropical spastic paraparesis)]; Cytomegalovirus (cytomegalic retinitis, HSV (herpetic keratitis)); Cardiovascular disease viruses [e.g., Coxsackievirus (pericarditis, myocarditis)]; Respiratory system / acute nasopharyngitis viruses / viral pneumonia: [African lymphocytic virus (herpes virus 4 infection / mononucleosis), Cytomegalovirus; SARS coronavirus (severe acute respiratory syndrome) Orthomyxovirus: Influenza virus A / B / C (influenza / avian influenza), Paramyxovirus: Human parainfluenza virus (parainfluenza), Respiratory syncytial virus (human respiratory syncytial virus), Lung virus]; Digestive system viruses [e.g., MUV (mumps), Cytomegalovirus (cytomegalovirus esophagitis); Adenovirus (adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus; HBV (hepatitis B virus), Coxsackievirus, Hepatitis A (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus)]; Genitourinary system viruses [e.g., BK virus, MUV (mumps)].

[0348] In accordance with still further objects, the present application also includes the conjugate components described above in combination with other possible pharmaceutical carriers for use as therapeutic agents for cancer and autoimmune diseases. The methods of the present application for treating cancer and autoimmune diseases include in vitro, in vivo or ex vivo therapy. Examples of in vitro therapy include the treatment of cells in culture with the agents to kill all cells except those that do not express the target antigen; or to kill cells that express an unwanted antigen. An example of ex vivo therapy is the treatment of hematopoietic stem cells in vitro to kill diseased or malignant cells and then returning the cells to the original patient. For example, cancer and autoimmune diseases can be treated clinically by ex vivo therapy to remove tumor cells or lymphocytes from the bone marrow of a patient and then returning the cells to the original patient, or to remove T cells and other lymphocytes from the bone marrow prior to transplantation to prevent immune rejection of the transplant. The method is carried out as follows: bone marrow cells are obtained from a patient or other individual and then incubated in serum-containing medium at 37°C with the conjugate of the present application at a concentration ranging from 1 pM to 0.1 mM for a period of time ranging from about 30 minutes to about 48 hours. The specific concentration and incubation time are determined by the skilled clinician. After incubation, the bone marrow cells are washed in serum-containing medium and returned to the patient by intravenous injection. If the patient requires other treatment, such as ablative chemotherapy or total body irradiation prior to obtaining the bone marrow cells and returning the treated cells, the treated bone marrow cells can be stored in a qualified liquid nitrogen medical facility.

[0349] For in vivo clinical use, the conjugates of the present application will be provided in solution or in lyophilized solid form that can be dissolved in sterile water prior to injection. Suitable methods of administration of the conjugates include, for example, intravenous injection of the conjugate once a week for a period of 4 to 12 weeks. The single dose is dissolved in 50 to 500 ml of normal saline, which can be supplemented with human serum albumin (e.g., 0.5 to 1 ml of 100 mg / ml concentrated human serum albumin). The dose of the agent is approximately 50 μg to 20 mg per kilogram of body weight per week, injected intravenously (10 ug to 200 mg / kg body weight per injection). After the period of 4 to 12 weeks of treatment, the patient can receive another round of treatment. The details of the method of treatment, including the route of administration, the excipient, the diluent, the dose of the agent, the period of treatment, etc., can be determined by the skilled surgeon.

[0350] Examples of diseases that can be treated by selective killing of a population of cells in vivo or ex vivo include any type of malignancy, autoimmune disease, graft rejection and infection (including viral, bacterial or parasitic).

[0351] The amount of conjugate required to achieve the desired biological effect will vary depending on a number of factors, including the nature of the compound, its therapeutic efficacy and bioavailability of the conjugate, the type of disease, the race of the patient, the state of the patient's health, the route of administration, all of which determine the appropriate dosage schedule and mode of administration.

[0352] In general, the conjugates of the present application can be administered in a physiologically acceptable buffer solution for parenteral administration at a concentration ranging from 0.1 to 10% mass / volume. Typical pharmaceutical dosage ranges from 1 ug to 0.1 g per kilogram of body weight per day; recommended pharmaceutical dosage ranges from 0.01 mg to 20 mg per kilogram of body weight per day or an equivalent dosage for children. The recommended dosage depends on a number of variables, including the type of disease or functional disorder, the overall health status of the patient, the relative biological efficacy of the conjugate drug, the formulation of the compound, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic properties under the selected mode of administration, and the rate of administration (single injection or continuous infusion) and the schedule of administration (number of doses over time).

[0353] The conjugates of the present application can also be administered in unit dosage form, where "unit dosage form" refers to a single dose of the drug for a single patient. Unit dosage forms can be packaged and used simply and conveniently, and are the active conjugate itself, maintained in a physically and chemically stable form, or in a pharmaceutically acceptable mixture as described below. Typical daily dosage ranges from 0.01 to 100 mg per kilogram of body weight. Generally, the unit dosage for humans ranges from 1 to 3000 mg per day. The recommended unit dosage is 1 mg to 500 mg, administered four times per day, or 10 mg to 500 mg, administered once per day. The conjugates of the present application can be formulated into pharmaceutical preparations by the addition of one or more pharmaceutically acceptable auxiliaries. The unit dosage form can be used for oral administration, such as tablets, simple capsules or soft capsules; or for intranasal administration, such as powders, nasal drops, or sprays; or for transdermal administration, such as ointments, creams, lotions, gels or sprays, or skin patches. The pharmaceutical preparation can be conveniently administered in unit dosage form and prepared by any of the methods known in the art, such as those described in Remington: The Science and Practice of Pharmacy, 21th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005.

[0354] Pharmaceutical dosage forms comprising the compounds of the present application include pharmaceutical compositions which are preferably administered orally or parenterally. For oral administration, dosage forms such as tablets, powders, capsules, troches and the like can contain one or more of the following ingredients: binders such as microcrystalline cellulose, gum tragacanth; diluents such as starch, lactose; dispersing agents such as starch and cellulose derivatives; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; flavoring agents such as peppermint, or methyl salicylate. Capsules can take the form of hard or soft capsules, generally comprising gelatin mixtures optionally with plasticizers, and starch capsules. Additionally, the physical form of the unit dosage form can be modified by the addition of various excipients, for example, sugar coatings, shellac or enteric coatings. Other oral dosage forms such as syrups or elixirs can contain sweetening agents, preservatives, colorants, flavorants and the like. Additionally, the active compounds can be processed through various treatments and formulations to make them into fast dissolving dosage forms, slow release dosage forms or sustained release dosage forms, with the sustained release dosage forms being the preferred dosage forms. Tablets preferably comprise dosage forms comprising combinations of lactose, corn starch, magnesium stearate, sodium croscarmellose, polyvinylpyrrolidone, magnesium stearate, talc and the like.

[0355] Liquid parenteral compositions include sterile aqueous and nonaqueous solutions, suspensions and emulsions. The liquid pharmaceutical compositions can also contain excipients such as buffers, preservatives, chelating agents, adjuvants and the like. Non-aqueous solvents include ethanol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic esters such as ethyl oleate. Aqueous solvents include water, mixtures of water and ethanol, buffers and salts, particularly biocompatible, biodegradable polymers of lactide, lactide / glycolide copolymers or polyethylene / polypropylene copolymers can be used as excipients to control the release of the active drug. Excipients in intravenous injections can include liquids and nutritional supplements, electrolyte supplements, and Ringer's dextrose based excipients, and the like. Other possible parenteral delivery systems for the active drugs of the present application include ethylene-vinyl acetate copolymer particles, implantable osmotic pumps and liposomes.

[0356] Other possible modes of administration include inhalation, including dry powder, aerosol, and water droplets. Inhalation can be in the form of a nasal drop, intranasal gel, buccal preparations including lozenges, troches, and the like, can contain flavoring agents such as sucrose, arabic gum, and other artificial or natural flavoring agents, and can also contain other excipients such as mannitol, sorbitol, and the like. Suppositories, suitable for unit dosage form, can be prepared from a variety of pthogenic materials, such as cocoa butter, and can contain salicylic acid. Topical formulations for the skin can be in the form of creams, lotions, liniments, patches, gels, sprays, aerosols, or oils. Petrolatum, lanolin, polyethylene glycols, alcohols, and mixtures thereof can serve as pharmaceutical carriers. Topical formulations can be in the form of patches, creams, buffered solutions, dissolved or dispersed in polymers or adhesives.

[0357] In particular, the conjugate of the present application can be used in combination with other known or unknown therapeutic agents, such as chemotherapeutic agents, radiotherapy, immunotherapy agents, autoimmune disease agents, anti-infective agents, or other antibody drug conjugates, to achieve synergistic effects. The synergistic agents or radiotherapy can be administered or performed before or after the administration of the conjugate of the present application. It can be 1 hour, 12 hours, one day, one week, one month, or several months before or after the administration of the conjugate of the present application.

[0358] In other embodiments, the synergistic agents include, but are not limited to:

[0359] 1). Chemotherapeutic drugs: a). Alkylating agents: such as [nitrogen mustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard); nitrosoureas: (carmustine, lomustine); alkyl sulfonates: (busulfan, treosulfan); triazenes: (dacarbazine); platinum-containing compounds: (carboplatin, cisplatin, oxaliplatin)]; b). Plant alkaloids: such as [vinca alkaloids: (vinblastine, vincristine, vindesine, vinorelbine); taxoids: (paclitaxel, docetaxel)]; c). DNA topoisomerase inhibitors: such as [epipodophyllotoxins: (9-aminocamptothecin, camptothecin, clitocine, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan); mitomycin: (mitomycin C)]; d). Antimetabolites: such as {[anti-folates: dihydrofolate reductase inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase inhibitors (mycophenolic acid, formylthiazolidine, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, desferrioxamine)]; [pyrimidine analogs: uracil analogs: (5-fluorouracil, doxifluridine, raltitrexed (Tomudex)); cytosine analogs: (cytarabine, arabinosylcytosine, fludarabine); purine analogs: (azathioprine, mercaptopurine, guanine)]; e). Hormones: such as {receptor antagonists: [anti-estrogens: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goserelin, leuprolide acetate); anti-androgens: (bicalutamide, flutamide)]; retinoids / triangular muscle: [vitamin D3 analogs (CB 1093, EB 1089 KH 1060, cholecalciferol, vitamin D2); photodynamic therapy: (porfimer sodium, phthalocyanine photosensitizers, PC4, metoxalen); cytokines: (interferon-alpha, interferon-gamma, tumor necrosis factor (tumor necrosis factor), human protein containing TNF domain)]} f). Kinase inhibitors, such as bibw 2992 (anti EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, ponatinib, vandetanib, flomatinib, e7080 (anti VEGFR2), motesanib, midostaurin, ponatinib (ap24534), HQP1351, bafetinib (INNO-406), bosutinib (SKI-606), sunitinib, cabozantinib, vatalanib, vemurafenib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, bevacizumab, sorafenib, trastuzumab, cetuximab, ranibizumab, panitumumab, ispinesib; g).Other classes: such as gemcitabine, epoxomicins (e.g. lenalidomide), bortezomib thalidomide, lenalidomide, pomalidomide, thalidomide, zybrestat, PLX4032, sta-9090, Stimuvax, allovectin-7, xegeva, Provenge, Yervoy, isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), dactinomycins (e.g. actinomycin D, dactinomycin), pingermycins (e.g. bleomycin bleomycin A2, B2, pepleomycin), anthracyclines (e.g. erythromycin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g. verapamil), Ca. 2+ ATPase inhibitors (e.g. thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD0103), belinostat, PCI-24781, entinostat, SB939, resminostat, givinostat, AR-42, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, 5 disulfiram, salinosporamide A.

[0360] 2). Anti-autoimmune agents include, but are not limited to: cyclosporine, cyclosporine A, azathioprine, aminoglutethimide, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, glucocorticoids (e.g. cortisone, betamethasone, budesonide, flunisolide, fluticasone propionate, hydrocortisone, dexamethasone, fluocortolone, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), dehydroepiandrosterone, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, sirolimus, tacrolimus, prednisone

[0361] 3) Anti-infective agents include, but are not limited to: a) Aminoglycosides: amikacin, Wuyimycin, gentamicin (netilmicin, sisomicin, isapamicin), hygromycin, kanamycin (amikacin, abekacin, aminodeoxykanamycin, dibekacin, tobramycin), neomycin (neomycin B, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, methylsodium thiosulfate; b) Aminoglycosides: chloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) Ansarmycins: gerdemycin, chloramphenicol; d) Carbapenems: biapenem, donipenem, ertapenem, imipenem / cilastatin, methylsodium thiosulfate ... Ropenem, Panipenem; e) Cephalosporins: Carbazocephalosporins (carbazocephalosporins), cefaclor, cefaclor, cefadroxil, cefadroxil, cefotaxime, cefalothin or cephalosporins, cefalexin, cefaram, cefamandole, cefepime, ceftriaxone, cefotaxime, cefoxitin, cefazolin, cefadroxil ... Imidazole, cefpirome, cefpirome, cefpodoxime, cefprozil, cefquinoxime, cefsulfuron, ceftazidime, cefterenol, cefbuprofen, cefotaxime, cefazolin, cefazolin, ceftriaxone, cefuroxime, cefazolin, cephalosporins (cefoxitin, cefotetan, cefmetazole), oxocephalosporins (fluoxocephalosporins, latamoxporins); f) Glycopeptides: bleomycin, vancomycin (olivacin, tervacin), teicoplanin (dapavancin), ramoranine, dapoxetine; g) Glycyl: such as tigecycline; h) β-lactamase inhibitors: penicillane (sulbactam, tazobactam), clavulanic acid; i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics (CDA); k) Macrolides: Azithromycin, Quinoerythromycin, Quinoerythromycin, Clarithromycin, Dierythromycin, Erythromycin, Fluerythromycin, Josamycin, Ketolides (Telithromycin, Quinoerythromycin, Quinoerythromycin, Quinoerythromycin, Quinoerythromycin), Midecamycin, Meokamycin, Rifampicin (Rifampin, Rifampin, Rifabutin, Rifapentine), Rotamycin, Roxithromycin, Spectinomycin, Spiramycin, Tacrolimus (FK506), Acetylosin, Telithromycin; l) Monocyclic β-lactam antibiotics: Aztreonam, Tegamona; m) Azolidinones: Linezolid; n)Penicillins: amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, penicillin, benzathine penicillin, phenoxymethylpenicillin, clometocillin, procaine penicillin, carbenicillin (carindacillin), cloxacillin, dicloxacillin, phenoxymethylpenicillin, flucloxacillin, mezlocillin (azidocillin), mezlocillin, methicillin, nafcillin, oxacillin, penamecillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) Polypeptides: bacitracin, polymyxin E, polymyxin B; p) Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, Floxin, gatifloxacin, gemifloxacin, grepafloxacin, trovafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, perfloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) Lincosamides: pristinamycin, quinupristin / dalfopristin); r) Sulfonamides: sulfonamides, sulfadiazine, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s) Steroidal antibacterials: fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, rolitetracycline, tetracycline, glycyl (e.g. tigecycline); u) Other types of antibiotics: anonitabelladonna, arsenophanes, bacterial terpene alcohol inhibitors (bacitracin), Dadal / AR inhibitors (cycloserine), dictyostatin, spongiolide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, furazidin, isoniazid, laulimalide, metronidazole, mupirocin, mycolactones, NAM synthesis inhibitors (e.g. fosfomycin), furantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), sulbactam metronidazole, bullatai.

[0362] 4) Anti-viral drugs: a) entry / fusion inhibitors: aplaviroc, maraviroc, vicriviroc, enfuvirtide (Fuzeon®), PRO 140, CD4 (ibalizumab); b) integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) maturation inhibitors: bevirimat, vivecon; d) neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir, clafamide, dexribavirin, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, floxuridine (5-FU), 3'-fluoro substituted 2',3'-dideoxynucleoside analogs (e.g., 3'-fluoro-2',3'- dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG) fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (such as beta-L-thymidine, beta-L-2'-deoxycytidine), penciclovir, racivir, ribavirin, stampidine, stavudine (d4T), telbivuine (vermiridine), telbivudine, tenofovir, valaciclovir trifluorothymidine, valganciclovir, zalcitabine (DDC), zidovudine (AZT); f) non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol, ethamivine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alpha, loxoribine, perhexiline, nevirapine, NOV-205, peginterferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g) protease inhibitors: amprenavir, atazanavir, boscalid, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950) tipranavir; h) other types of anti-viral drugs: abacavir, calanolides A, cerulenin, cyanophycin-N, diarylpyrimidines, epigallocatechin gallate (EGCG), foscarnet sodium, griffithsin, heptavirin (vermiridine), hydroxyurea, KP-1461, miltefosine, placenvir, synthetic inhibitors, ribavirin, seliciclib;

[0363] 5) other immunotherapeutic agents: such as imiquimod, interferons (e.g., alpha, beta), granulocyte colony-stimulating factor, cytokines, interleukins (IL-1 to IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, ocrelizumab), protein binding drugs (e.g., Abraxane), an antibody binding drug selected from the group consisting of a calicheamicin derivative, a maytansinoid derivative (DM1 and DM4), CC-1065 and duocarmycin minor groove agents, an effective taxol derivative, doxorubicin, an auristatin antimitotic drug (e.g., trastuzumab-DM1, Inotuzumab ozogamicin, brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab mertansine, AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, IMGN529, IMGN853, milatuzumab-doxorubicin, SGN-75 (anti-CD70), anti-CD22-MCC-DM1).

[0364] As a further object of the present application, the present application is also directed to the preparation of the antibody drug conjugates of the present application. The conjugates of the present application can be prepared by a variety of methods well known in the art, for example, the antimitotic agents of the conjugates of the present application can be synthesized according to the following methods or modifications of the following methods. These modifications are readily apparent to one skilled in the art and are well known in the art and obvious from the scientific literature. In particular, these methods are described in detail in Comprehensive Organic Transformations (R. C. Larock, 1999, Wiley-VCH, 2ndEdition).

[0365] During the reactions described herein, it can be necessary to protect reactive functional groups, for example, hydroxyl, amino, imino, thio and carboxyl groups, where these are desired to be present in the final product, to avoid their unwanted participation in a reaction. Conventional protecting groups can be used according to standard practice (see for example, P. G. Wuts and T. W. Greene, Greene's Protective Groups in Organic Synthesis, 4th edition, Wiley-Interscience, 2006). Some of the reactions can be performed in a suitable solvent in the presence of an acid or a base. The acid, base and solvent for such reactions are not particularly limited, and any conventional acid, base and solvent can be used herein, provided that they have no adverse effect on the reaction. Furthermore, the reactions can be performed over a wide range of temperatures. However, generally, the reaction temperature is usually between -80 °C to 150 °C (more preferably between room temperature and 100 °C) for easier handling. The reaction time required can also vary widely, depending on many factors, especially the reaction temperature and the nature of the solvent. Generally, a reaction time of 3 to 20 hours is suitable for a more desirable reaction.

[0366] Work-up after the reaction can be performed according to the conventional method. For example, the reaction product can be recovered by distilling off the solvent from the reaction system. Alternatively, if necessary, after distilling off the solvent, the residue can be poured into water and extracted with a water-immiscible organic solvent. Finally, after distilling off the extraction solvent, the reaction product can be obtained. Furthermore, if a higher purity is required, further purification can be performed by various conventional methods, for example, recrystallization, precipitation or various chromatographic methods. Generally, column chromatography and preparative thin plate chromatography are more commonly used.

[0367] Examples

[0368] The application is further illustrated by the following examples, which are not intended to limit the scope of the application. In the examples, cell lines, unless otherwise specified, were maintained under standard conditions according to the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ), or the Shanghai Cell Culture Center of the Chinese Academy of Sciences. Cell culture reagents, unless otherwise specified, were from Invitrogen. All anhydrous reagents were obtained commercially and stored in Sure-Seal bottles. Other reagents and solvents were purchased at the highest available purity and used without further purification. Preparative HPLC separations were performed on a Varain PreStar HPLC. NMR spectra were obtained on a Bruker 500 MHz instrument, chemical shifts are in ppm with respect to tetramethylsilane (0 ppm) as reference, and coupling constants (J) are in Hz. Mass spectrometry data were obtained on a Waters Xevo Q Tof mass spectrometer coupled to a Waters Acquity UPLC high performance liquid chromatograph and TUV detector.

[0369] Example 1 Synthesis of Compound 1

[0370]

[0371] Diethoxyacetonitrile (1.00 kg, 7.74 mol) was dissolved in methanol (6.0 L) in a 10-L reaction kettle, and (NH4)2S (48% aqueous solution, 1.41 kg, 9.29 mol) was added at room temperature. The temperature of the kettle rose to 33 °C and then returned to room temperature. After stirring overnight, the reaction was concentrated. Ethyl acetate (5 L) was added to the residue, which was washed with saturated NaHC03solution (4 x 1.0 L), and the aqueous phase was back-extracted with ethyl acetate (5 x 1.0 L). The combined organic phases were washed with saturated brine (3 L), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was slurried with petroleum ether, vacuum filtered, and the solid was collected and washed with petroleum ether. The filtrate was concentrated and then slurried with petroleum ether, and the resulting solid was collected to give a total of 1.1 kg (87% yield) of the target product as a white or light yellow solid. 1 H NMR (500 MHz, CDC13) δ 7.81 (d, J = 71.1 Hz, 2H), 5.03 (s, 1H), 3.73 (dq, J = 9.4, 7.1 Hz, 2H), 3.64 (dq, J = 9.4, 7.0 Hz, 2H), 1.25 (t, J = 7.1 Hz, 6H).

[0372] Example 2 Synthesis of Compound 2

[0373]

[0374] Equip a reflux condenser and a constant-pressure dropping funnel onto a 5-L three-necked round-bottom flask. Add molecular sieves ( An ethanol solution (3 L) of 500 g of thioamide 2 (350 g, 2.14 mol) and ethyl 3-bromopyruvate (80% purity, 404 mL, 2.57 mol) was added dropwise over 30 minutes. The internal temperature rose slightly during the addition. The reaction solution was then heated to reflux and stirred for 30 minutes. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth to remove insoluble matter, and the filter cake was washed with ethyl acetate. The crude product obtained by concentrating the filtrate was mixed with silica gel (1.5 kg) and purified by column chromatography (10 kg) using a silica gel column (10-20% ethyl acetate / petroleum ether gradient elution) to obtain a brown oily substance, which was the target compound (509 g, 92% yield).

[0375] Example 3 Synthesis of Compound 3

[0376]

[0377] A solution of acetal (300 g, 1.16 mol) in acetone (3.0 L) was heated to reflux, and 250 mL of 4N HCl solution was added dropwise over 1 hour. TLC showed that the starting material reacted completely. The reaction solution was concentrated under reduced pressure and the two phases were separated. The organic phase was diluted with ethyl acetate (1.5 L) and washed successively with saturated NaHCO3 aqueous solution (1.0 L), water (1.0 L), and brine (1.0 L), and then dried over anhydrous sodium sulfate. All aqueous phases were combined and back-extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After filtering out the drying agent, the organic phase was concentrated, and the crude product was slurried with petroleum ether / ethyl acetate (5:1) solution. The precipitated solid was collected by vacuum filtration and washed with petroleum ether / ethyl acetate (10:1) solution. The filtrate was concentrated and purified by column chromatography (0-15% ethyl acetate / petroleum ether). All solids were combined to give 40 g (43% yield) of the target product as a white or bright yellow solid. 1 H NMR (500MHz, CDCl3) δ10.08–10.06 (m, 1H), 8.53–8.50 (m, 1H), 4.49 (q, J=7.1Hz, 2H), 1.44 (t, J=7.1Hz, 3H). MS ESI m / z C7H8NO3S[M+H] + Calculated value: 186.01, measured value: 186.01.

[0378] Example 4 Synthesis of Compound 4

[0379]

[0380] To a solution of (S)-tert-butylsulfinylamine (100 g, 0.825 mol) in tetrahydrofuran (1 L) was added Ti(OEt)4(345 mL, 1.82 mol) and 3-methyl-2-butanone (81 mL, 0.825 mol) at room temperature under N2protection. The reaction was heated to reflux for 16 h, then cooled to room temperature, and poured into ice water (1 L). The mixture was filtered and the filter cake was washed with ethyl acetate. The organic phase was separated from the filtrate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was distilled under reduced pressure (15-20 torr, 95 °C) to give the target product 4 (141 g, 90% yield) as a yellow oil. 1 H NMR (500 MHz, CDC13) δ 2.54 - 2.44 (m, 1H), 2.25 (s, 3H), 1.17 (s, 9H), 1.06 (dd, J = 6.9, 5.1 Hz, 6H). MS ESI m / z C9H 19 NaNOS[M+Na] + : Calc. 212.12, Found 212.11.

[0381] Example 5 Synthesis of compound 5

[0382]

[0383] To a solution of diisopropylamine (264 mL, 1.87 mol) in tetrahydrofuran was added n-butyllithium solution (2.5 M, 681 mL, 1.70 mol) at -78 °C under N2protection. The reaction was allowed to warm to 0 °C over 30 min and then re-cooled to -78 °C. Compound 10 (258 g, 1.36 mol) was added to the reaction and rinsed with tetrahydrofuran (50 mL). After stirring for 1 h, ClTi(O i Pr)3(834 g, 3.17 mol) in tetrahydrofuran (1.0 L). After 1 h from the completion of the addition, a solution of compound 4 (210 g, 1.13 mol) in tetrahydrofuran (500 mL) was added slowly over 1 h. The resulting solution was stirred at -78 °C for an additional 3 h. After monitoring the reaction completion by TLC, the reaction was quenched with a mixture of acetic acid and tetrahydrofuran (1:1, 300 mL), and then poured into brine (2 L) and extracted with ethyl acetate (8 x 1 L). The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (dichloromethane / ethyl acetate / petroleum ether 2:1:2) to give compound 5 (298 g, 74% yield) as a colorless oil. 1H-NMR (500 MHz, CDC13) δ 8.13 (s, 1H), 6.63 (d, J = 8.2 Hz, 1H), 5.20-5.11 (m, 1H), 4.43 (q, J = 7.0 Hz, 2H), 3.42-3.28 (m, 2H), 2.89 (dt, J = 13.1, 6.5 Hz, 1H), 1.42 (t, J = 7.1 Hz, 3H), 1.33 (s, 9H), 1.25-1.22 (m, 6H). MS ESI m / z C 16 H 26 NaN2O4S2[M+Na] + : Calculated 397.13, Found 397.11.

[0384] Example 6 Synthesis of compound 6

[0385]

[0386] Compound 5 (509 g, 1.35 mol) was dissolved in tetrahydrofuran (200 mL) and cooled to -78 °C, to which Ti(OEt)4(570 mL, 2.72 mol) was added slowly, and stirred for 1 hour after the addition. Then NaBH4(51.3 g, 1.36 mol) was added in portions over 90 minutes, and the reaction was stirred at -78 °C for 3 hours. TLC monitoring found that the starting material was still remaining. Ethanol (50 mL) was added slowly, and after 1.5 hours of continued stirring, the reaction was poured into saturated brine (2 L, containing 250 mL acetic acid), and raised to room temperature. The organic phase was separated after filtration through celite. The organic phase was washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (ethyl acetate / petroleum ether 1:1) to give compound 6 (364 g, 71% yield) as a white solid. 1 H NMR (500 MHz, CDC13) δ 8.10 (s, 1H), 5.51 (d, J = 5.8 Hz, 1H), 5.23-5.15 (m, 1H), 4.41 (q, J = 7.0 Hz, 2H), 3.48-3.40 (m, 1H), 3.37 (d, J = 8.3 Hz, 1H), 2.29 (t, J = 13.0 Hz, 1H), 1.95-1.87 (m, 1H), 1.73-1.67 (m, 1H), 1.40 (t, J = 7.1 Hz, 3H), 1.29 (s, 9H), 0.93 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 7.2 Hz, 3H). MS ESI m / z: Calculated C 16 H 28 NaN2O4S2[M+Na] + : 399.15, Found 399.14.

[0387] Example 7 Synthesis of compound 7

[0388]

[0389] To a solution of compound 6 (600 g, 1.60 mol) in ethanol (590 mL) was added a solution of 4N HC1 in 1,4-dioxane (590 mL) at 0 °C. After the reaction was allowed to warm to room temperature, it was stirred for 2.5 h. The white solid that precipitated was collected by filtration and washed with ethyl acetate. The filtrate was concentrated and slurried with ethyl acetate. The white solid obtained from both times was combined, 446 g (90% yield).

[0390] Example 8 Synthesis of compound 8

[0391]

[0392] To a solution of sodium azide (740 g, 11.4 mol) in water (2.0 L) was added dichloromethane (2.0 L) and cooled to 0 °C. To this solution was added Tf20 (700 mL, 4.10 mol) over 1.5 h. After the addition was complete, the reaction was stirred at 0 °C for an additional 3 h. The organic phase was separated and the aqueous phase was extracted with dichloromethane (2 x 500 mL). The organic phases were combined and washed with saturated NaHC03(3 x 1.0 L). The dichloromethane solution was added to a mixture of (L)-isoleucine (300 g, 2.28 mol), potassium carbonate (472 g, 3.42 mol), copper sulfate pentahydrate (5.7 g, 22.8 mmol) in a mixture of methanol / water (1 : 1 v / v, 6.0 L) at room temperature. The temperature of the reaction mixture increased slightly during the addition. The mixture was stirred at room temperature for 16 h, the solvent was removed under reduced pressure, and the aqueous phase was acidified with concentrated HC1 (approximately 280 mL) to pH 6-6.5 (approximately 280 mL) and then diluted with phosphate buffer (0.25 M, pH 6.2, 6.0 L). The sulfonamide byproduct was washed out with ethyl acetate (6 x 2.0 L). The aqueous phase was acidified with concentrated HC1 (approximately 400 mL) to pH 3 and extracted with ethyl acetate (4 x 2.0 L). The organic phases were combined, washed with saturated brine (2.0 L), dried over anhydrous sodium sulfate, filtered, and concentrated to give product 8 (320 g, 89% yield) as a bright yellow oil. 1 H NMR (500 MHz, CDC13) δ 12.01 (s, 1H), 3.82 (d, J = 5.9 Hz, 1H), 2.00 (ddd, J = 10.6, 8.6, 5.5 Hz, 1H), 1.54 (dqd, J = 14.8, 7.5, 4.4 Hz, 1H), 1.36 - 1.24 (m, 1H), 1.08 - 0.99 (m, 3H), 0.97 - 0.87 (m, 3H).

[0393] Example 9 Synthesis of compound 9

[0394]

[0395] Compound 7 (150 g, 0.49 mmol) was added in portions. After stirring at 0 °C for 30 min, the temperature was gradually raised to room temperature and stirring was continued for 2 h. The reaction was quenched with ice water at 0 °C and extracted with ethyl acetate three times. The combined organic phase was washed with 1 N HC1, saturated NaHC03, and brine, dried over anhydrous sodium sulfate. Filtration and concentration gave a residue, which was purified by column chromatography (0-30% ethyl acetate / petroleum ether) to give a white solid (140 g, 70% yield). 1 H NMR (500 MHz, CDC13) δ 8.14 (s, 1H), 6.57 (d, J = 8.9 Hz, 1H), 4.91 (d, J = 11.1 Hz, 1H), 4.44 (dd, J = 13.2, 6.3 Hz, 2H), 4.08 - 3.95 (m, 2H), 2.21 (dd, J = 24.4, 11.5 Hz, 2H), 1.90 - 1.79 (m, 3H), 1.42 (t, J = 6.6 Hz, 3H), 1.37 - 1.27 (m, 2H), 1.11 (d, J = 6.4 Hz, 3H), 1.01 - 0.94 (m, 9H). MS ESI m / z C 18 H 30 N5O4S [M+H] + : Calc. 412.19, Found 412.19.

[0396] Example 10 Synthesis of compound 10

[0397]

[0398] To a solution of compound 9 (436 g, 1.05 mol) in dichloromethane (50 mL) was added imidazole (94 g, 1.37 mmol) and triethylsilyl chloride (222 mL, 1.32 mol) sequentially at 0 °C. After the reaction was raised to room temperature over 1 h, stirring was continued for 1 h. The reaction was quenched with saturated brine and the organic phase was separated. The aqueous phase was extracted with ethyl acetate. The combined organic phase was dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate / petroleum ether) to give product 10 (557.4 g, 95% yield) as a colorless oil. 1H NMR (500 MHz, CDC13) δ 8.12 (s, 1H), 6.75 (d, J = 8.0 Hz, 1H), 5.20 - 5.12 (m, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.06 - 3.97 (m, 1H), 3.87 (d, J = 3.8 Hz, 1H), 2.14 (d, J = 3.8 Hz, 1H), 2.01 - 1.91 (m, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.34 - 1.25 (m, 2H), 1.06 (d, J = 6.8 Hz, 3H), 1.00 - 0.93 (m, 18H), 0.88 (dd, J = 19.1, 6.8 Hz, 6H). MS ESI m / z C 24 H 44 N5O4SSi [M+H] + : Calculated 526.28, Found 526.28.

[0399] Example 11 Synthesis of Compound 11

[0400]

[0401] To a solution of compound 10 (408 g, 0.77 mol) and iodomethane (145 mL, 2.32 mol) in tetrahydrofuran (4 L) was added sodium hydride (60%, 62.2 g, 1.55 mol) at 0 °C. The resulting reaction was stirred at 0 °C overnight, then poured into vigorously stirred saturated aqueous ammonium chloride (5 L). Extracted with ethyl acetate (3 x 500 mL). The combined organic phase was dried, filtered, concentrated and purified by column chromatography (15-35% ethyl acetate / petroleum ether) to give product 11 (388 g, 93% yield) as a bright yellow oil. 1 H NMR (500 MHz, CDC13) δ 8.12 (s, 1H), 6.75 (d, J = 8.0 Hz, 1H), 5.20 - 5.12 (m, 1H), 4.44 (q, J = 7.0 Hz, 2H), 4.06 - 3.97 (m, 1H), 3.87 (d, J = 3.8 Hz, 1H), 2.14 (d, J = 3.8 Hz, 1H), 2.01 - 1.91 (m, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.34 - 1.25 (m, 2H), 1.06 (d, J = 6.8 Hz, 3H), 1.00 - 0.93 (m, 18H), 0.88 (dd, J = 19.1, 6.8 Hz, 6H). MS ESI m / z C 25 H 46 N5O4SSi [M+H] +Calcd 540.30, Found 540.30.

[0402] Example 12 Synthesis of Compound 12

[0403]

[0404] A mixture of 2-methylalanine (500 g, 4.85 mol), formaldehyde (37% in water, 1.0 L, 12.1 mol) and formic acid (1.0 L) was heated to reflux (80 °C). After stirring for 3.0 h, the reaction was cooled to room temperature, 6 N HCl (850 mL) was added, and the reaction was concentrated. The resulting solid was collected by filtration and washed with ethyl acetate (1.0 L) three times. The solid was dissolved in water (1.5 L) and neutralized to pH 7 with 4 N NaOH (about 1.0 L). The solution was concentrated and azeotroped with ethanol (2.0 L) to remove water. The residue was dissolved in methanol (2.0 L), filtered to remove NaCl solids, and washed with ethyl acetate. The filtrate was concentrated to give a white solid, 639.2 g, containing a small amount of NaCl, which was used directly without further purification.

[0405] Example 13 Synthesis of Compound 13

[0406]

[0407] To a solution of compound 12 (97 g, 0.74 mol) in ethyl acetate (1 L) was added pentafluorophenol (163 g, 0.88 mol) and DIC (126 mL, 0.81 mol). The reaction was stirred at room temperature for 24 h, filtered through celite, and washed with 10 mL of ethyl acetate. The filtrate was used directly without further purification.

[0408] Example 14 Synthesis of Compound 14

[0409]

[0410] To a solution of the above pentafluorophenyl ester 13 in ethyl acetate was added compound 11 (200 g, 0.37 mol) and dry Pd / C (10 wt%, 10 g). The reaction was stirred under hydrogen (1 atm) for 27 h. The reaction was filtered through celite and washed with ethyl acetate. The combined organic phase was concentrated and purified by column chromatography (0-5% methanol / ethyl acetate) to give compound 14 (184 g, 79% yield). MS ESI m / z C 31 H 58 N4O5SSi[M+H] + Calcd 627.39, Found 627.39.

[0411] Example 15 Synthesis of Compound 15

[0412]

[0413] Compound 14 (200 g, 0.32 mmol) was dissolved in a mixture of acetic acid / water / tetrahydrofuran (v / v / v 3:1:1, 638 mL) and stirred at room temperature for 4 days. The reaction was concentrated and azeotroped with toluene to dryness. This step was repeated twice to give compound 15 which was used directly in the next step. MS ESI m / z C 25 H 45 N4O5S [M+H] + Calcd 513.30, Found 513.30.

[0414] Example 16 Synthesis of Compound 16

[0415]

[0416] A solution of lithium hydroxide (0.4 N, 600 mL, 2.55 mol) in water was added to a solution of compound 15 (160 g, 0.319 mol, 1.0 eq.) in methanol (1.2 L) at 0 °C. The reaction was stirred at room temperature for 2 h and concentrated. Purification by column chromatography (100% dichloromethane to 80:20:1 dichloromethane / methanol / ammonia) gave compound 16 (140 g, 91% over two steps) as an amorphous white solid. MS ESI m / z C 23 H 40 N4O5S [M+H] + Calcd 485.27, Found 485.27.

[0417] Example 17 Synthesis of Compound 17

[0418]

[0419] Compound 16 (143 g, 0.30 mol) and DMAP (0.36 g, 2.95 mmol) were dissolved in a mixture of dry tetrahydrofuran (1.4 L) and dry DMF (75 mL). The reaction was cooled to 0 °C and triethylamine (82.2 mL, 0.59 mol) and acetic anhydride (56 mL, 0.59 mol) were added. The reaction was allowed to warm to room temperature and stirred for 24 h. Purification by column chromatography (5-50% methanol / dichloromethane) gave compound 17 (147 g, 95% yield) as an amorphous white solid. 1H NMR (500 MHz, DMSO) δ 8.37 (s, 1H), 7.63 (d, J = 9.5 Hz, 1H), 5.54 (dd, J = 11.2, 2.5 Hz, 1H), 4.64 (dd, J = 9.4, 7.2 Hz, 1H), 4.34 (s, 1H), 2.95 (s, 3H), 2.27 - 2.19 (m, 1H), 2.19 - 2.12 (m, 1H), 2.11 (s, 6H), 2.08 (s, 3H), 1.82 - 1.66 (m, 2H), 1.54 - 1.42 (m, 1H), 1.10 (s, 3H), 1.06 - 0.95 (m, 1H), 0.99 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H), 0.65 (d, J = 6.6 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 175.35, 172.78, 169.70, 169.58, 162.23, 148.03, 128.29, 69.51, 63.00, 55.10, 52.37, 38.86, 36.46, 33.83, 29.25, 28.82, 23.64, 21.09, 20.60, 19.96, 19.40, 18.38, 15.65, 10.77. MS ESI m / z C 25 H 44 N4O6S [M+H] + Calcd 527.3, Found 527.4.

[0420] Example 18 Synthesis of compound 18

[0421]

[0422] To a solution of compound 17 (41.0 g, 77.9 mmol, 1.0 eq) in anhydrous dichloromethane (600 mL) was added EDC HCI (44.8 g, 233 mmol, 3.0 eq) and pentafluorophenol (35.9 g, 194 mmol, 2.5 eq) at room temperature. The mixture was stirred at room temperature for 2 hours, washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (25-100% ethyl acetate / n-hexane elution) to give a white solid (43.0 g, yield 80%). 1H NMR (500 MHz, DMSO) δ 9.06 (s, 1H), 7.65 (d, J = 9.4 Hz, 1H), 5.60 (dd, J = 11.0, 2.8 Hz, 1H), 4.64 (dd, J = 9.4, 7.2 Hz, 1H), 4.35 (s, 1H), 2.97 (s, 3H), 2.34 - 2.16 (m, 2H), 2.12 (s, 6H), 2.11 (s, 3H), 1.88 - 1.65 (m, 2H), 1.57 - 1.37 (m, 1H), 1.11 (s, 3H), 1.06 - 0.96 (m, 1H), 1.00 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H), 0.66 (d, J = 6.6 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 175.24, 172.78, 171.75, 169.81, 156.32, 141.69, 141.56, 139.71, 138.59, 136.60, 134.68, 69.49, 63.11, 55.16, 52.41, 38.83, 36.40, 33.64, 29.42, 28.82, 23.62, 21.01, 20.55, 19.93, 19.39, 18.35, 15.62, 10.73. MS ESI m / z C 31 H 42 F5N4O6S [M+H] + Calcd 693.3, Found 693.3.

[0423] Example 19 Synthesis of Compound 19

[0424]

[0425] Sodium hydride (60%, 8 g, 200 mmol) was added to a solution of HO-PEG9-OMe (42.8 g, 100 mmol) in tetrahydrofuran (1 L) at room temperature. After stirring for 30 min, tert-butyl bromoacetate (48.8 g, 250 mmol) was added, stirred at room temperature for 1 h, then poured into ice water, extracted with dichloromethane, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate. Purified by silica gel column chromatography (0-5% methanol / dichloromethane) to give compound 19 as a yellow oil (32 g, 59% yield).

[0426] Example 20 Synthesis of Compound 20

[0427]

[0428] Compound 432 (40 g, 73.8 mmol) was dissolved in dichloromethane (400 mL), then formic acid (600 mL) was added, stirred at 25 °C overnight. All volatiles were removed by distillation under reduced pressure to give yellow oil (36 g, about 100% yield). ESI m / z C 21 H 43 O 12 [M+H] + : Calc. 487.27, Found 487.24.

[0429] Example 21 Synthesis of compound 21

[0430]

[0431] Compound 20 (36 g, 73.8 mmol) was dissolved in dichloromethane (640 mL), oxalyl chloride (100 mL) and DMF (52 g, 0.74 mmol) were added successively. The resulting solution was stirred at room temperature for 4 hours, all volatiles were removed by distillation under reduced pressure to give yellow oil.

[0432] Example 22 Synthesis of compound 22

[0433]

[0434] Z-L-Lys-OH (41.4 g, 147.6 mmol), sodium carbonate (23.4 g, 221.4 mmol) and NaOH (5.9 g, 147.6 mmol) were dissolved in water (720 mL), cooled to 0 °C, then a tetrahydrofuran solution (20 mL) of compound 21 (37.2 g, 73.8 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, THF was removed by distillation under reduced pressure, adjusted to pH 3 with concentrated hydrochloric acid in ice bath. The solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate to give yellow oil (55 g, 99% yield). ESI m / z C 35 H 60 N2O 15 [M+H] + : Calc. 749.40, Found 749.39.

[0435] Example 23 Synthesis of compound 23

[0436]

[0437] To a solution of Boc-L-tyrosine methyl ester (2.2 kg, 7.45 mol), potassium carbonate (1.54 kg, 11.2 mol) and potassium iodide (48 g, 0.29 mol) in acetonitrile (8.8 L) was added benzyl bromide (1.33 kg, 7.78 mol) slowly. Stirred at room temperature overnight, added water (8 L) to dissolve the solid, extracted with ethyl acetate (2 x 4 L). The combined organic phase was washed with water (4 L), saturated brine (4 L), dried over anhydrous sodium sulfate, filtered, concentrated and slushed with petroleum ether (20 L) to give white solid 98 (2.73 kg, 95% yield). 1 H NMR (500 MHz, CDC13) δ 7.43 (d, J = 7.0 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 5.04 (s, 2H), 4.55 (d, J = 6.9 Hz, 1H), 3.71 (s, 3H), 3.03 (qd, J = 14.0, 5.8 Hz, 2H), 1.43 (s, 9H). MS ESI m / z C 22 H 28 NO5[M+H] + : Calc. 386.19, Found 386.19.

[0438] Example 24 Synthesis of compound 24

[0439]

[0440] To a solution of compound 23 (616 g, 1.6 mol) in dichloromethane (2.4 L) was added NaBH4(122 g, 3.2 mol) and LiCl (136 g, 3.2 mol) in a mixture solvent of 2.4 L of ethanol and 2.4 L of dichloromethane, and cooled to 0 °C. After the addition, 2.4 L of dichloromethane was added to the reaction solution, and the reaction solution was naturally warmed to room temperature, and a large amount of bubbles was generated. The reaction solution was stirred overnight. The reaction solution was diluted with water (6 L), and stirred for 30 minutes. The aqueous phase was extracted with dichloromethane (2 L x 2), and the combined organic phase was washed with water (2 L) and brine (2 L), dried, filtered, and concentrated to give white solid 542 g (yield 95%).

[0441] Example 25 Synthesis of compound 25

[0442]

[0443] Dissolve oxalyl chloride (1.02 kg, 8.0 mol) in dichloromethane (4 L) and cool to -75 °C. Add a solution of DMSO (1.25 kg, 16 mol) in dichloromethane (400 mL) dropwise, maintaining the temperature below -65 °C. After the addition is complete, stir for 30 minutes. Add a solution of compound 24 (1.90 kg, 5.33 mol) in dichloromethane (8 L) dropwise. After the addition is complete, allow the solution to warm to approximately -65 °C. Stir for 30 minutes and then add triethylamine (1.62 kg, 16 mol) dropwise, maintaining the temperature below -50 °C. After the addition is complete, stir for 15 minutes. Allow the reaction to warm slowly and continue stirring for approximately 1 hour. Allow the reaction to warm to approximately -30 °C. TLC monitoring indicates that the reaction is complete. Add water (6 L) to the reaction and separate the layers after stirring. Wash the aqueous layer with dichloromethane (2 L) and combine the organic layers. Wash the combined organic layers with 10% HCl (4 L) and brine (2 L) each. Dry and concentrate the combined organic layers. Slurry the concentrate with 5:1 petroleum ether / ethyl acetate and vacuum filter to give compound 25 as a light yellow solid (1.36 kg, 72% yield).

[0444] Example 26 Synthesis of compound 26

[0445]

[0446] Stir a solution of tert-butyl 2-bromopropionate (255 g, 1.22 mol) and triphenylphosphine (320 g, 1.22 mol) in dry acetonitrile (1 L) at room temperature for 18 hours. Remove the acetonitrile under reduced pressure and add toluene to precipitate the white solid. Decant the toluene and dissolve the white solid in dichloromethane (1 L) and transfer to a separatory funnel. Add 10% aqueous NaOH (1 L) and the organic layer quickly turns yellow upon shaking. Separate the organic layer and back extract the aqueous layer with dichloromethane (1 L). Combine the dichloromethane layers, wash with saturated brine (400 mL), dry over anhydrous sodium sulfate, filter and concentrate to give the ylide 26 (280 g, 58%) as a yellow solid.

[0447] Example 27 Synthesis of compound 27

[0448]

[0449] Add ylide 26 (546 g, 1.40 mmol) to a solution of compound 25 (450 g, 1.27 mol) in dry dichloromethane (3 L). Stir at room temperature overnight. Purify by column chromatography (10-50% ethyl acetate / petroleum ether) after TLC monitoring indicates the reaction is complete to give compound 27 (444 g, 75% yield) as a white solid. ESI m / z C 28 H 38 NO5[M+H] + : Calculated 468.27, found 468.22.

[0450] Synthesis of compound 28

[0451]

[0452] Compound 27 (63 g, 0.13 mol) was dissolved in methanol (315 mL), Pd / C (10 wt%, 6.3 g) was added, and the mixture was stirred at room temperature under hydrogen (1 atm) overnight. After filtration to remove the catalyst, the filtrate was concentrated to give compound 28 (45.8 g, 93% yield).

[0453] Synthesis of compound 29

[0454]

[0455] To a solution of compound 28 (390 g, 1.03 mol) in tetrahydrofuran (4 L) was added tert-butyl nitrite (1.06 kg, 10.3 mol) at room temperature. After stirring overnight, tetrahydrofuran was removed by concentration under reduced pressure, and the residue was purified by column chromatography (10-50% ethyl acetate / petroleum ether) to give compound 29 (314 g, 72% yield) as a bright yellow solid.

[0456] Synthesis of compound 30

[0457]

[0458] To a solution of compound 30 (166 g, 0.392 mol) in ethyl acetate (500 mL) was added Pd / C (10 wt%, 16 g) under nitrogen protection. Hydrogen was bubbled in and replaced with vacuum for three times. The reaction was stirred at room temperature under hydrogen (1 atm) for 16 hours. After filtration with celite, compound 30 (146 g, 97% yield) was obtained as a yellow foam solid. 1 H NMR (400 MHz, CDC13) δ 6.62 (d, J = 7.9 Hz, 1H), 6.55 (s, 1H), 6.43 (d, J = 7.3 Hz, 1H), 4.39 (dd, J = 53.0, 44.2 Hz, 1H), 3.77 (s, 4H), 2.72 - 2.29 (m, 3H), 1.83 - 1.58 (m, 1H), 1.40 (d, J = 7.6 Hz, 18H), 1.24 (s, 1H), 1.06 (t, J = 5.7 Hz, 3H). MS ESI m / z C 21 H 35 N2O5[M+H] + Calculated 394.25, found 395.25.

[0459] Synthesis of compound 31

[0460]

[0461] HATU (39.9 g, 105 mmol) was added to a solution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (26.1 g, 110 mmol) in DMF (300 mL). After stirring at room temperature for 30 min, the reaction mixture was added to a solution of compound 30 (39.4 g, 100 mmol) and triethylamine (20.2 g, 200 mmol) in DMF (300 mL). The reaction was stirred at room temperature for 2 h, diluted with water, extracted with ethyl acetate, and the organic layer was washed with saturated brine, dried over sodium sulfate. After concentration, it was purified by silica gel column chromatography (20-70% ethyl acetate / petroleum ether) to give a white solid (45 g, 73% yield). ESI m / z C 33 H 48 N3O8[M+H] + : Calc. 614.34, Found 614.15.

[0462] Synthesis of compound 32

[0463]

[0464] Compound 31 (100 g, 163 mmol) was dissolved in methanol (500 mL), Pd / C catalyst (10 wt%, 10 g) was added, and the reaction was hydrogenated (1 atm H2) at room temperature overnight. After the catalyst was filtered off, the filtrate was concentrated under reduced pressure to give brown foamy solid 32 (75.8 g, 97% yield). 1 H NMR (400 MHz, CDC13) δ 7.11 (s, 1H), 6.83 (d, J = 10.3 Hz, 2H), 5.04 - 4.52 (m, 6H), 3.90 - 3.56 (m, 1H), 2.81 (d, J = 5.3 Hz, 2H), 2.63 (dd, J = 12.5, 6.1 Hz, 2H), 2.54 - 2.26 (dd, J = 14.0, 7.6 Hz, 4H), 1.94 - 1.64 (m, 3H), 1.44 - 1.36 (m, 18H), 1.08 (d, J = 6.9 Hz, 3H). ESI m / z C 25 H 42 N3O6[M+H] + : Calc. 480.30, Found 480.59.

[0465] Synthesis of compound 33

[0466]

[0467] To a solution of compound 22 (130 g, 174 mmol) in DMF (500 mL) was added triethylamine (66 mL, 474 mmol) and HATU (72 g, 190 mmol) at 0 °C, then the reaction mixture was warmed to room temperature and stirred for 2 h. A solution of compound 32 (75.8 g, 158 mmol) in DMF (500 mL) was added to the above solution at 0 °C, and the reaction was stirred at room temperature for 1 h. The reaction was poured into water (4 L) and extracted with ethyl acetate (3 x 500 mL), the organic layers were combined and washed with saturated brine (2 L), dried over sodium sulfate, filtered and concentrated. The crude 33 (190 g) was used directly in the next step. ESI m / z C 60 H 100 N5O 20 [M+H] + : Calculated 1210.69, Found 1210.69.

[0468] Example 34 Synthesis of compound 34

[0469]

[0470] The crude 33 (190 g) from the previous reaction was dissolved in methanol (900 mL), Pd / C catalyst (10 wt%, 19 g) was added, and the reaction was hydrogenated (1 atm H2) at room temperature overnight. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure and purified on a silica gel column (0-10% methanol / dichloromethane) to give a brown oil (105 g, 62% yield over two steps). ESI m / z C 52 H 95 N5O 18 [M+H] + : Calculated 1077.65, Found 1077.65.

[0471] Example 35 Synthesis of compound 35

[0472]

[0473] To a solution of compound 34 (105 g, 97.1 mmol) in EtOH (5.3 L) at room temperature was added compound 4-maleimidobutyric acid-N-succinimidyl ester (54.4 g, 194.2 mmol) and 0.1 N sodium phosphate dibasic solution (1.1 L) and the reaction was stirred at room temperature overnight. The EtOH was removed by distillation under reduced pressure and the residual aqueous solution was poured into water (3 L) and then extracted with ethyl acetate (4 x 500 mL) and the combined organic phases were washed with saturated brine (2 L), dried over sodium sulfate, concentrated and the crude product was purified on a silica gel column (0-10% methanol / dichloromethane) to give a yellow oil (100 g, 83% yield). 1 H NMR (500 MHz, DMSO) δ 9.53 (s, 0.7 H), 9.52 (s, 0.3 H), 9.22 (s, 0.7 H), 9.21 (s, 0.3 H), 7.95 - 7.87 (m, 2 H), 7.65 (t, J=5.9 Hz, 1 H), 7.51 - 7.44 (m, 1 H), 6.99 (s, 2 H), 6.77 - 6.66 (m, 2 H), 6.65 - 6.57 (m, 1 H), 4.13 (dt, J=5.4, 8.1 Hz, 1 H), 3.84 (s, 2 H), 3.55 (s, 2 H), 3.52 (s, 2 H), 3.51 - 3.45 (m, 30 H), 3.42 (dd, J=5.8, 3.7 Hz, 2 H), 3.38 (t, J=6.9 Hz, 2 H), 3.23 (s, 3 H), 3.14 - 3.01 (m, 4 H), 2.64 - 2.44 (m, 1 H), 2.41 - 2.22 (m, 4 H), 2.16 - 2.04 (m, 2 H), 1.76 - 1.64 (m, 4 H), 1.64 - 1.52 (m, 2 H), 1.52 - 1.35 (m, 2 H), 1.37 (s, 3 H), 1.35 (s, 6 H), 1.31 (s, 9 H), 0.97 (t, J=8.5 Hz, 3 H). 13C NMR (126 MHz, DMSO) δ 175.35 (minor), 174.88, 171.69, 171.42, 171.29, 171.10, 169.04, 155.19 (minor), 155.05, 145.97, 134.47, 129.36, 126.00, 125.20, 122.92, 115.69, 79.32 (minor), 79.17, 77.35 (minor), 77.27, 71.29, 70.25, 69.95, 69.79, 69.60, 69.53, 58.06, 52.57, 50.13, 49.55 (minor), 41.25, 38.12, 37.94, 37.45, 36.84, 36.80 (minor), 33.38, 32.37, 31.86, 28.96, 28.28, 28.23, 27.69, 27.57, 25.42, 24.19, 22.86, 18.04, 16.49 (minor). ESI m / z C 60 H 101 N6O 21 [M+H] + : calculated 1241.7, found 1241.8.

[0474] Example 36 Synthesis of compound 36

[0475]

[0476] Compound 35 (31.5 g, 25.4 mmol) was dissolved in dichloromethane (125 mL), trifluoroacetic acid (125 mL) was added, after addition, the reaction was carried out at room temperature for 3 hours. After the reaction was completed, the reaction liquid was concentrated on a rotary evaporator until no solvent was distilled out. Then vacuum concentration was carried out on a vacuum oil pump until the weight was basically unchanged to obtain a crude product. Ethyl ether (200 mL) was added to the crude product to wash and separate the product layer, and concentrated under reduced pressure until no solvent was distilled out. Vacuum concentration was carried out again on a vacuum oil pump until the weight was basically unchanged to obtain compound 36 (36.0 g, containing solvent). 1H NMR (500 MHz, DMSO) δ 9.18 (s, 1H), 7.97 - 7.87 (m, 2H), 7.79 (s, 2H), 7.71 - 7.61 (m, 2H), 7.00 (s, 2H), 6.85 - 6.73 (d, J=5.4 Hz, 2H), 4.17 - 4.07 (m, 1H), 3.84 (s, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 3.50 (s, 30H), 3.42 (dd, J=5.8, 3.7 Hz, 2H), 3.39 (t, J=7.0 Hz, 2H), 3.32 - 3.23 (m, 1H), 3.23 (s, 3H), 3.14 - 3.01 (m, 4H), 2.82 - 2.71 (m, 1H), 2.71 - 2.61 (m, 1H), 2.58 - 2.50 (m, 1H), 2.38 (t, J=7.3 Hz, 2H), 2.11 (dt, J=7.8, 3.0 Hz, 2H), 1.88 - 1.77 (m, 1H), 1.76 - 1.64 (m, 4H), 1.59 (dt, J=15.1, 5.8 Hz, 1H), 1.53 - 1.32 (m, 4H), 1.31 - 1.11 (m, 2H), 1.05 (d, J=7.0 Hz, 2.1H), 1.00 (d, J=6.9 Hz, 0.9H). 13 C NMR (126 MHz, DMSO) δ 176.78 (minor), 176.55, 171.74, 171.42, 171.34, 171.12, 169.07, 146.63 (minor), 146.57, 134.49, 126.51, 126.31, 125.19, 122.85, 115.80, 71.31, 70.27, 69.96, 69.81, 69.61, 69.55, 58.07, 54.93 (minor), 52.64, 50.72, 50.13 (minor), 38.30 (minor), 38.14, 37.95, 36.85, 35.75, 35.38 (minor), 34.87, 34.81 (minor), 33.46, 32.38, 31.83, 28.98, 25.40, 24.21, 22.89, 17.49, 16.74 (minor). ESI m / z C 51 H 85 N6O 19 [M+H] + : Calculated 1085.6, Found 1085.4.

[0477] Example 37 Synthesis of Compound 37

[0478]

[0479] To a reaction flask was charged with compound 36 (36.0 g, 25.4 mmol) in DMF (60 mL) and cooled to 5 °C in an ice water bath. To the reaction flask was added compound 18 (19.3 g, 27.9 mmol) in DMF (150 mL) followed by the dropwise addition of DIPEA (25 mL, 139 mmol). After the addition was complete, the ice water bath was removed and the reaction was allowed to warm to room temperature and stirred for 18 hours. After the reaction was complete, the reaction was concentrated to remove the solvent under vacuum using an oil pump. After the concentration was complete, the concentrated solution was diluted with dichloromethane, cooled to 5 °C in an ice water bath, and formic acid was added slowly dropwise while the pH was adjusted to 3.0-4.0. After this, the solution was concentrated to remove the solvent, and the residue was transferred to a silica gel column and eluted with n-hexane / ethyl acetate / formic acid and dichloromethane / methanol / formic acid. The purified crude was concentrated to give a light yellow foam. The foam was dissolved in water / methanol / formic acid and purified further by preparative HPLC using water / acetonitrile / formic acid as the eluent. The appropriate fractions were collected, concentrated, and diluted with water. The solution was divided equally into lyophilization vials and lyophilized to give a light yellow foam (24 g, 60% yield). 1H NMR (500 MHz, DMSO) δ 9.60 (bs, 1H), 9.20 (s, 1H), 8.19 (s, 0.33H), 8.17 (s, 0.67H), 8.02 (d, J = 9.0 Hz, 0.33H), 7.98 (d, J = 9.0 Hz, 0.67H), 7.94 - 7.83 (m, 2H), 7.65 (t, J = 5.8 Hz, 1H), 7.63 (s, 1H), 7.56 (s, 0.33H), 7.55 (s, 0.67H), 6.99 (s, 2H), 6.82 - 6.74 (m, 1H), 6.74 - 6.67 (m, 1H), 5.62 - 5.54 (m, 1H), 4.69 - 4.59 (m, 1H), 4.39 (s, 1H), 4.25 - 4.05 (m, 2H), 3.85 (s, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 3.50 (s, 30H), 3.44-3.36 (m, 4H), 3.23 (s, 3H), 3.14 - 3.02 (m, 4H), 2.96 (s, 3H), 2.83 - 2.71 (m, 1H), 2.71 - 2.57 (m, 1H), 2.44 - 2.32 (m, 3H), 2.32 - 2.14 (m, 2H), 2.14 - 2.05 (m, 2H), 2.12 (s, 6H), 2.09 (s, 3H), 1.99 - 1.65 (m, 7H), 1.64 - 1.53 (m, 2H), 1.53 - 1.32 (m, 5H), 1.31 - 1.14 (m, 2H), 1.11 (s, 3H), 1.05 (d, J = 7.2 Hz, 2H), 1.03 (d, J = 6.9 Hz, 1H), 0.99 (s, 3H), 0.93 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H), 0.67 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 177.52 (minor), 177.01, 175.44, 172.75, 171.69, 171.43, 171.29, 171.09, 169.74, 169.55 (minor), 169.46, 169.04, 159.92 (minor), 159.88, 149.86, 149.74 (minor), 146.07, 134.46, 129.13 (minor), 129.08, 126.12 (minor), 126.07, 125.18, 124.28 (minor), 124.11, 122.95, 122.86 (minor), 115.67, 71.30, 70.26, 69.96, 69.80, 69.60, 69.54, 69.48, 63.01, 58.06, 55.09, 52.58, 52.39, 49.03, 47.96 (minor), 40.35, 38.89, 38.11, 37.94, 37.39, 36.84, 36.53, 36.02, 35.78 (minor), 33.87, 33.38, 32.38, 31.86, 29.02, 28.97, 25.37, 24.19, 23.60, 22.86, 21.15, 20.62, 19.99, 19.41, 18.34, 18.11, 16.17 (minor), 15.69, 10.81. ESI m / z C 76 H 125 N 10 O 24 S[M+H] + : Calcd 1593.9, Found 1593.8.

[0480] Example 38 Synthesis of Compound 38

[0481]

[0482] (S)-4-isopropyl oxazolidin-2-one (400 g, 3.09 mol) was dissolved in dry tetrahydrofuran (8 L) under nitrogen and cooled to about -70 °C. n-Butyllithium (2.5 M in n-hexane, 1.36 L, 3.4 mol) was added dropwise to the reaction vessel and the reaction was allowed to stir at -70 °C for 1 h after the addition was complete. Propionyl chloride (315 g, 3.4 mol) was then added dropwise and the reaction was allowed to stir at -70 °C for 1 h after the addition was complete. The reaction was allowed to warm to room temperature slowly and the reaction was poured into ice cold saturated aqueous brine solution (7 L). The reaction was extracted with ethyl acetate (3 x 2 L) and the combined organic layers were washed with water (2 L) and saturated aqueous brine solution (2 L) once each. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (pure petroleum ether to 5:1 petroleum ether / ethyl acetate) to give 500 g of colorless oil in 87% yield. MS ESI m / z C9H 16 NO3[M+H] + : Calculated 186.10, Found 186.10. 1 H NMR (400 MHz, CDC13) δ 4.48 - 4.39 (m, 1H), 4.27 (t, J = 8.7 Hz, 1H), 4.21 (dd, J = 9.1, 3.1 Hz, 1H), 3.06 - 2.82 (m, 2H), 2.38 (dtd, J = 14.0, 7.0, 4.0 Hz, 1H), 1.17 (t, J = 7.4 Hz, 3H), 0.90 (dd, J = 17.0, 7.0 Hz, 6H).

[0483] Example 39 Synthesis of Compound 39

[0484]

[0485] Compound 38 (92.6 g, 0.50 mol) was dissolved in anhydrous dichloromethane (1.5 L) under nitrogen protection, and the solution was cooled to -10 °C. Diisopropylethylamine (70.5 g, 0.54 mol) and n-Bu2BOTf (1.0 M dichloromethane solution, 500 mL, 0.50 mol) were added dropwise into the reaction flask. The reaction was carried out at 0 °C for 1 h, and then the solution was cooled to -78 °C. A solution of compound 25 (161 g, 0.45 mol) in dichloromethane (1 L) was added dropwise into the reaction flask, and the temperature of the solution was not allowed to exceed -70 °C. After the addition was completed, the reaction was carried out at -78 °C for 2 h, and then the solution was slowly warmed to room temperature and stirred overnight. On the next day, phosphate buffer (0.1 N, pH 7.0, 2 L) was added into the reaction flask, and the solution was partitioned. The aqueous phase was extracted with dichloromethane (2 x 500 mL), and the combined organic phase was washed once with saturated aqueous NaCl solution (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was dissolved in methanol (2 L), and the solution was cooled to 0 °C. H2O2 (30% aqueous solution, 500 mL) was added dropwise, and the reaction was carried out at 5 °C for 1 h. Water (3 L) was added, and the solution was extracted with dichloromethane (3 x 800 mL). The combined organic phase was washed with water (500 mL), saturated NaHCO3 solution (500 mL), and saturated aqueous NaCl solution (500 mL) once, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (pure petroleum ether to 5:1 petroleum ether / ethyl acetate) to give compound 39 as a white solid (150 g, 60% yield). 1 H NMR (400 MHz, CDC13) δ 7.36 (ddd, J = 24.2, 14.2, 7.1 Hz, 5H), 7.12 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.02 (s, 2H), 4.69 (d, J = 9.0 Hz, 1H), 4.45 (d, J = 4.1 Hz, 1H), 4.33 (t, J = 8.4 Hz, 1H), 4.15 (d, J = 8.6 Hz, 1H), 3.90 (dd, J = 16.6, 8.0 Hz, 1H), 3.85 - 3.77 (m, 2H), 2.81 (d, J = 7.6 Hz, 2H), 2.27 (dd, J = 11.4, 6.7 Hz, 1H), 1.35 (s, 9H), 0.89 (dd, J = 14.3, 6.9 Hz, 6H). MS ESI m / z C 30 H 41 N2O7[M+H] + : Calcd 541.28, Found 541.30.

[0486] Example 40 Synthesis of compound 40

[0487]

[0488] Compound 39 (200 g, 0.37 mol) was dissolved in dry tetrahydrofuran (3.5 L) under nitrogen and dithio carbonyl imidazole (198 g, 1.11 mol) was added and the reaction refluxed for 8 h, additional dithio carbonyl imidazole (65 g, 0.37 mol) was added and the reaction left to run overnight. The reaction was cooled to room temperature the following day and the solvent removed under reduced pressure, the residue was purified by silica gel column chromatography (neat petroleum ether to 5:1 petroleum ether / ethyl acetate) to give an oil (170 g, 83% yield). 1 H NMR (400 MHz, CDC13) δ 8.41 (s, 1H), 7.67 (s, 1H), 7.36 (dt, J = 16.0, 6.9 Hz, 6H), 7.09 (s, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.32 (d, J = 9.5 Hz, 1H), 5.01 (s, 2H), 4.56 - 4.43 (m, 2H), 4.32 (ddd, J = 16.2, 15.6, 7.8 Hz, 3H), 4.19 (d, J = 8.7 Hz, 1H), 2.96 (dd, J = 14.6, 4.4 Hz, 1H), 2.49 (dd, J = 14.5, 10.5 Hz, 1H), 2.29 (td, J = 13.4, 6.7 Hz, 1H), 1.73 (s, 1H), 1.29 (s, 9H), 0.91 (dd, J = 13.9, 6.9 Hz, 6H). MS ESI m / z C 34 H 43 N4O7S[M+H] + : Calculated 651.27, Found 651.39.

[0489] Example 41 Synthesis of compound 41

[0490]

[0491] Compound 40 (210 g, 323 mmol) was dissolved in dry toluene (3 L) under nitrogen and tri-n-butylstannane (182 g, 646 mmol) and azobisisobutyronitrile (0.5 g, 3.23 mmol) were added under nitrogen and the reaction refluxed for 2 h, cooled to room temperature and spun dry, purified by silica gel column chromatography (neat petroleum ether to 5:1 petroleum ether / ethyl acetate) to give an oil (141 g, 83% yield). 1H NMR (400 MHz, CDC13) δ 7.36 (ddd, J = 24.5, 14.5, 7.1 Hz, 5H), 7.08 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 5.04 (d, J = 5.1 Hz, 2H), 4.48 (d, J = 4.2 Hz, 1H), 4.33 (t, J = 8.4 Hz, 1H), 4.22 (d, J = 9.7 Hz, 1H), 4.15 (d, J = 8.8 Hz, 1H), 3.81 (s, 2H), 2.73 (dd, J = 14.1, 5.9 Hz, 1H), 2.61 (dd, J = 14.0, 7.2 Hz, 1H), 2.29 (dq, J = 13.5, 6.8 Hz, 1H), 2.11 - 2.00 (m, 1H), 1.60 (dd, J = 15.2, 6.2 Hz, 2H), 1.35 (s, 9H), 1.20 (d, J = 6.9 Hz, 3H), 0.89 (dd, J = 14.0, 6.9 Hz, 6H). MS ESI m / z C 30 H 41 N2O6[M+H] + : Calculated 525.28, Found 525.37.

[0492] Example 42 Synthesis of compound 42

[0493]

[0494] Compound 41 (208 g, 390 mmol) was dissolved in a mixture of tetrahydrofuran (2.1 L) and water (0.7 L), and a solution of LiOH (23.7 g, 0.99 mol) in H2O2(30% in water, 336 mL, 2.97 mol) was added dropwise with ice bath cooling, controlling the temperature not to exceed 5 °C. After 3 hours of reaction, a solution of sodium sulfite (1.5 M, 2 L) was added dropwise, and the pH was adjusted to 4 with 2 N HCl. The organic phase was extracted with ethyl acetate (3 x 800 mL), combined, washed with water (500 mL) and saturated brine (500 mL) once, dried over anhydrous sodium sulfate, filtered, concentrated, and purified on a silica gel column (pure petroleum ether to 3:1 petroleum ether / ethyl acetate) to give an oil (158 g, 96% yield). 1H NMR (400 MHz, CDC13) δ 7.46 - 7.28 (m, 5H), 7.07 (d, J = 7.7 Hz, 2H), 6.91 (d, J = 7.8 Hz, 2H), 5.04 (s, 2H), 4.52 (d, J = 8.5 Hz, 1H), 3.87 (d, J = 41.8 Hz, 1H), 2.82 - 2.43 (m, 3H), 1.85 (t, J = 12.2 Hz, 1H), 1.41 (s, 9H), 1.17 (d, J = 6.9 Hz, 3H). MS ESI m / z C 24 H 32 NO5[M+H] + : Calculated 414.22, Found 414.21.

[0495] Example 43 Synthesis of compound 43

[0496]

[0497] Compound 42 (158 g, 0.38 mmol) was dissolved in methanol (1.5 L), Pd / C (10 wt%, 15 g) was added, and the reaction was catalytically hydrogenated (1 atm H2) for 16 h, filtered, and the filtrate was concentrated to an oil (123 g, 100% yield). 1 H NMR (400 MHz, CDC13) δ 7.46 - 7.28 (m, 5H), 7.07 (d, J = 7.7 Hz, 2H), 6.91 (d, J = 7.8 Hz, 2H), 5.04 (s, 2H), 4.52 (d, J = 8.5 Hz, 1H), 3.87 (d, J = 41.8 Hz, 1H), 2.82 - 2.43 (m, 3H), 1.85 (t, J = 12.2 Hz, 1H), 1.41 (s, 9H), 1.17 (d, J = 6.9 Hz, 3H). MS ESI m / z C 17 H 26 NO5[M+H] + : Calculated 324.17, Found 324.16.

[0498] Example 44 Synthesis of compound 44

[0499]

[0500] Compound 43 (113 g, 0.35 mol) was dissolved in anhydrous tetrahydrofuran (1.5 L), and tert-butylnitrite (360 g, 3.5 mol) was added dropwise. The reaction was stirred at room temperature for 3 h, and upon completion, the reaction was concentrated and purified by silica gel column (pure petroleum ether to 2:1 petroleum ether / ethyl acetate) to give a yellow solid (85 g, 61% yield). 1H NMR (400 MHz, DMSO) δ 12.00 (s, 1H), 10.68 (s, 1H), 7.67 (s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.9 Hz, 1H), 3.56 (d, J = 3.8 Hz, 1H), 2.67 (dd, J = 13.5, 5.1 Hz, 1H), 2.41 (dd, J = 13.8, 6.6 Hz, 1H), 1.78 - 1.65 (m, 1H), 1.27 (s, 9H), 1.18 (s, 1H), 1.05 (d, J = 7.1 Hz, 3H). MS ESI m / z C 17 H 25 N2O7[M+H] + : Calculated 369.15, Found 369.14.

[0501] Example 45 Synthesis of compound 45

[0502]

[0503] Compound 44 (80 g, 217 mmol) was dissolved in methanol (500 mL), Pd / C (10 wt%, 2.0 g) was added, and catalytic hydrogenation (1 atm H2) was performed for 1 hour, filtered, and rotary evaporated to give a white solid (73 g, 93% yield). MS ESI m / z C 17 H 27 N2O5[M+H] + : Calculated 339.18, Found 339.17. 1 H NMR (400 MHz, MeOD) δ 6.60 (d, J = 7.9 Hz, 2H), 6.44 (d, J = 7.3 Hz, 1H), 3.71 (d, J = 6.3 Hz, 1H), 2.62 - 2.37 (m, 3H), 1.83 (ddd, J = 13.7, 9.9, 3.7 Hz, 1H), 1.39 (s, 9H), 1.13 (d, J = 7.1 Hz, 3H).

[0504] Example 46 Synthesis of compound 46

[0505]

[0506] Dissolve monomethyl ether of octaethylene glycol (115.2 g, 0.3 mol) in dry tetrahydrofuran (3 L). Add sodium hydride (60 wt%, 24 g, 0.6 mol) at room temperature. After stirring the reaction for 1 hour, add tert-butyl bromoacetate (146.3 g, 0.75 mol). Stir the reaction at room temperature for 1 hour. Add the reaction to 4 L of dichloromethane and add 2 kg of crushed ice while stirring. Separate the aqueous phase and extract with 1 L of dichloromethane. Combine the organic phases, wash with water, concentrate, and purify by column chromatography (20% ethyl acetate / petroleum ether, then 0 to 5% methanol / dichloromethane) to give the product 108 g (72% yield).

[0507] Example 47 Synthesis of compound 47

[0508]

[0509] Add compound 46 (210 g, 0.422 mol) to a mixture of dry formic acid (1 L) and dichloromethane (500 mL). Stir at room temperature overnight and concentrate to give the product 200 g (100% yield).

[0510] Example 48 Synthesis of compound 48

[0511]

[0512] Dissolve compound 48 (198 g, 0.422 mol) in 2.6 L of dichloromethane. Add 0.5 mL of DMF and oxalyl chloride (275 mL) dropwise at room temperature. Stir the reaction for 3 hours and concentrate to give the product 210 g.

[0513] Example 49 Synthesis of compound 49

[0514]

[0515] Mix compound Cbz-L-lysine (236.3 g, 0.844 mol), sodium carbonate (89.5 g, 0.844 mol), and sodium hydroxide (33.8 g, 0.844 mol) in 1.6 L of water. Cool to 0°C using an ice-salt bath and add a tetrahydrofuran (160 mL) solution of compound 48 (210 g, crude, 0.422 mol). After the addition is complete, stir the reaction at room temperature for 1 hour. Add ethyl acetate (1 L) and separate the aqueous phase after stirring. Adjust the pH to 3 to 4 using concentrated hydrochloric acid and extract with dichloromethane. Dry the organic phase over anhydrous sodium sulfate. Filter and concentrate to give the product 290 g (97% yield).

[0516] Example 50 Synthesis of compound 50

[0517]

[0518] Compound 49 (182.5 g, 0.26 mol) was dissolved in 2 L of dichloromethane. Pentafluorophenol (95.4 g, 0.52 mol) and DIC (131 g, 1.04 mol) were added at room temperature. The mixture was stirred for 1 hour, and the product was concentrated to obtain 430 g of crude product.

[0519] Example 51 Synthesis of Compound 51

[0520]

[0521] 62 g (0.39 mol) of tert-butyl 4-aminobutyrate was dissolved in 1.5 L of DMF. The mixture was cooled to ice water, and DIPEA (134.2 g, 1.04 mol) was added. Compound 50 (430 g, crude product, 0.26 mol) was slowly added while maintaining the temperature at 10-20°C. The reaction was carried out at room temperature for 1 hour. The mixture was concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 0.2 N hydrochloric acid, then with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (25%-100% ethyl acetate / petroleum ether, then 0-5% methanol / dichloromethane) yielded 180 g of the product, 82% yield.

[0522] Example 52 Synthesis of Compound 52

[0523]

[0524] Compound 51 (78 g, 92.3 mmol) and palladium on carbon (10 wt%, 13 g) were mixed in 500 mL of methanol. The mixture was reacted overnight under a hydrogen balloon at room temperature. The mixture was filtered, the filtrate was concentrated, and purified by column chromatography (0 to 20% methanol / dichloromethane) to give 70.2 g of the product (92% yield).

[0525] Example 53 Synthesis of Compound 53

[0526]

[0527] Compound 52 (17.3 g, 94.2 mmol) was dissolved in 500 mL of dichloromethane. Pentafluorophenol (34.7 g, 188.5 mmol) and DIC (47.5 g, 377 mmol) were added sequentially at room temperature. The mixture was stirred for 1 hour, and the solution was concentrated to give 105 g of crude product.

[0528] Example 54 Synthesis of Compound 54

[0529]

[0530] Compound 52 (67 g, 94.2 mmol) was dissolved in 0.75 L of DMF, cooled with ice water, and DIPEA (48.6 g, 376.8 mmol) was added. The reaction mixture was kept at 10°C to 20°C, and compound 53 (105 g, crude product, 94.2 mmol) was slowly added. The reaction was carried out at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 0.2 N hydrochloric acid, then with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (50%–100% ethyl acetate / petroleum ether, then 10% methanol / dichloromethane) yielded 80.5 g of the product, 98% yield.

[0531] Example 55 Synthesis of Compound 55

[0532]

[0533] Compound 54 (80.5 g, 91.9 mmol) was added to a mixed solvent of anhydrous formic acid (400 mL) and dichloromethane (200 mL). The reaction was allowed to proceed overnight at room temperature. The mixture was concentrated, diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (0% to 20% methanol / dichloromethane) to give 70 g of the product (93% yield).

[0534] Example 56 Synthesis of Compound 56

[0535]

[0536] Compound 55 (104 g, 0.127 mol) was dissolved in dichloromethane (1000 mL), and N-hydroxysuccinimide (16 g, 0.14 mol) and EDC·HCl (37 g, 0.2 mol) were added sequentially at room temperature. The reaction was allowed to proceed for 1 hour at room temperature. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 120 g of the product (100% yield).

[0537] Example 57 Synthesis of Compound 57

[0538]

[0539] Compound 56 (120 g, 127 mmol) and compound 45 (45.0 g, 133 mmol) were mixed in 1 L of tetrahydrofuran. The mixture was heated to reflux overnight, concentrated, diluted with 2 L of dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. 1.5 L of water and 1.5 L of ethyl acetate were added to the crude product, and the mixture was stirred for half an hour. The aqueous phase was separated, and the ethyl acetate layer was extracted twice with water (600 mL × 2). The aqueous phases were combined and washed with ethyl acetate (700 mL) to remove impurities. Sodium chloride was added to the aqueous phase until saturated, and the mixture was extracted with dichloromethane (1 L × 2). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by column chromatography (0% to 20% methanol / dichloromethane) to give 83.6 g (58% yield). 1 H NMR (600MHz, DMSO) δ9.51 (bs, 1H), 9.23 (s, 1H), 7.97–7.83 (m, 1H), 7.64 (t, J=5.8Hz, 1H), 7.46 (s, 1H), 6.99 (s, 1H), 6.73 (s, 1H), 6.61 (d, J= 8.7Hz, 0H), 4.14 (dt, J=5.4, 8.3Hz, 1H), 3.84 (s, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 3.51–3.46 (m, 26H), 3.42 (dd, J=5.7, 3.8Hz, 2H), 3.39 (t, J=7 ... 9Hz, 2H), 1.75–1.64 (m, 4H), 1.64–1.54 (m, 1H), 1.52–1.43 (m, 1H), 1.4 3–1.35 (m, 2H), 1.32 (s, 9H), 1.32–1.15 (m, 4H), 1.02 (d, J=7.1Hz, 3H). 13C NMR (151MHz, DMSO) δ177.18, 171.68, 171.43, 171.28, 171.08, 169.02, 155.15, 14 5.99, 134.45, 129.41, 125.91, 125.30, 123.04, 115.71, 77.29, 71.29, 70.25, 69.9 5, 69.79, 69.59, 69.53, 58.05, 52.57, 50.33, 40.71, 38.12, 37.93, 37.64, 36.83, 35.92, 33.35, 32.37, 31.85, 28.95, 28.26, 27.86 (minor), 25.39, 24.18, 22.85, 18.10. ESI m / z C 54 H 89 N6O 20 [M+H] + Calculated value: 1141.6, measured value: 1141.9.

[0540] Example 58 Synthesis of Compound 58

[0541]

[0542] Compound 57 (59.0 g, 51.7 mmol) was dissolved in dichloromethane (345 mL), and trifluoroacetic acid (172 mL) was added. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated on a rotary evaporator until no solvent was distilled off. Then, it was concentrated under vacuum on a vacuum pump until the weight remained essentially constant, yielding a crude product. The crude product was extracted with diethyl ether (600 mL), and the product layer was separated and concentrated under reduced pressure until no solvent was distilled off. Then, it was concentrated under vacuum on a vacuum pump until the weight remained essentially constant, yielding compound 58 (75.5 g, containing solvent). 1H NMR (600MHz, DMSO) δ9.82 (s, 1H), 9.18 (s, 1H), 7.95-7.88 (m, 2H), 7.83 (s, 2H) ), 7.67 (s, 1H), 7.66 (t, J = 6.1Hz, 1H), 6.99 (s, 2H), 6.84-6.78 (m, 2H), 4.16– 4.09 (m, 1H), 3.84 (s, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 3.52–3.44 (m, 26H), 3.4 2(dd, J=5.6, 3.9Hz, 2H), 3.39(t, J=7.0Hz, 2H), 3.33–3.25(m, 1H), 3.23(s, 3H ), 3.14–3.02(m, 4H), 2.76(dd, J=13.9, 6.1Hz, 1H), 2.65(dd, J=13.9, 6.1Hz, 1H), 2.55(dq, J=14.0, 7.0Hz, 1H), 2.38(t, J=7.3Hz, 2H), 2.14–2.08(m, 2H), 1.82 (ddd, J=14.1, 8.7, 5.5Hz, 1H), 1.74–1.65 (m, 4H), 1.64–1.55 (m, 1H), 1. 52–1.43 (m, 2H), 1.43–1.34 (m, 2H), 1.30–1.13 (m, 2H), 1.05 (d, J=7.0Hz, 3H). 13 C NMR (151MHz, DMSO) δ176.54, 171.73, 171.43, 171.34, 171.11, 169.07, 14 6.59, 134.48, 126.50, 126.33, 125.19, 122.85, 115.84, 71.30, 70.27, 69. 96, 69.81, 69.61, 69.55, 58.06, 52.65, 50.72, 38.31, 38.13, 37.95, 36.8 5, 35.75, 34.88, 33.45, 32.38, 31.82, 28.97, 25.40, 24.20, 22.88, 17.48. ESI m / z C 49 H 81 N6O 18 [M+H] + Calculated value: 1041.6, measured value: 1041.7.

[0543] Example 59 Synthesis of Compound 59

[0544]

[0545] Add a 160 mL solution of DMF containing 75.5 g (51.7 mmol) of compound 58 to the reaction flask and cool to 5°C in an ice-water bath. Add a 240 mL solution of DMF containing 35.8 g (51.7 mmol) of compound 18, followed by dropwise addition of DIPEA (36.8 g, 285 mmol). After the addition is complete, remove from the ice-water bath, bring to room temperature, and stir for 10 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure using a vacuum oil pump until no solvent evaporates. After concentration, dilute the concentrate with dichloromethane, cool to 5°C in an ice-water bath, and slowly add formic acid to adjust the pH to 3.0-4.0. Then, concentrate on a rotary evaporator until no solvent evaporates. Transfer the residue to a silica gel column and elute with n-hexane / ethyl acetate / formic acid and dichloromethane / methanol / formic acid. Concentrate to obtain the purified crude product. The crude product was dissolved and purified by water / methanol / formic acid, and further purified by preparative HPLC. The product was eluted with water / acetonitrile / formic acid, and a suitable eluent was collected. After concentration, the concentrate was diluted with water and evenly distributed into lyophilization flasks for lyophilization to obtain a pale yellow foamy solid (48 g, 60% yield). 1H NMR(600MHz,DMSO)δ9.20(s,1H),8.17(s,1H),8.03-7.95(m,1H),7.95–7.86(m,2H),7.77-7.61(m,2H),7.55(s,1H),6.98(s,2H),6.77(d,J=8.1Hz,1H),6.71(d,J=8.1Hz,1H),5.58(d,J=10.7Hz,1H),4.65(dd,J=9.3,7.1Hz,1H),4.40(s,1H),4.18–4.08(m,2H),3.85(s,2H),3.55(s,2H),3.52(s,2H),3.51-3.47(m,26H),3.44–3.36(m,4H),3.23(s,3H),3.14–3.02(m,4H),2.96(s,3H),2.75(dd,J=13.5,6.7Hz,1H),2.63(dd,J=13.5,6.7Hz,1H),2.43–2.32(m,3H),2.31–2.23(m,1H),2.23–2.09(m,3H),2.13(s,6H),2.09(s,3H),1.88–1.73(m,3H),1.75–1.65(m,4H),1.65–1.53(m,2H),1.53–1.44(m,3H),1.44–1.34(m,2H),1.31–1.15(m,2H),1.12(s,3H),1.05(d,J=6.9Hz,3H),1.01(s,3H),0.93(d,J=6.4Hz,3H),0.86(d,J=6.6Hz,3H),0.82(t,J=7.3Hz,3H),0.67(d,J=6.1Hz,3H)。 13C NMR (151MHz, DMSO) δ177.05, 175.28, 172.76, 171.74, 171.47, 171.34, 171.11, 16 9.78, 169.49, 169.09, 159.91, 149.88, 146.10, 134.47, 129.10, 126.11, 125.23, 124.12, 122.97, 115.72, 71.33, 70.30, 69.99, 69.83, 69.63, 69.57, 69.52, 63.21, 58.08, 55.05, 52.63, 52.49, 49.07, 40.37, 38.89, 38.14, 37.97, 37.4 3, 36.87, 36.50, 36.06, 33.91, 33.41, 32.41, 31.87, 29.06, 28.99, 25.40, 24 .22, 23.66, 22.88, 21.08, 20.63, 20.00, 19.43, 18.41, 18.13, 15.68, 10.81. ESI m / z C 74 H 121 N 10 O 23 S[M+H] + Calculated value: 1549.8, measured value: 1550.2.

[0546] Example 60 Synthesis of Compound 60

[0547]

[0548] Octadecyl glycol monomethyl ether (10 g, 26 mmol, 1.0 eq) was dissolved in 100 mL of anhydrous dichloromethane. DMAP (32 mg, 0.26 mmol, 0.01 eq) was added, followed by the dropwise addition of triethylamine (10.5 g, 104 mmol, 4.0 eq) and TsCl (14.9 g, 78 mmol, 3.0 eq) solid under ice bath conditions. After completion, the reaction was allowed to proceed for 10 min, then heated to room temperature and reacted overnight. The next day, the reaction solution was washed with 1 N HCl (100 mL), water (100 mL), and brine (100 mL), dried over anhydrous sodium sulfate, evaporated to dryness, dissolved in a small amount of dichloromethane, and loaded onto a column. The eluent was 5%–100% petroleum ether / ethyl acetate and 1%–3% methanol / dichloromethane, yielding 11.6 g of a yellow oily substance, with a yield of 83%. ESI m / z C 24 H 43 O 11 S[M+H] + Calculated value: 539.2, measured value: 539.2.

[0549] Example 61 Synthesis of Compound 61

[0550]

[0551] Compound 60 (11.6 g, 21.5 mmol, 1.0 eq) was dissolved in 20 mL of anhydrous DMF, and dibenzylamine (5.5 g, 27.8 mmol, 1.5 eq) was added. The mixture was reacted overnight at 100 °C. The next day, the solution was diluted with 300 mL of dichloromethane, washed with water (300 mL × 3), washed with brine (300 mL), dried over anhydrous sodium sulfate, evaporated to dryness, dissolved in a small amount of dichloromethane, loaded onto a column, and eluent was 5%–100% petroleum ether / ethyl acetate to give 8.2 g of a pale yellow oil, yield 66%. ESI m / z C 31 H 50 NO8[M+H] + Calculated value: 564.3, measured value: 564.3.

[0552] Example 62 Synthesis of Compound 62

[0553]

[0554] Compound 61 (8.6 g, 15.2 mmol, 1.0 eq) was dissolved in 100 mL of anhydrous methanol, and dry palladium on carbon catalyst (0.9 g, 10 wt%) was added. The mixture was heated to reflux and reacted overnight. The next day, the reaction solution was filtered, washed with methanol, and evaporated to dryness to give 5.3 g of a colorless oil, with a yield of 90%. (ESI m / z C) 17 H 38 NO8[M+H] + Calculated value: 384.3, measured value: 384.3.

[0555] Example 63 Synthesis of Compound 63

[0556]

[0557] ZL-tert-butyl glutamate (0.96 g, 2.86 mmol) and compound 62 (1.1 g, 2.86 mmol) were dissolved in DMF (20 mL). HATU (1.2 g, 3.15 mmol) and DIPEA (1 mL, 6.3 mmol) were added at 0 °C, and the mixture was slowly brought to room temperature for 1 hour. The reaction mixture was poured into ice water and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with water (30 mL), saturated sodium bicarbonate (30 mL), and saturated brine (30 mL), dried over sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane) to give product 63 (1.5 g, 75% yield). ESI m / z C 34 H 59 N2O13 [M+H] + Calculated value: 703.4, measured value: 703.4.

[0558] Example 64 Synthesis of Compound 64

[0559]

[0560] Compound 63 (0.66 g, 0.93 mmol) was dissolved in methanol (10 mL), and Pd / C (10 wt%, 60 mg) was added. The reaction was carried out under a hydrogen balloon for 2 hours. The mixture was then filtered and concentrated to give compound 64 (450 mg, 85% yield). ESI m / z C 26 H 53 N2O 11 [M+H] + Calculated value: 569.4, measured value: 569.4.

[0561] Example 65 Synthesis of Compound 65

[0562]

[0563] Compound 64 (0.45 g, 0.79 mmol) and 4-maleimide butyric acid-N-succinimide ester (0.33 g, 1.18 mmol) were dissolved in ethanol (5 mL), and NaH₂PO₄ (0.1 N, 1 mL) was added. The mixture was reacted overnight at room temperature. After removing most of the ethanol by vacuum concentration, water (20 mL) was added, and the mixture was extracted with dichloromethane (3 × 30 mL). The organic phases were combined, washed with water (20 mL), washed with brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (mobile phase: methanol / dichloromethane) to give compound 65 (380 mg, 65% yield). ESI m / z C 34 H 60 N3O 14 [M+H] + Calculated value: 734.4, measured value: 734.4.

[0564] Example 66 Synthesis of Compound 66

[0565]

[0566] Compound 65 (230 mg, 0.31 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added. The reaction was carried out at room temperature for 1 hour. After concentration, the solution was carried out three times with dichloromethane and then dried using an oil pump to give compound 66 (208 mg, 100% yield). ESI m / z C 30 H 52 N3O 14 [M+H]+ Calculated value: 678.3, measured value: 678.3.

[0567] Example 67 Synthesis of Compound 67

[0568]

[0569] Compound 66 (208 mg, 0.3 mmol) was dissolved in dichloromethane (5 mL), and pentafluorophenol (113 mg, 0.6 mmol) and EDC·HCl (117 mg, 0.6 mmol) were added. The reaction was allowed to proceed overnight at room temperature. The solution was diluted with dichloromethane (20 mL), washed with water (5 mL), dried over sodium sulfate, filtered, and concentrated to give compound 67 (252 mg, 100% yield). ESI m / z C 36 H 51 F5N3O 14 [M+H] + Calculated value: 844.3, measured value: 844.3.

[0570] Example 68 Synthesis of Compound 68

[0571]

[0572] Compound 45 (7.3 g, 21.7 mmol) and N-Boc-alanine hydroxysuccinimide ester (7.2 g, 21.7 mmol) were dissolved in ethanol (300 mL), and 0.1 N NaH₂PO₄ (150 mL) was added. The mixture was reacted overnight at room temperature. The reaction solution was concentrated, and water (100 mL) was added. The mixture was extracted with ethyl acetate (3 × 50 mL), and the organic phases were combined, washed with brine (50 mL), dried over sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether) to give compound 68 (6.7 g, 55% yield). ESI m / z C 29 H 40 N3O8[M+H] + Calculated value: 558.3, measured value: 558.3.

[0573] Example 69 Synthesis of Compound 69

[0574]

[0575] Compound 68 (6.7 g, 12 mmol) was dissolved in methanol (100 mL), and Pd / C (10 wt%, 0.67 g) was added. The mixture was reacted under a hydrogen balloon for 2 hours. The mixture was filtered, and the filtrate was concentrated to give compound 69 (5 g, 100% yield). ESI m / z C 21 H 34 N3O6[M+H] +Calculated value: 424.2, measured value: 424.2.

[0576] Example 70 Synthesis of Compound 70

[0577]

[0578] Compound 67 (252 mg, 0.3 mmol) and compound 69 (190 mg, 0.45 mmol) were dissolved in DMF (5 mL), cooled to 0 °C, and DIPEA (0.13 mL, 0.75 mmol) was added. After the addition was complete, the mixture was slowly brought to room temperature and reacted for 1 hour. The mixture was diluted with water (20 mL), extracted with dichloromethane (3 × 10 mL), and the organic phases were combined. The mixture was washed with water (10 mL), 1N HCl (10 mL), and brine (10 mL), dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (mobile phase: methanol / dichloromethane) to give compound 70 (180 mg, 55% yield). ESI m / z C 51 H 83 N6O 19 [M+H] + Calculated value: 1083.6, measured value: 1083.6.

[0579] Example 71 Synthesis of Compound 71

[0580]

[0581] Compound 70 (8.2 g, 7.6 mmol) was dissolved in dichloromethane (56.8 mL), and trifluoroacetic acid (18.9 mL) was added. The mixture was reacted at room temperature for 2 hours, concentrated, and then concentrated twice more with dichloromethane. The mixture was then dried under an oil pump. Diethyl ether (100 mL) was added to the residual liquid, and the mixture was stirred vigorously for 1 hour. After standing, the supernatant was discarded, and the process was repeated twice. The lower layer was concentrated on a rotary evaporator and dried under an oil pump to give compound 71 (9.2 g, >100% yield). ESI m / z C 46 H 75 N6O 17 [M+H] + Calculated value: 983.51, measured value: 983.37.

[0582] Example 72 Synthesis of Compound 72

[0583]

[0584] Compound 71 (8.2 g, 7.6 mmol) was dissolved in DMF (80 mL). The reaction flask was cooled to 0-5°C using an ice-water bath. 20 mL of DMF solution containing 18 (5.2 g, 7.6 mmol) was added, followed by slow dropwise addition of DIPEA (4 mL, 22.8 mmol). The dropping rate was controlled to maintain the reaction solution temperature between 5-10°C throughout the addition process. After the addition was complete, the ice-water bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for 1.5 hours. Concentrate the extract and add dichloromethane (100 mL). Adjust the pH to 3-4 with formic acid under ice bath conditions. Concentrate again and purify by silica gel column chromatography. The eluent is 20-100% ethyl acetate / n-hexane and 0-20% methanol / dichloromethane (each containing 0.1% formic acid). The crude product (11.44 g) obtained from silica gel column chromatography is further purified by preparative HPLC with 20-30% acetonitrile / water (each containing 0.1% formic acid). Concentrate and lyophilize to give compound 72 (6.8 g, yield 60%). ESI m / z C 71 H 115 N 10 O 22 S[M+H] + Calculated value: 1491.78, measured value: 1492.01.

[0585] Example 73 Synthesis of Compound 73

[0586]

[0587] Compound 63 (22.4 g, 0.03 mol) was added to a mixed solvent of anhydrous formic acid (500 mL) and dichloromethane (250 mL). The mixture was stirred overnight at room temperature and concentrated to give 19 g of the product (100% yield).

[0588] Example 74 Synthesis of Compound 74

[0589]

[0590] Compound 73 (19.0 g, 0.03 mol) was dissolved in dichloromethane (200 mL). Pentafluorophenol (11.0 g, 0.06 mol) and DIC (15.1 g, 0.12 mol) were added at room temperature. The mixture was stirred for 1 hour, and the product was concentrated to give 45 g of crude product.

[0591] Example 75 Synthesis of Compound 75

[0592]

[0593] 6.40 g (0.04 mol) of tert-butyl 4-aminobutyrate was dissolved in 500 mL of DMF. The mixture was cooled to ice water, and DIPEA (15.5 g, 0.12 mol) was added. Compound 74 (45 g, crude product, 0.03 mol) was slowly added while maintaining the temperature between 10 and 20°C. The reaction was allowed to proceed at room temperature for 1 hour. The mixture was concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 0.2 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (25%–100% ethyl acetate / petroleum ether, followed by 0–5% methanol / dichloromethane) yielded 19.5 g of the product, 83% yield.

[0594] Example 76 Synthesis of Compound 76

[0595]

[0596] Compound 75 (19.5 g, 24.8 mmol) and palladium on carbon (10 wt%, 5 g) were mixed in 200 mL of methanol. The mixture was reacted overnight at room temperature under a hydrogen balloon. The mixture was filtered, and the filtrate was concentrated to give 16.7 g of product (100% yield).

[0597] Example 77 Synthesis of Compound 77

[0598]

[0599] Compound 76 (16.7 g, 24.8 mmol) was dissolved in 200 mL of DMF, cooled with ice water, and DIPEA (12.9 g, 0.10 mol) was added. The reaction mixture was kept at 10°C to 20°C, and compound 53 (30 g, crude product, 24.8 mmol) was slowly added. The reaction was carried out at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with 0.2 N hydrochloric acid, then with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (50%–100% ethyl acetate / petroleum ether, followed by 10% methanol / dichloromethane) yielded 20 g of product, 98% yield.

[0600] Example 78 Synthesis of Compound 78

[0601]

[0602] Compound 77 (16.8 g, 20.5 mmol) was dissolved in dichloromethane (60 mL), and anhydrous formic acid (120 mL) was added. The reaction was carried out overnight at room temperature, concentrated, diluted with ethyl acetate (150 mL), extracted with water (300 mL), and the organic phase was discarded. The aqueous phase was saturated with solid sodium chloride, then extracted with dichloromethane (200 mL × 2), and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to give compound 78 (16.4 g, yield >100%, containing some formic acid). ESI m / z C 34 H 59 O 15 N4[M+H] + Calculated value: 763.39, measured value: 763.29.

[0603] Example 79 Synthesis of Compound 79

[0604]

[0605] Compound 78 (15.6 g, 20.5 mol) was dissolved in dichloromethane (200 mL). At room temperature, N-hydroxysuccinimide (NHS, 3.7 g, 32.3 mol) and EDC·HCl (8.3 g, 43 mol) were added sequentially. The reaction was allowed to proceed for 30 minutes at room temperature. The mixture was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 79 (17.6 g, 100% yield). ESI m / z C 38 H 62 O 17 N5[M+H] + Calculated value: 860.41, measured value: 860.29.

[0606] Example 80 Synthesis of Compound 80

[0607]

[0608] Method 1:

[0609] Compound 79 (8.8 g, 10.2 mmol) and compound 45 (3.5 g, 10.2 mmol) were dissolved in 200 mL of tetrahydrofuran. The mixture was heated to reflux and stirred overnight. The solution was concentrated, and water (300 mL) and ethyl acetate (100 mL) were added to the crude product. The mixture was stirred, and the aqueous phase was separated. Sodium chloride was added to the aqueous phase until saturated, and the mixture was extracted with dichloromethane (2 × 150 mL). The dichloromethanes were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The solution was purified by column chromatography (0–20% methanol / dichloromethane) to give compound 80 (4.0 g, 36% yield). ESI m / z C 51 H 83 O 19N6[M+H] + Calculated value: 1083.56, measured value: 1083.47.

[0610] Method 2:

[0611] Compound 79 (4.4 g, 5.12 mmol) and compound 45 (1.73 g, 5.12 mmol) were dissolved in 100 mL of EtOH. Then, 20 mL of 0.1 N NaH₂PO₄ solution was added, and the mixture was stirred overnight. The solution was concentrated, and water (200 mL) and ethyl acetate (100 mL) were added to the crude product. The mixture was stirred, and the aqueous phase was separated. Sodium chloride was added to the aqueous phase until saturated, and the mixture was extracted with dichloromethane (2 × 100 mL). The dichloromethanes were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The solution was purified by column chromatography (0–20% methanol / dichloromethane) to give compound 80 (2.0 g, 36% yield).

[0612] Method 3:

[0613] Compound 79 (4.4 g, 5.12 mmol) and compound 45 (1.73 g, 5.12 mmol) were dissolved in 100 mL of acetonitrile. Then, 20 mL of 0.1 N NaH₂PO₄ solution was added, and the mixture was stirred overnight. The solution was concentrated, and water (200 mL) and ethyl acetate (100 mL) were added to the crude product. The mixture was stirred, and the aqueous phase was separated. Sodium chloride was added to the aqueous phase until saturated, and the mixture was extracted with dichloromethane (2 × 100 mL). The dichloromethanes were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The solution was purified by column chromatography (0–20% methanol / dichloromethane) to give compound 80 (2.2 g, 40% yield).

[0614] Example 81 Synthesis of Compound 73

[0615]

[0616] Compound 49 (20 g, 28.4 mmol, 1.0 eq.) was dissolved in 350 mL of anhydrous dichloromethane and cooled on an ice-water bath. NHS (3.9 g, 34.1 mmol, 1.2 eq.) and EDC (27 g, 142.0 mmol, 5.0 eq.) were added sequentially. The reaction mixture was stirred overnight at room temperature, then washed with water (200 mL × 2) and brine (200 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of dichloromethane and loaded onto a silica gel column, eluted with methanol / ethyl acetate / dichloromethane in a ratio of 2:49:49 to 4:48:48. The product was a yellow oil (14.2 g, 62% yield). ESI m / z C 37 H 60 N3O 16 [M+H] +Calculated value: 802.4, Measured value: 802.4.

[0617] Example 82 Synthesis of compound 74.

[0618]

[0619] Compound 73 (12.7 g, 15.9 mmol, 1.0 eq) was added to a mixture of 40 mL ethanol and 10 mL 0.1 M NaH₂PO₄ to compound 69 (6.4 g, 15.1 mmol, 1.0 eq). The reaction mixture was stirred overnight, concentrated, and dissolved in dichloromethane. The solution was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (3–5% methanol / dichloromethane) to give a white foam (11.7 g, 70% yield). ESI m / z C 54 H 88 N5O 19 [M+H] + Calculated value, measured value: 1110.6, measured value: 1110.6.

[0620] Example 83 Synthesis of compound 75.

[0621]

[0622] Compound 74 (4.2 g, 3.79 mmol, 1.0 eq.) and palladium on carbon (0.4 g, 10 wt%) were mixed in 5 mL of methanol. The mixture was stirred overnight under a hydrogen balloon at room temperature. The catalyst was filtered off and washed with methanol. The filtrate was concentrated to give 0.32 g of crude product, which was used directly in the next step (yield 87%). ESI m / z C 46 H 82 N5O 17 [M+H] + Calculated value: 1997.1, Actual measured value: 1997.1.

[0623] Example 84 Synthesis of compound 76.

[0624]

[0625] In a 500 mL flask, H₂N-PEG₄-CH₂CH₂CO₂H (3.0 g, 11.3 mmol, 1.0 eq.) and K₂CO₃ (4.7 g, 33.93 mmol, 3.0 eq.) were dissolved in 50 mL of water and cooled on an ice-water bath. A 50 mL solution of Boc₂O (3.2 g, 14.7 mmol, 1.3 eq.) in tetrahydrofuran was added dropwise. The reaction mixture was warmed to room temperature and then stirred overnight. The reaction mixture was adjusted to pH 4–5 with 1 N KHSO₄, extracted with dichloromethane (200 mL × 1, 100 mL × 3), washed with water (500 mL × 1) and brine (500 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of dichloromethane, then loaded onto a silica gel column and eluted with 2-4% methanol / dichloromethane. The fractions were combined and concentrated to give 3.8 g of a colorless oil (93% yield). ESI m / z C 16 H 32 NO8[M+H] + Calculated value: 366.2, measured value: 366.2.

[0626] Example 85 Synthesis of Compound 77

[0627]

[0628] In a 50 mL single-necked flask, BocHN-PEG4-CH2CH2CO2H (0.81 g, 2.22 mmol, 1.0 eq.), K2CO3 (0.92 g, 6.66 mmol, 3.0 eq.), and NaI (0.033 g, 0.222 mmol, 0.1 eq.) were mixed in 10 mL of DMF and cooled in an ice-water bath. BnBr (0.57 g, 3.33 mmol, 1.5 eq.) was added dropwise, and the mixture was warmed to room temperature and stirred overnight. The reaction mixture was diluted with 100 mL of water, extracted with dichloromethane (100 mL × 2), washed with water (200 mL × 1) and brine (200 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of dichloromethane, loaded onto a silica gel column, and eluted with 70-90% ethyl acetate / petroleum ether to give 0.69 g of a colorless oil (69% yield). ESI m / z C 23 H 38 NO8[M+H] + Calculated value: 446.3, measured value: 446.3.

[0629] Example 86 Synthesis of Compound 78

[0630]

[0631] 6 mL of dichloromethane and 3 mL of TFA solution containing BocHN-PEG4-CH2CH2CO2Bn (0.69 g, 1.5 mmol, 1.0 eq.) were stirred at room temperature for 30 minutes. The solvent was removed, and the residue was co-concentrated three times with dichloromethane. The crude product was then placed on a high vacuum pump and used directly in the next reaction. ESI m / z C 18 H 30 NO6[M+H] + Calculated value: 356.2, measured value: 356.2.

[0632] Example 87 Synthesis of Compound 79

[0633]

[0634] To a solution of BocHN-PEG4-CH2CH2CO2H (3.8 g, 10.4 mmol, 1.0 eq.) in 50 mL of anhydrous dichloromethane, NHS (1.4 g, 12.5 mmol, 1.2 eq.) and EDC (10.0 g, 52.0 mmol, 5.0 eq.) were added, and the mixture was stirred overnight at room temperature. The mixture was then washed with water (50 mL × 2) and brine (100 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The crude product was used directly in the next step. ESI m / z C 20 H 35 N2O 10 [M+H] + Calculated value: 463.2, Actual measured value: 463.2.

[0635] Example 88 Synthesis of Compound 80

[0636]

[0637] In a 300 mL flask, H₂N-PEG₄-CH₂CH₂CO₂H (2.8 g, 10.4 mmol, 1.0 eq.) and K₂CO₃ (4.3 g, 31.2 mmol, 3.0 eq.) were dissolved in 40 mL of water. The mixture was cooled in an ice-water bath, and a tetrahydrofuran solution (40 mL) of compound 79 (3.8 g, 10.4 mmol, 1.0 eq.) was added dropwise. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was adjusted to pH 4–5 using 1 N KHSO₄, extracted with dichloromethane (150 mL × 1, 100 mL × 2), washed with water (200 mL × 1) and brine (200 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in a small amount of dichloromethane and loaded onto a silica gel column, eluted with 4–6% methanol / dichloromethane, to give a colorless oil (5.18 g, 81% yield). ESI m / z C 27 H53 N2O 13 [M+H] + Calculated value: 613.3, Measured value: 613.3.

[0638] Example 89 Synthesis of Compound 81

[0639]

[0640] H2N-PEG4-CH2CH2CO2Bn (from the crude product of the previous step) was dissolved in 3 mL of DMF and cooled on an ice / water bath. DIPEA (0.78 g, 6.0 mmol, 4.0 eq.) was added dropwise, followed by a solution of compound 80 (0.93 g, 1.5 mmol, 1.0 eq.) in DMF (7 mL) and HATU (1.72 g, 4.5 mmol, 3.0 eq.). The reaction mixture was stirred on an ice bath for 2 hours and diluted with 100 mL of water. Extraction was performed with dichloromethane (100 mL × 3), followed by washing with 1 N KHSO4 (200 mL × 1), saturated sodium bicarbonate (200 mL × 1), and brine (200 mL × 1). The mixture was dried over anhydrous sodium sulfate and concentrated. The residue was dissolved in a small amount of dichloromethane, loaded onto a silica gel column, and eluted with 0–5% methanol / dichloromethane. The samples were combined and concentrated to give 1.0 g of a pale yellow oil (71% yield). ESI m / z C 45 H 80 N3O 18 [M+H] + Calculated value: 950.5, measured value: 950.5.

[0641] Example 90 Synthesis of Compound 82

[0642]

[0643] In a DMF (50 mL) solution of benzyl 11-aminoundecanoate (2.91 g, 10.0 mmol) and Boc-Glu(OBzl)-OH (3.37 g, 10.0 mmol), EDC (1.91 g, 12.0 mmol) and triethylamine (3.5 mL, 25.0 mmol) were added. The mixture was stirred at room temperature for 8 hours, diluted with water (100 mL), and extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed once with 100 mL of brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / dichloromethane, 1:15) to give the title compound as a colorless oil (5.37 g, 88% yield).

[0644] Example 91 Synthesis of Compound 83

[0645]

[0646] Compound 82 (0.64 g, 1.05 mmol, 1.0 eq.) was stirred in a mixture of 5 mL dichloromethane and 2 mL TFA for 2 hours at room temperature, and then concentrated. The residue was co-concentrated three times with dichloromethane under high vacuum. The crude product was redissolved in 3 mL DMF and cooled on an ice-water bath. A DMF solution (7 mL) of compound 80 (0.64 g, 1.05 mmol, 1.0 eq.) was added, followed by DIPEA (0.54 g, 4.20 mmol, 4.0 eq.) and HATU (1.2 g, 3.15 mmol, 3.0 eq.). The reaction mixture was stirred on an ice bath for 1 hour, then 100 mL of water was added, and the mixture was extracted with dichloromethane (150 mL × 1, 100 mL × 1). The organic phase was washed with 1N KHSO4 (200 mL × 1), saturated sodium bicarbonate (200 mL × 1), and brine (200 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in a small amount of dichloromethane and loaded onto a silica gel column, then eluted with 0–10% methanol / dichloromethane. The fractions were combined and concentrated to give 0.94 g of a pale yellow oil (81% yield). ESI m / z C 57 H 92 N4O 17 [M+H] + Calculated value: 1104.6, measured value: 1104.6.

[0647] Example 92 Synthesis of Compound 84

[0648]

[0649] At 0 °C, HATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol) were added to a DMF (18 mL) solution of tert-butyl 4-aminobutyrate (1.03 g, 6.12 mmol) and compound 49 (3.91 g, 5.56 mmol). The reaction was stirred for 1 h, 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 the title compound (5.10 g, 99% yield). ESI MS m / z 846.50 ([M+H) + )

[0650] Example 93 Synthesis of Compound 85

[0651]

[0652] Compound 84 (1.0 g, 1.18 mmol) and Pd / C (10 wt%, 0.10 g) were added to a hydrogenation flask containing methanol (50 mL). The mixture was shaken for 2 hours, filtered through diatomaceous earth (filter aid), and the filtrate was concentrated to give compound 85 (0.93 g, yield >100%). ESI MS m / z 712.50 ([M+H) + ).

[0653] Example 94 Synthesis of Compound 86

[0654]

[0655] Compound 85 was dissolved in 95% EtOH (50 mL) and NaH₂PO₄ solution (0.1 M, pH 5.0, 10 mL), and N-succinimide-4-maleimide-butyrate (0.50 g, 1.77 mmol, 1.5 eq) was added. The mixture was stirred overnight, then concentrated, 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). The title compound was given as a pale yellow oil (0.82 g, 80%).

[0656] Example 95 Synthesis of compound 87.

[0657]

[0658] Compound 86 (0.82 g, 0.94 mmol) was dissolved in 50 mL of HCOOH and stirred at room temperature for 1 hour. The reaction mixture was concentrated twice with toluene, and the residue was placed on a vacuum pump to give compound 87 (0.80 g, crude product). ESI MS m / z ([M+H) + Value: 820.45.

[0659] Example 96 Synthesis of Compound 88

[0660]

[0661] To a DMA (5.0 mL) solution of compound 87 (0.80 g, crude, 0.94 mmol), NHS (0.12 g, 1.03 mmol) and EDC·HCl (0.27 g, 1.41 mmol) were added, and the reaction mixture was stirred for 2 hours. The mixture was 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 chromatography (10–50% ethyl acetate / petroleum ether) to give a colorless oily compound (0.67 g, 78% yield). ESI MS m / z ([M+H)) + Value: 918.55

[0662] Example 97 Synthesis of Compound 89

[0663]

[0664] A mixture of N-Boc-ethylenediamine (5.6 mL, 35.4 mmol, 1.1 eq.) and saturated NaHCO3 (60 mL) was cooled to 0 °C, and N-methoxycarbonylmaleimide (5.00 g, 32.2 mmol, 1.0 eq.) was added in portions. The mixture was stirred at 0 °C for 30 min, then warmed to room temperature and stirred for 1 h. The precipitate was collected by filtration, washed with cold water, dissolved in ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated to give a white solid (6.69 g, 87% yield). ESI MS m / z ([M+H)) + Value: 241.12.

[0665] Example 98 Synthesis of Compound 90

[0666]

[0667] In a high-pressure tube, a solution of compound 89 (6.00 g, 25.0 mmol), furan (18.0 mL), and toluene (120 mL) was heated to reflux and stirred for 16 hours. The colorless solution turned yellow during the reaction. The mixture was then cooled to room temperature and concentrated. The resulting white solid was slurried with diethyl ether to give compound 90 (6.5 g, 84% yield). ESI MS m / z ([M+H)) + Value: 309.13.

[0668] Example 99 Synthesis of Compound 91

[0669]

[0670] A solution of compound 90 (9.93 g, 32.2 mmol) in dioxane (15 mL) was reacted with concentrated HCl (15 mL) for 3 hours at room temperature. The resulting solid was collected by filtration and the filter cake was washed with ethyl acetate. The solid was dried overnight in an oven (50 °C) to give compound 91 (6.94 g, 88% yield). ESI MS m / z ([M+H]+) value: 206.05.

[0671] Example 100 Synthesis of Compound 92

[0672]

[0673] At -10°C, POCl3 (0.47 mL, 5 mmol) was added to a tetrahydrofuran (10 mL) solution of compound 91 (1.22 g, 5 mmol). After stirring for 10 min, 2,5,8,11,14,17,20,23,26-nonaoxooctadecane-28-amine (2.14 g, 5 mmol) was added, followed by DIPEA (0.87 mL, 5 mmol). The reaction was heated to 0°C and stirred for 3 h, then concentrated. The residue was diluted with dichloromethane (10 mL), filtered through diatomaceous earth, and the filtrate was used directly for the next step. ESI MS m / z ([M+H) + Value: 716.29.

[0674] Example 101 Synthesis of Compound 93

[0675]

[0676] 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 by silica gel column chromatography, eluting with 5–25% ethyl acetate / dichloromethane to give the title compound (12.5 g, 83% yield). ESI MS m / z ([M+Na)) + Value: 242.42.

[0677] Example 102 Synthesis of Compound 94

[0678]

[0679] To a solution of compound 93 (12.0 g, 49.56 mmol) in anhydrous pyridine (350 mL), methanesulfonyl chloride (20.0 g, 175.4 mmol) was added. After stirring overnight, the mixture was concentrated, diluted with ethyl acetate (350 mL), washed with cold 1 M NaH₂PO₄ (2 × 300 mL), dried over MgSO₄, filtered, and concentrated to give the crude product (18.8 g, >100% yield). The crude product was ready for use in the next step without further purification. ESI MS m / z ([M+H) + Value: 376.06

[0680] Example 103 Synthesis of Compound 95

[0681]

[0682] Furan (10.0 mL, 137.4 mmol) was added to a toluene (200 mL) solution of maleimide (10.0 g, 103.0 mmol). The mixture was heated at 100 °C for 8 hours, cooled to room temperature, concentrated, and crystallized in ethyl acetate / hexane. The solid was washed with methanol to give 16.7 g (99%) of the title compound. 1 H NMR (CDC) l3 ):11.12(s,1H),6.68~6.64(m,2H),5.18~5.13(m,2H),2.97~2.92(m,2H). ESI MS m / z([M+Na] + Value: 188.04.

[0683] Example 104 Synthesis of Compound 96

[0684]

[0685] To a DMA (350 mL) solution of compound 94 (freshly prepared, 90% pure, 8.5 g, approx. 20 mmol), compound 95 (10.2 g, 61.8 mmol), sodium carbonate (8.0 g, 75.5 mmol), and sodium iodide (0.3 g, 2.0 mmol) were added. The mixture was stirred overnight at room temperature, concentrated, diluted with ethyl acetate (350 mL), and washed with saturated NaHCO3 solution (300 mL), saturated NaCl solution (300 mL), and 1 M NaH2PO4 (300 mL). The organic layer was dried over sodium sulfate, filtered, concentrated, and loaded onto a silica gel column. Elution with 10–30% ethyl acetate / n-hexane gave the title compound (7.9 g, 77% yield). ESI MS m / z ([M+Na)) + Value: 536.4.

[0686] Example 105 Synthesis of Compound 97

[0687]

[0688] A solution of compound 96 (3.0 g, 5.8 mmol) and trimethyltinyl alcohol (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 passed through a short silica gel column. The residue was eluted with dichloromethane / methanol to remove excess trimethyltin hydroxide. The fractions were combined, concentrated, 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). ESI MS m / z ([M+Na)) + Value: 386.2.

[0689] Example 106 Synthesis of Compound 98

[0690]

[0691] EDC·HCl (0.81 g, 4.20 mmol) was added to a DMA (20 mL) solution of compound 97 (1.62 g, 4.20 mmol) and compound 85 (2.71 g, 3.82 mmol). The reaction was stirred at room temperature. The mixture was allowed to react overnight, then poured into water (50 mL) and extracted with ethyl acetate (3 × 40 mL). The combined organic phases were 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). ESI MS m / z ([M+H) + Value: 1057.85.

[0692] Example 107 Synthesis of Compound 99

[0693]

[0694] A solution of compound 98 (3.20 g, 3.03 mmol) in formic acid (10 mL) was stirred overnight at room temperature. The solution was then concentrated and co-concentrated three times with toluene to give a colorless oil (3.00 g, crude product), which could be used without further purification. ESI MS m / z ([M+H)) + Value: 1001.50.

[0695] Example 108 Synthesis of Compound 100

[0696]

[0697] To a DMA (15.0 mL) solution of compound 99 (3.00 g, crude product, 3.03 mmol), NHS (0.38 g, 3.33 mmol) and EDC·HCl (0.87 g, 4.55 mmol) were added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was then diluted with water (50 mL) and extracted with ethyl acetate (3 × 30 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 chromatography (10–50% ethyl acetate / petroleum ether) to give a colorless oil (2.90 g, 90% yield). ESI MS m / z ([M+H)) + Value: 1098.50.

[0698] Example 109 Synthesis of Compound 101

[0699]

[0700] Sodium (0.1 g) was added to a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 eq.) in anhydrous tetrahydrofuran (200 mL), and the mixture was stirred until the Na disappeared. 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 HCl solution (20.0 mL, 1 N) at 0 °C. Tetrahydrofuran was removed by rotary condensation, followed by extraction with brine (300 mL) and 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 ([M+H) + Value: 278.17.

[0701] Example 110 Synthesis of Compound 102

[0702]

[0703] At 0 °C, TsCl (41.37 g, 217.0 mmol, 2.0 eq.) and TEA (30.0 mL, 217.0 mmol, 2.0 eq.) were added to a solution of tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate (30.20 g, 108.5 mmol, 1.0 eq.) in anhydrous dichloromethane (220 mL). The mixture was stirred overnight at room temperature, then washed with water (3 × 300 mL) and brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (3:1 hexane / ethyl). A colorless oil (39.4 g, yield 84.0%) was given. MS ESI m / z ([M+H) + Value: 433.28.

[0704] Example 111 Synthesis of Compound 103

[0705]

[0706] To a solution of tert-butyl propionate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF (100 mL), NaN3 (20.67 g, 316.6 mmol, 3.5 eq.) was added. The mixture was stirred overnight at room temperature. Water (500 mL) was added and the mixture was 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 chromatography (5:1 hexane / ethyl acetate) to give a colorless yellow oil (23.8 g, 85.53% yield). MS ESI m / z ([M+Na) + Value: 326.2.

[0707] Example 112 Synthesis of Compound 104

[0708]

[0709] Raney-Ni (7.5 g, suspended in water) was washed with water (three times) and isopropanol (three times) and mixed with compound 103 (5.0 g, 16.5 mmol) in isopropanol. The mixture was stirred at room temperature under a hydrogen balloon for 16 hours, then filtered through a diatomaceous earth pad, the pad was washed with isopropanol and the filtrate was concentrated and purified by column chromatography (5-25% methanol / dichloromethane) to give a pale yellow oil (2.60 g, 57% yield). MS ESI m / z ([M+H) + Value: 279.19.

[0710] Example 113 Synthesis of Compound 105

[0711]

[0712] To a solution of tetradecanoic acid (2.06 g, 8 mmol) in DMF (30 mL), K₂CO₃ (1.1 g, 8 mmol) and benzyl bromide (1.36 g, 8 mmol) were added. The mixture was stirred overnight at room temperature, then concentrated and purified by column chromatography (ethyl acetate / petroleum ether) to give the title compound 105 (1.2 g, 45% yield). ESI MS m / z ([M+H) + Value: 349.23.

[0713] Example 114 Synthesis of Compound 106

[0714]

[0715] EDC·HCl (2.15 g, 11.2 mmol) and DIPEA (3.6 mL, 20.6 mmol) were added to a dichloromethane (50 mL) solution of compound 104 (2.60 g, 9.35 mmol) and compound 105 (3.91 g, 11.2 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 (4.94 g, 87% yield). ESI m / z ([M+H)) + Value: 608.40.

[0716] Example 115 Synthesis of Compound 107

[0717]

[0718] TFA (20 mL) was added to a solution of compound 106 (4.94 g, 8.14 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 1 hour, then concentrated to dryness and co-concentrated twice with dichloromethane. The residue was then placed on a pump to give compound 107 (4.50 g, crude product). ESI MS m / z ([M+H)) + Value: 552.35.

[0719] Synthesis of Compound 116 and Compound 108

[0720]

[0721] To a solution of compound 107 (4.50 g, crude product, 8.14 mmol) and compound 104 (1.95 g, 7.00 mmol) in dichloromethane (50 mL), EDC·HCl (1.56 g, 8.14 mmol) and DIPEA (2.7 mL, 15.4 mmol) were added. 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 title compound 108 (5.22 g, 92% yield). ESI m / z ([M+H)) + Value: 811.52.

[0722] Synthesis of Compound 117 and Compound 109

[0723]

[0724] TFA (5 mL) was added to a solution of compound 108 (5.22 g, 6.44 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 1 hour, then concentrated to dryness and co-concentrated twice with dichloromethane. The residue was then placed on a pump to give compound 109 (4.90 g, crude product). ESI MS m / z ([M+H)) + The value is 755.46.

[0725] Example 118 Synthesis of Compound 110

[0726]

[0727] To a solution of compound 109 (4.90 g, crude product, 6.44 mmol) in dichloromethane (30 mL), NHS (0.81 g, 7.08 mmol), EDC·HCl (1.85 g, 9.66 mmol), and DIPEA (2.8 mL, 16.1 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then diluted with water (50 mL) and extracted with ethyl acetate (3 × 30 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 chromatography (10–50% ethyl acetate / petroleum ether) to give colorless oil 236 (4.90 g, 90% yield). ESI MS m / z ([M+H)) + Value: 852.48.

[0728] Example 119 Synthesis of Compound 111

[0729]

[0730] In a hydrogenation flask, Pd / C (10 wt%, 0.20 g) was added to a tetrahydrofuran (20 mL) solution of compound 110 (4.90 g, 5.75 mmol). The mixture was stirred overnight at 1 atm hydrogen, filtered through diatomaceous earth (filter aid), and the filtered solution was concentrated to give compound 111 (4.50 g, >100% yield). ESI MS m / z ([M+H) + Value: 762.44.

[0731] Example 120 Synthesis of Compound 112

[0732]

[0733] At 0 °C, HATU (0.50 g, 1.32 mmol) and triethylamine (0.06 mL, 1.32 mmol) were added to a solution of compound 111 (1.00 g, 1.32 mmol) in dichloromethane (10 mL). The reaction was stirred at 0 °C for 30 min, then Z-Lys-OH (0.40 g, 1.43 mmol) was added, and the mixture was stirred at room temperature for 1 h. The mixture was then diluted with water (20 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 chromatography (0–10% methanol / dichloromethane) to give a colorless oily compound 112 (1.28 g, 95% yield). ESI MS m / z ([M+H)) + Value: 1017.60.

[0734] Example 121 Synthesis of Compound 113

[0735]

[0736] To a solution of compound 112 (1.28 g, 1.26 mmol) in dichloromethane (10 mL), NHS (0.17 g, 1.51 mmol) and EDC·HCl (0.29 g, 1.51 mmol) were added, followed by triethylamine (0.38 mL, 2.77 mmol). The reaction was stirred at room temperature for 2 h, 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 chromatography (0–10% methanol / dichloromethane) to give a colorless oily compound 113 (1.28 g, 91% yield). ESI MS m / z ([M+H)) + Value: 1114.62.

[0737] Example 122 Synthesis of Compound 114

[0738]

[0739] A solution of tert-butyl acrylate (12.81 g, 0.10 mmol) and ethyl-1,2-diamine (24.3 g, 0.40 mol) in tetrahydrofuran (150 mL) was stirred at 45 °C for 24 h. The mixture was concentrated and purified on an Al₂O₃ gel column, eluting with methanol / dichloromethane (triethylamine) (5%:15%:80%) to give the title compound (17.50 g, 92% yield). ESI MS m / z ([M+H) + Value: 189.20.

[0740] Example 123 Synthesis of Compound 115

[0741]

[0742] A solution of tert-butyl 3-((2-aminoethyl)amino)propionate (17.00 g, 90.33 mmol) in 1,4-dioxane (50 mL) was stirred with concentrated hydrochloric acid (15 mL) at room temperature for 30 min, concentrated, and diluted with pure water (150 mL) and ethyl acetate / 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 under vacuum to give the title compound (18.70 g, 100% yield, 96% purity by LC-MS). ESI MS m / z ([M+H) + Value: 133.20.

[0743] Example 124 Synthesis of Compound 116

[0744]

[0745] At 0 °C, maleic anhydride (8.85 g, 90.33 mmol) was added to a tetrahydrofuran (150 mL) solution of 3-((2-aminoethyl)amino)propionic acid (18.70 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-oxobut-2-enoic acid. Toluene (150 mL) and DMA (50 mL) were then added to the mixture, and the mixture was stirred at 90 °C and refluxed using a Dean-Stark separator. After collecting 30 mL of the solvent in a trap, HMDS (hexamethyldisilazane, 9.0 mL, 43.15 mmol) and ZnCl2 (16 mL, 1.0 M ether solution) were added. The mixture was heated to 115–125 °C, and toluene was collected using a Dean-Stark separator. The reaction mixture was heated at 120 °C for 6 hours. During this process, 2 × 40 mL of anhydrous toluene was added to maintain a mixture volume of approximately 50 mL. The mixture was then cooled and 1 mL of 1:10 HCl (conc.) / methanol was added. The mixture was concentrated, purified on a silica gel column, eluted with water / acetonitrile (1:15), concentrated, and dried under vacuum to give 14.75 g of the title compound (yield 77.0%). ESI MS m / z ([M+H)) + Value: 213.10.

[0746] Example 125 Synthesis of Compound 117

[0747]

[0748] Compound 60 (57.30 g, 0.106 mol) was added to a mixture of tetrahydrofuran (300 mL), DIPEA (50 mL), and HSAc (10.0 g, 0.131 mol). The mixture was stirred overnight, concentrated, and purified on a silica gel column. The purified product was eluted with ethyl acetate / dichloromethane (1:2 to 4:1), concentrated, and dried under vacuum to give 40.51 g (86% yield) of the title compound. ESI MS m / z ([M+H) + Value: 443.35.

[0749] Example 126 Synthesis of Compound 118

[0750]

[0751] Compound 117 (40.40 g, 0.091 mol) was added to a mixture of acetic acid (200 mL) and 30% H₂O₂ (100 mL). The mixture was stirred overnight at 35 °C. 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, concentrated, and dried under vacuum to give 40.50 g of the title compound (99% yield, 95% purity by LC-MS). ESI MS m / z ([M+H)) + Value: 449.30

[0752] Example 127 Synthesis of Compound 119

[0753]

[0754] To a mixture of compound 118 (20.0 g, 44.62 mmol) in tetrahydrofuran (100 mL) and dichloromethane (100 mL), oxalyl chloride (25.21 g, 200.19 mmol) and DMF (0.015 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours, concentrated, co-concentrated with dichloromethane / toluene (1:1, 2 × 50 mL), and then dissolved in tetrahydrofuran (50 mL). A solution of compound 116 (7.50 g, 35.36 mmol) in tetrahydrofuran (100 mL) was added. The mixture was stirred overnight, concentrated under vacuum, and purified on a silica gel column, eluted with methanol / dichloromethane (1:6 to 1:5), and dried under vacuum to give 14.76 g (65% yield) of the title compound. ESI MS m / z ([M+H) + Value: 643.35.

[0755] Example 128 Synthesis of Compound 120

[0756]

[0757] A solution of compound 119 (7.50 g, 11.67 mmol), N-hydroxysuccinimide (1.50 g, 13.04 mmol), and EDC (10.10 g, 52.60 mmol) in tetrahydrofuran (100 mL) was stirred overnight, concentrated under vacuum, and purified on a silica gel column. The solution was eluted with ethyl acetate / dichloromethane (1:4 to 2:1) and dried under vacuum to give 6.30 g (73% yield) of the title compound. ESI MS m / z ([M+H) + Value: 740.40

[0758] Example 129 Synthesis of Compound 121

[0759]

[0760] DIPEA (3 mL) was added to a DMF (15 mL) solution of 2-(2-(2-(2-aminoacetamido)acetamido)acetamido)acetic acid (gly-gly-gly) (0.50 g, 2.03 mmol) and compound 120 (1.65 g, 2.22 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h, and then stirred at room temperature for 4 h. The reaction mixture was then concentrated and purified by silica gel chromatography (mobile phase: acetonitrile / water = 95:5, containing 0.1% formic acid) to give title compound 121 (1.04 g, 63% yield). MS-ESI m / z C 32 H 56 N5O 17 S[M+H] + The calculated value is 14.33, and the measured value is 814.46.

[0761] Example 130 Synthesis of Compound 122

[0762]

[0763] A solution of compound 121 (0.70 g, 0.86 mmol), N-hydroxysuccinimide (0.20 g, 1.73 mmol), and EDC (1.21 g, 6.36 mmol) in tetrahydrofuran (20 mL) was stirred overnight at room temperature, concentrated under vacuum, and purified by silica gel column chromatography. The solution was eluted with ethyl acetate / dichloromethane (1:4 to 2:1) and dried under vacuum to give 0.540 g (69% yield) of the title compound. MS-ESI m / z C 36 H 59 N6O19S[M+H] + Calculated value: 911.34, measured value: 911.42.

[0764] Example 131 Synthesis of Compound 123

[0765]

[0766] At 0 °C, DIPEA (2 mL) was added to a solution of (2S,4R)-5-(3-amino-4-hydroxyphenyl)-4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,3,3,8-tetramethyl-4,7,13-trioxo-12-oxa-2,5,8-triazaundecane-11-yl)thiazo-4-carboxamido)-2-methylpentanoic acid (Tub-039, R. Zhao, et al, PCT / CN2017 / 120454; R. Zhao, et al, 14th PEGS Boston, Boston, MA, USA, 3rd May 2018) (83 mg, 0.106 mmol) and compound 122 (122 mg, 0.134 mmol) in DMF (8 mL). The reaction mixture was stirred at 0°C for 0.5 h, and then stirred at room temperature for 4 h. The reaction mixture was then concentrated and purified by preparative HPLC (mobile phase: acetonitrile / water = 10% to 80%, containing 0.1% formic acid) to give compound 123 (95.5 mg, 58% yield). MS-ESI m / z C 69 H 112 N 11 O 24 S[M+H] + Calculated value: 1542.72, measured value: 1542.76.

[0767] Example 132 Synthesis of Compound 124

[0768]

[0769] A solution of (S)-1-benzyl-5-tert-butyl-2-aminopentanoic acid salt (8.70 g, 26.39 mmol), 14-(benzyloxy)-14-oxotetradecanoic acid (9.19 mmol), DIPEA (8.0 mL, 46.0 mmol), and EDC (15.3 g, 80.50 mmol) in dichloromethane (200 mL) was stirred at room temperature for 6 hours. The mixture was diluted with water (100 mL) and separated. The aqueous phase was extracted with dichloromethane (100 mL). The organic phases were combined, washed with brine, dried over sodium sulfate, filtered, concentrated, and purified on a silica gel column (dichloromethane / ethyl acetate = 20:1 to 5:1) to give the title compound 314. MS-ESI m / z C 37 H 54 NO7[M+H] + Calculated value: 624.38, measured value: 624.38.

[0770] Example 133 Synthesis of Compound 125

[0771]

[0772] Compound 124 (12.50 g, 20.05 mmol) was dissolved in dioxane (30 mL) at 4 °C and stirred with hydrochloric acid (10 mL, 36%) for 0.5 h. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL) and concentrated at 15 °C to give the title compound (11.26 g, 99% yield). MS-ESI m / z C 33 H 46 NO7[M+H] + Calculated value: 568.32, measured value: 568.34.

[0773] Example 134 Synthesis of Compound 126

[0774]

[0775] A solution of compound 125 (10.70 g, 18.86 mmol), 1-amino-15-oxo-3,6,9,12,19,22,25,28-octoxy-16-azatriacontane-31-tert-butyl ester hydrochloride (11.45 g, 18.93 mmol), EDC (9.51 g, 50.01 mmol), and DIPEA (4.00 mL, 23.00 mol) in dichloromethane (200 mL) was stirred overnight at room temperature, diluted with brine (100 mL), and separated. The aqueous phase was extracted with dichloromethane (100 mL). The combined organic phases were washed with brine, dried over sodium sulfate, filtered, concentrated, and purified on a silica gel column (dichloromethane / ethyl acetate = 10:1 to 4:1) to give the title compound (18.15 g, 86% yield). MS-ESI m / z C 59 H 96 N3O 17 [M+H] + Calculated value: 1118.67, measured value: 1118.80.

[0776] Example 135 Synthesis of Compound 127

[0777]

[0778] Compound 126 (10.50 g, 9.39 mmol) was dissolved in dioxane (45 mL) at 4 °C and stirred with hydrochloric acid (15 mL, 36%) for 0.5 h. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL), concentrated at 15 °C, and purified by silica gel column chromatography (dichloromethane / methanol = 10:1 to 6:1) to give the title compound (8.67 g, 87% yield). MS-ESI m / z C 55 H88 N3O 17 [M+H] + Calculated value: 1062.60, measured value: 1062.68.

[0779] Example 136 Synthesis of Compound 128

[0780]

[0781] A solution of compound 127 (8.50 g, 8.01 mmol), N-hydroxysuccinimide (3.20 g, 27.82 mmol), EDC (10.28 g, 54.10 mmol), and DIPEA (6.00 mL, 34.51 mmol) in tetrahydrofuran (150 mL) was stirred at room temperature for 6 hours, then concentrated under vacuum to give the NHS ester, which could be used in the next step without purification. The N-succinimide ester prepared above was added in four portions over one hour to a mixed solution of (S)-6-amino-2-((tert-butoxycarbonyl)amino)hexanoate (2.75 g, 9.73 mmol) in DMF (100 mL) and 1.0 M Na₂PO₄ (pH 7.5, 55 mL). The reaction mixture was stirred at room temperature for 3 hours. After concentration, the residue was purified on a silica gel column (dichloromethane / methanol = 10:1 to 4:1) to give the title compound (8.16 g, 79% yield). MS-ESI m / z C 66 H 108 N5O 20 [M+H] + Calculated value: 1289.75, measured value: 1289.90.

[0782] Example 137 Synthesis of Compound 129

[0783]

[0784] Compound 128 (8.10 g, 6.28 mmol) was dissolved in dioxane (40 mL) at 4 °C and stirred with hydrochloric acid (15 mL, 36%) for 0.5 h. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL) and concentrated at 15 °C to give the title compound (7.71 g, 100% yield), which was used for the next step without further purification. MS-ESI m / z C 61 H 88 N3O 17 [M+H]+: Calculated value 1190.70, measured value 1190.78.

[0785] Example 138 Synthesis of Compound 130

[0786]

[0787] DIPEA (10 mL) was added to a DMF (50 mL) solution of 4-maleimide-N-succinamide ester (7.10 g, 25.35 mmol) and alanine (3.01 g, 33.80 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h, and then stirred at room temperature for 1 h. The reaction mixture was then concentrated and purified on a silica gel column (mobile phase: dichloromethane / methanol = 10:1, containing 0.1% formic acid) to give compound 130 (5.21 g, 81% yield). MS-ESI m / z C 11 H 14 N₂O₅[M+H] + Calculated value: 255.09, measured value: 255.15.

[0788] Example 139 Synthesis of Compound 131

[0789]

[0790] A solution of compound 130 (5.15 g, 20.26 mmol), N-hydroxysuccinimide (2.80 g, 24.34 mmol), EDC (10.28 g, 54.10 mmol), and DIPEA (5.50 mL, 31.63 mmol) in dichloromethane (70 mL) was stirred at room temperature for 6 hours, concentrated under vacuum, and purified on a silica gel column (mobile phase: dichloromethane / ethyl acetate = 10:1) to give compound 131 (5.83 g, 82% yield). MS-ESI m / z C 15 H 17 N3O7[M+H] + Calculated value: 351.11, measured value: 351.20.

[0791] Example 140 Synthesis of Compound 132

[0792]

[0793] DIPEA (7 mL) was added to a 40 mL solution of DMF containing compounds 129 (7.61 g, 6.39 mmol) and 131 (2.90 g, 8.280 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h, then at room temperature for 1 h. The reaction mixture was then concentrated and purified on a silica gel column (mobile phase: dichloromethane / methanol = 10:1, containing 0.1% formic acid) to give compound 132 (7.10 g, 78% yield). MS-ESI m / z C 11 H 14 N₂O₅[M+H]+ Calculated value: 1426.7782, measured value: 1426.7820.

[0794] Example 141 Synthesis of Compound 133

[0795]

[0796] A solution of compound 132 (7.05 g, 4.94 mmol), N-hydroxysuccinimide (0.92 g, 8.00 mmol), EDC (3.01 g, 15.84 mmol), and DIPEA (1.00 mL, 5.75 mmol) in tetrahydrofuran (50 mL) was stirred at room temperature for 6 hours and then concentrated under vacuum to obtain NHS ester, which could be used in the next step without purification.

[0797] The above compound was added in four portions over one hour to a mixed solution of 2-(2-(2-aminoacetamido)acetamido)acetic acid (gly-gly-gly) hydrochloride (1.67 g, 7.40 mmol) in DMF (40 mL) and 1.0 M Na₂PO₄ (pH 7.5, 15 mL). The reaction mixture was stirred at room temperature for 3 hours. After concentration, the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10:1 to 7:1) to give the title compound (8.16 g, 79% yield). MS-ESI m / z C 11 H 14 N₂O₅[M+H] + Calculated value: 597.84, measured value: 1597.84.

[0798] Example 142 Synthesis of Compound 134

[0799]

[0800] EDC (100 mg, 0.526 mmol) was added to a solution of compound 133 (150.3 mg, 0.0935 mmol), Tub-039 (60.2 mg, 0.0769 mmol), and DIPEA (0.030 mL, 0.172 mmol) in DMA (5 mL). The reaction mixture was stirred at room temperature for 6 hours, concentrated under vacuum, and then dissolved in methanol / dichloromethane (0.5 mL: 3 mL). The solution was then passed through a short silica gel column, eluted with methanol / dichloromethane (1:3), and concentrated under vacuum to give the crude compound for the next step. MS-ESI m / z value: 2326.25.

[0801] A dichloromethane (1 mL) solution of the above compound was stirred with TFA (3 mL) for 1 hour. The reaction mixture was diluted with toluene (3 mL) and DMF (3 mL), concentrated, and purified by preparative HPLC (mobile phase: 2% to 50% aqueous acetonitrile containing 0.1% formic acid) to give compound 134 (69.0 mg, 72% yield). MS-ESI m / z C 11 H 14 N₂O₅[M+H] + Calculated value: 2146.1497, measured value: 2146.1588.

[0802] Example 143 Synthesis of Compound 135

[0803]

[0804] EDC (11 mg, 0.059 mmol) and pentafluorophenol (10.8 mg, 0.059 mmol) were added to a solution of (S)-30-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butamido)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontane-31-acid (20 mg, 0.029 mmol) in dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 2 hours, concentrated, and purified on a silica gel column by elution with ethyl acetate / dichloromethane (1:4) to give the title compound (24 mg, 100% yield). MS-ESI m / z C 36 H 50 F5N3O 14 [M+H] + Calculated value: 844.32, measured value: 844.32.

[0805] Example 144 Synthesis of Compound 136

[0806]

[0807] A methanol solution (100 mL) of (S)-2-(((S)-2-(((benzyloxy)carbonyl)amino)propamido)tert-butyl propionate (10 g, 0.028 mol) and 10% palladium on carbon (1.0 g) were stirred under hydrogen (5 psi) for 3 hours. The solid was filtered off, and the filtered solution was concentrated to give a colorless oily product (6.1 g, 100% yield). ESI m / z C 10 H 20 N₂O₃[M+H] + Calculated value: 217.15, measured value: 217.15.

[0808] Example 145 Synthesis of Compound 137

[0809]

[0810] EDC (59 mg, 0.309 mmol) and pentafluorophenol (PFP) (57 mg, 0.309 mmol) were added to a solution of (S)-30-(((benzyloxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacont-31-acid (100 mg, 0.154 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature for 2 hours, diluted with dichloromethane (20 mL), washed with water (5 mL), dried over sodium sulfate, filtered, and concentrated. The residue was redissolved in DMF (5 mL), and then compound 136 (49 mg, 0.23 mmol) and DIPEA (90 mg, 0.69 mmol) were added. The mixture was stirred at room temperature for 1 hour, concentrated, and purified on a short silica gel column by elution with methanol / dichloromethane (1:10) to give title compound 137 (80 mg, 61% yield). ESI m / z C 40 H 68 N4O 15 [M+H] + Calculated value: 845.47, measured value: 845.47.

[0811] Example 146 Synthesis of Compound 138

[0812]

[0813] A methanol (5 mL) solution of compound 137 (80 mg, 0.094 mmol) and 10% palladium on carbon (10 mg) were stirred at hydrogen (5 psi) for 2 hours. The solid was filtered off, and the filtrate was concentrated to give a colorless oily product (66 mg, 100% yield), which was used for the next step without further purification. MS-ESI m / z C 32 H 62 N4O 13 [M+H] + Calculated value: 711.43, measured value: 711.43.

[0814] Example 147 Synthesis of Compound 139

[0815]

[0816] To compound 138 (66 mg, 0.094 mmol), ethanol (5 mL), 2,5-dioxopyrrolidone-1-yl 4-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)butyrate (39 mg, 0.141 mmol), and PBS (0.1 M, pH 7.5, 1.0 mL) were added. The reaction mixture was stirred overnight, concentrated, and purified on a silica gel column (dichloromethane / methanol = 100:0 to 10:1) to give title compound 139 (37 mg, 45% yield). ESI m / z C 40 H 69 N5O = [M + H] + Calculated value: 876.47, measured value: 876.47.

[0817] Example 148 Synthesis of Compound 140

[0818]

[0819] A solution of compound 139 (50 mg, 0.057 mmol) in dichloromethane (3 mL) was stirred with TFA (1 mL) at room temperature for 2 hours. The reaction mixture was concentrated to dryness and then redissolved in dichloromethane (5 mL), to which EDC (16 mg, 0.084 mmol) and pentafluorophenol (15 mg, 0.084 mmol) were added. The mixture was stirred at room temperature for 4 hours, concentrated, and purified on a silica gel column (dichloromethane / ethyl acetate = 100:10 to 3:1) to give title compound 140 (41 mg, 73% yield). ESI m / z C 42 H 60 F5N5O 16 [M+H] + Calculated value: 986.40, measured value: 986.42.

[0820] Example 149 Synthesis of Compound 141

[0821]

[0822] To a solution of 4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)amino)-4-oxobutyric acid (100 mg, 0.27 mmol) in dichloromethane, 5 mL of EDC (210 mg, 1.10 mmol) and pentafluorophenol (101 mg, 0.55 mmol) were added. The mixture was stirred at room temperature for 3 hours, concentrated, and purified on a silica gel column (dichloromethane / ethyl acetate = 20:1 to 5:1) to give title compound 141 (114 mg, 80% yield). MS-ESI m / z C 22 H 16F5N3O7[M+H] + Calculated value: 530.0, measured value: 530.09.

[0823] Example 150. A general method for preparing a conjugate by reducing the antibody disulfide bond to a Tubulysin derivative.

[0824] To a 2.0 mL solution of HER2 antibody at a pH of 6.0–8.0 (10 mg / mL), add 0.70 mL–2.0 mL of 100 mM phosphate (PBS), pH 6.5–8.5 buffer, or TCEP (16–20 μL, 20 mM aqueous solution). Incubate at room temperature to 37.5 °C for 0.5–4 hours. Add an equivalent amount of azide compound (azidobenzoic acid, or 2-(2-(2-hydroxyethoxy)ethoxy)ethoxyazide compound) and incubate at room temperature to 37.5 °C for 1–4 hours. Then add a Tubulysin derivative molecule (28–32 μL, 20 mM DMA solution) that reacts with a thiol group (e.g., compounds 39, 57, 72, 123, or 134). Incubate the mixture at room temperature to 37.5 °C for 2–18 hours, then add DHAA (135 μL, 50 mM). After overnight continuous culture at RT, the mixture was purified to the conjugated compound (75%–99% yield) using a G-25 column, a cation exchange column, or an anion exchange column with a buffer solution of 10–100 mM phosphoric acid or citric acid and 50–200 mM NaCl at pH 6–7.5. This purification step can also be performed by dialysis filtration with a buffer solution of 10–100 mM phosphoric acid or citric acid and 50–200 mM NaCl at pH 6–7.5, at a volume of 3–30 times the dialysis volume. The drug / antibody ratio (DAR) was 3.1–4.9 as determined by liquid chromatography-mass spectrometry (LC-MS / MS), and the monomer content was 95–99% as determined by HPLC (Tosoh Bioscience, Tskgel G3000SW column, inner diameter 7.8 mm x 30 cm, 0.5 mL / min, 100 min). The structures of the antibody-conjugates prepared using compounds 39, 57, 72, 123, or 134 are as follows:

[0825]

[0826] C-37

[0827]

[0828] C-59 or

[0829]

[0830] C-72

[0831]

[0832] Example 151. Another method for preparing the coupling compound.

[0833] Cell-binding molecules (antibodies) can be conjugated to the compounds of this patent via amides, thioethers, or disulfide bonds. Antibodies (>5 mg / mL) are diluted with PBS buffer (pH 8.0) containing 50 mM sodium borate, and dithiothreitol is added (final concentration 10 mM). After treatment at 35°C for 30 minutes, free thiol groups are released from the antibodies. Following gel filtration chromatography on a G-25 column (PBS buffer with 1 mM EDTA added), and determination using Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], approximately 8 thiol groups are conjugated to each antibody. Antibodies can also release thiol groups when reacting with Traut's reagent (2-iminothiophene) (Jue, R., et al. Biochem. 1978, 17(25): 5399-5405), or under pH 7-8 conditions, react with different linkers such as SATP (N-succinimide-S-acetylthiopropionate) or N-succinimide-S-acetyl (thiotetraoxoethylene) (SAT(PEG)4), and then form thiol groups after hydroxylamine treatment (Duncan, R., et al. Anal. Biochem. 1983, 132, 68-73, Fuji, N. et al. Chem. Pharm. Bull. 1985, 33, 362-367). Basically, each antibody molecule is linked with 5-9 thiol molecules.

[0834] At 4°C, add a drug containing maleimide or bromoacetamide groups (requiring 0.5M sodium borate solution (pH 9) to ice-cold dimethylacetamide (DMA) (2–20% v / v) containing free thiol groups (the molar ratio of drug to thiol group should be 1.2–1.5:1). After 1–2 hours, add excess cysteine ​​to terminate the reaction; obtain the concentrated conjugated product by ultrafiltration, gel chromatography (G-25, PBS buffer), and sterile filtration. Determine the protein concentration and the number of drug molecules linked to each antibody by measuring absorbance at 280 nm and 252 nm. Size exclusion HPLC can be used to determine the proportion of monomeric form of the conjugate, while unbound drug below 0.5% can be determined by RP-HPLC. For monomeric drugs formed by thioether linkages, each antibody molecule will, on average, link 3.2–4.8 tubulysin derivatives.

[0835] Connectors can be classified into dimethyl (phenyl)silyl (DMPS), SMDP, 4-succinimide-oxycarbonyl-methyl-α-(2-pyridyl disulfide)toluene (SMPT), N-succinimide-4-(2-pyridinyl thio)valerate (SPP), N-succinimide-4-(2-pyridinyl thio)propionate (SPDP), N-succinimide-4-(2-pyridinyl thio)butyrate (SPDB), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide-(polyethylene glycol)n-maleimide (SM(PEG)n), etc. Antibodies (>5 mg / mL) are diluted in buffer (pH 6.5–7.5, 5 mM PBS, 50 mM NaCl, 1 mM EDTA) and reacted with the linker for 2 hours, with a molar ratio of linker to antibody of 6–10 or greater. The reaction mixture can be separated by Sephadex G25 gel chromatography, removing lower molecular weight molecules. This purification step can also be performed using a cation exchange column or anion exchange column, with a buffer solution of 10–100 mM phosphate or citric acid, 50–200 mM NaCl, pH 6–7.5, to purify the conjugated compound (75%–99% yield). This purification step can also be performed by dialysis filtration, with a buffer solution of 10–100 mM phosphate or citric acid, 50–200 mM NaCl, pH 6–7.5, at a volume of 3–30 times the dialysis buffer, to purify the conjugated compound (75%–99% yield). The antibody concentration was determined spectrophotometrically, and the conjugate contained pyridine thiols. The extinction coefficient of the antibody at 280 nm was 2067550 M. -1 cm -1 The modified antibody was treated with an excess of dithiothreitol (more than 20 equivalents), and the extinction coefficients of the released 2-thiopyridine groups at 343 and 280 nm were measured to be 8080 and 5100 M, respectively. -1 cm -1 1.2–1.5 equivalents of a thiol-containing Tubulysin derivative molecule were added to the modified antibody. The reaction was carried out at room temperature for 5–18 hours. The reaction mixture was subjected to Sephadex G25 gel chromatography to remove unlinked drug or other low molecular weight substances. The concentration of the ligation product was determined by measuring the absorbance at 280 nm and 252 nm. The ligation product was in monomeric form, with an average of 3.2–4.8 drug molecules linked to each antibody molecule.

[0836] Example 152. In vitro cytotoxicity evaluation of Her2 antibody conjugates C-37, C-59, C-72, C-123 and C-134 (compared to T-DM1 control):

[0837] The cell line used for the cytotoxicity assay was the human gastric cancer cell line NCI-N87; 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 and 5% CO2 for 24 hours. Next, cells were treated with different concentrations of the test compound (20 μL) in appropriate cell culture medium (total volume, 0.2 mL). Control wells contained cells and culture medium but no test compound. The plates were incubated at 37°C and 5% CO2 for 120 hours, then MTT (5 mg / mL, 20 μL) was added to the wells, and the plates were incubated at 37°C for 1.5 hours. The culture medium was carefully removed, and then DMSO (180 μL) was added. The plates were shaken for 15 minutes, and absorbance was measured at 490 nm and 570 nm, with 620 nm as a reference. The inhibition rate was calculated using the following formula: Inhibition rate % = [1 - (analytical value - blank control value) / (control value - blank control value)] × 100. The cytotoxicity results of the compounds are summarized in Table 1.

[0838] Table 1. Cytotoxicity of Her-amatoxin analogue conjugates in this invention application

[0839] Compound DAR (drug / antibody ratio) NCI-N87 cell IC 50 (nM)C-373.90.22C-594.00.11C-723.80.10C-1237.80.15C-1343.90.25T-DM13.50.32

[0840] Example 153: In vivo antitumor activity study in BALB / c nude mice loaded with NCI-N87 xenograft tumors.

[0841] The in vivo efficacy of conjugates C-37, C-49, C-72, C-123, C-134, and T-DM1 was evaluated in a human gastric cancer N-87 cell line xenograft model. Sixty-six five-week-old female BALB / c nude mice were induced to undergo xenografting of N-87 cancer cells (5 × 10⁻⁶ cells) in the right subscapular region. 6 Subcutaneous inoculation was performed using cells / mouse. Tumors grew to an average size of 140 mm after 8 days. 3The animals were then randomly divided into 10 groups (6 animals per group). The first group of mice served as the control group and was injected with phosphate-buffered saline. The remaining 6 groups were injected intravenously with conjugates C-37, C-49, C-72, C-123, C-134 and T-DM1 at a dose of 6 mg / kg, respectively. The three-dimensional dimensions of the tumor were measured every 3 or 4 days, and the tumor volume was calculated using the formula: tumor volume = 1 / 2 (length × width × height), while the animal's weight was also measured. Mice were euthanized when any of the following criteria were met: (1) weight loss exceeding 20% ​​of pre-injection weight, (2) tumor volume greater than 1500 mm. 3 (3) The disease is so severe that the mouse is unable to eat or drink, or (4) the skin is necrotic. If the tumor is imperceptible, the mouse is considered to be tumor-free.

[0842] The results are plotted in Figure 7. At a dose of 6.0 mg / kg, none of the six conjugates caused weight loss in the animals. All conjugates showed antitumor activity compared to the control PBS solution. All tested Tubulysin conjugates showed superior antitumor activity compared to T-DM1.

[0843] Example 154. Toxicity study of Her2 antibody conjugate of Tubulysin B derivative (compared to T-DM1).

[0844] Changes in animal body weight (typically a decrease) are a macroscopic reflection of drug toxicity. Fifty-six 6-7 week old female ICR mice were divided into seven groups of eight animals each, and administered intravenously at doses of 150 mg / kg of drugs C-37, C-49, C-72, C-123, C-134, and T-DM1, respectively. The control group (eight animals) received PBS-buffered saline. As shown in Figure 8, during the 12-day experiment, the body weight of mice in all conjugate groups except the control and T-DM1 groups did not decrease by more than 5%. Conversely, the body weight of animals in the T-DM1 group showed a sustained decrease, with a maximum reduction of 24% from pre-administration levels, and no recovery trend was observed at the end of the study. These results indicate that animals tolerated these Her2 antibody conjugates containing branched linkers of Tubulysin B derivatives better than those containing T-DM1 with a conventional single linker.

[0845] The foregoing has merely illustrated the principles of the invention. It should be understood that the scope of the invention is not intended to be limited to the exemplary aspects described herein, but should include all currently known and future-developed equivalents. Furthermore, it should be noted that several improvements and modifications can be made without departing from the technical principles of the invention, and these improvements and modifications should also be considered within the scope of the invention.

Claims

1. An antibody-Tubulysin B derivative (homolog) conjugate, It is characterized in that The conjugate has a structure as shown in the following formula (I): Or a pharmaceutically acceptable salt, hydrate or hydrated salt having the structure shown in Formula I as the parent, a polymorph having the structure shown in Formula I, an optical isomer of the structure shown in Formula I, one or more deuterium ( 2 H) atoms replace hydrogen ( 1 H) atom, or one or more of the structures shown in formula I 13 C atom replacement 12 C atoms; wherein, P 1 is H, COCH 3 , COH, PO(OH) 2 , CH 2 OPO(OH) 2 , CONHCH 3 , CON(CH 3 ) 2 , CON(CH 2 CH 2 ) 2 NCH 3 , CON(CH 2 CH 3 ) 2 or CON(CH 2 CH 2 ) 2 CHN(CH 2 CH 2 ) 2 CH 2； R 1 , R 2 , R 3 and R 4 are independently H, C 1 -C 6 Alkyl, C 1 -C 6 Olefin group, C 1 -C 6 Alkyl ether group, C 1 -C 6 Alkylcarbonyl, C 1 -C 6 Alkyl ester, C 1 -C 6 Alkyl carboxyl or C 1 -C 6 Alkylamide; Or, R 1 and R 2 Together, R. 1 and R 3 Together, R. 2 and R 3 Together, or R 3 and R 4 Together they form C 2 -C 7 Heterocyclic or C 2 -C 7 Cycloalkyl structure; R 5 Yes H, OC 1 -C 6 Alkyl, C(O)-H, C(O)-C 1 -C 6 (straight or branched) alkane, C(O)-NH-C 1 -C 6 (straight or branched) alkyl or C(O)-N(C 1 -C 6 (straight or branched) alkyl) 2 ; R 6 , R 7 and R 8 are independently H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl ether group, C 1 -C 6 Alkylcarbonyl, C 1 -C 6 Alkyl ester, C 1 -C 6 Alkyl carboxyl or C 1 -C 6 Alkylamide; preferably R 6 , R 7 and R 8 are independently H or CH 3 ; mAbs are antibodies, antibody fragments, monoclonal antibodies, polyclonal antibodies, nanobodies, prodrug antibodies, or antibodies and antibody fragments modified with synthetic molecules or proteins; L is a linker containing a hydrophilic side chain, and its main structure is C 2 -C 100 The peptide unit (1 to 12 natural or unnatural amino acids), a hydrazone bond group, a disulfide group, an ester group, an oxime group, an amide group or a thioether bond group; n＝1-30。 2. The conjugate according to claim 1, It is characterized in that The structure of L is: wherein Aa is an L- or D-natural or unnatural amino acid; r is an integer between 0 and 12; when r is not 0, (Aa) r It is a peptide unit composed of the same or different amino acids; m 1 = an integer between 1 and 18; m 2 = integer between 1 and 100; m 3 = an integer between 1 and 8; m 4 = integer between 0 and 8; m 5 = an integer between 1 and 8; Y is NHC(=O), NHS(O 2 ), NH(SO), NHS(O 2 )NH, NHP(O)(OH)NH or C(O)NH; R 9 is H, (O=)CR 1 、(O=)CNHR 1 , R 1 COOH, R 1 (COCH 2 NH) m2 H.R 1 (Aa) r or R 1 (COCH 2 NCH 3 ) m2 H, and R 1 、m 2 and (Aa) r Definitions As defined in claim 1 and above.

3. The conjugate according to any one of claims 1 to 2, It is characterized in that The synthesis of the conjugate comprises one or more of the following steps: Wherein P in the structural formula (II) 1 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 And mAb as described in any one of claims 1-2; The structure of L' is: Where m 1 、m 2 、m 3 、m 4 、m 5 , Aa, r and R 9 As described in any one of claims 1-2.

4. The conjugate according to claim 3, It is characterized in that The structure of L' is: Where m 1 、m 2 、m 3 、m 4 、m 5 , Aa, r and R 9 As described in any one of claims 1-2.

5. The conjugate according to any one of claims 3 to 4, It is characterized in that The preparation method of mAb-SH includes any one of the following a) to c): a). Reducing the disulfide bonds between the heavy and light chains, between the heavy and heavy chains, or between the self-chains of antibodies, antibody fragments, monoclonal antibodies, polyclonal antibodies, nanobodies, prodrug antibodies, or antibodies and antibody fragments modified with synthetic molecules or proteins by a reducing agent (preferably, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), dithiopentaerythritol (DTE), L-glutathione (GSH), 2-mercaptoethylamine (β-MEA) or / and β-mercaptoethanol (β-ME, 2-ME)); b) Prepared by reacting Traut's reagent or thiolactone with the amine of the antibody molecule to form a thiol: c). Under the conditions of the buffer system, a disulfide bond group that is easier to reduce is introduced into the antibody through a biochemical reaction, and then reduced with TCEP, DTT, GSH, β-MEA, and β-ME:

6. The conjugate according to any one of claims 3 to 5, It is characterized in that The buffer system used in the synthesis of the conjugate is: a buffer solution of phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (trishydroxymethylaminomethane), benzoic acid or triethanolamine, or a mixture thereof, with a pH of 5.0 to 9.5 and a concentration of 1 mM to 1000 mM, and contains a water-soluble organic solvent of 0% to 35% by volume: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA or DMSO. The coupling reaction temperature is controlled at 0° C. to 45° C., and the coupling reaction is 5 minutes to 96 hours.

7. The conjugate according to any one of claims 3 to 6, It is characterized in that After the coupling reaction is completed, ultrafiltration or column chromatography is used for purification to obtain a conjugate with structural formula (I).

8. The conjugate according to claim 7, It is characterized in that The column chromatography includes a molecular sieve column, a cation column, an anion column, a hydrophobic (HIC) column, a reverse phase column or a protein A or G affinity column.

9. The conjugate according to any one of claims 3 to 8, It is characterized in that The compound of structural formula (II) is obtained by condensation reaction of a Tubulysin B derivative of structural formula (III) and a compound of structural formula (L'): wherein X is OH, halogen (F, Cl, Br, or I), phenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3'-sulfonic acid, Self-anhydride or anhydride formed with other anhydrides such as acetic anhydride and formic anhydride; or polypeptide condensation reaction intermediate or Mitsunobu reaction intermediate; in, The condensation reaction is carried out in an organic solvent containing 1% to 100% by volume of pyridine, triethylamine or diisopropylethylamine in dichloromethane, dichloroethane, DMF, DMA, tetrahydrofuran (THF), DMSO, acetone, isopropanol, n-butanol or acetonitrile, or a mixed solvent of two or more of the above solvents, with or without the protection of an inert gas (nitrogen, argon, helium), the temperature is controlled at -20°C to 150°C, and the reaction time is 5 minutes to 120 hours; or The condensation reaction is carried out in the following buffer system and conditions: the buffer system is: pH 5.0-9.5, concentration of 1 mM-1000 mM phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (trishydroxymethylaminomethane), benzoic acid or triethanolamine, or a mixture thereof, and contains a water-soluble organic solvent with a volume ratio of 0%-35%: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA or DMSO, the coupling reaction temperature is controlled at 0°C-45°C, and the coupling reaction is 5 minutes-96 hours.

10. The conjugate according to claim 9, It is characterized in that NH in the structural formula (III) 2 The groups are condensed in the form of trifluoroacetate, hydrochloride, formates, acetates, sulfates, phosphates, nitrates, citrates, succinates, benzoates, sulfonates.

11. The conjugate according to any one of claims 9 to 10, It is characterized in that When X is OH, the above condensation reaction needs to be carried out with the aid of a condensation reagent, which is: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC or CME-CDI), carbonyldiimidazole (CDI), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (B OP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl pyrocarbonate (DEPC), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholinyl)methylene]-1[1,2,3]triazolo[4,5-b]1-pyridine-3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), bis(tetramethylene)fluoroformamide (B TFFH), N,N,N',N'-tetramethyl-thio-(1-oxo-2-pyridyl)thiouronium hexafluorophosphate, 2-(2-pyridone-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU), thio-(1-oxo-2-pyridyl)-N,N,N',N'-tetramethylthiouronium hexafluorophosphate, O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiouronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylenaminooxy)dimethylamino-morpholinium-carbonium hexafluorophosphate (COMU), (benzotriazol-1-yloxy)dipyrrolidine carbon Hexafluorophosphate (HBPyU), N-benzyl-N′-cyclohexylcarbodiimide (or supported on a polymer), dipyrrolidinyl (N-succinimidyloxy) carbonium hexafluorophosphate (HSPyU), 1-(chloro-1-pyrrolidinylmethylene) pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolium tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipyridinium carbonium hexafluorophosphate (HBPipU), 6-chlorobenzotriazol-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), tris(dimethylamino)phosphine bromide hexafluorophosphate (BrOP), 1-n-propylphosphoric anhydride (PPACA, ), 2-isocyanoethylmorpholine (MEI), N,N,N',N'-tetramethylurea-oxy-(N-succinimidyl) hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthiourea tetrafluoroborate (TOTU), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl Aminyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)uronium tetrafluoroborate (TDBTU), dipiperidinium dicarboxylate (ADD), bis(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD) or diethyl azodicarboxylate (DEAD).

12. The conjugate according to any one of claims 9 to 11, It is characterized in that The synthesis of Tubulysin B derivatives of formula (III) comprises one or more of the following steps: R 5 'It is H, C 1 -C 6 Alkyl, C 1 -C 6 Alkyl, or C 1 -C 6 (Straight or branched) amine alkyl; 13. The conjugate according to claim 12, It is characterized in that The synthesis of Tubulysin B derivatives of formula (III) comprises one or more of the following steps: Step 1. Stir diethoxyacetonitrile and an aqueous solution of ammonium sulfide at room temperature to obtain compound 1, namely 2,2-diethoxythioacetamide; Step 2. Compound 1 and bromopyruvate are heated in an anhydrous solvent (such as anhydrous tetrahydrofuran, dichloromethane, acetonitrile, N,N-dimethylformamide, methanol, isopropanol) to condense to obtain compound 2; Step 3. Dissolve compound 2 in a solvent (such as tetrahydrofuran, dichloromethane, ethyl acetate, n-heptane, dioxane, acetonitrile), and use a Lewis acid or a protonic acid (including hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, pyridine p-toluenesulfonate, AlCl 3 、FeCl 3 、ZnCl 2 , BF 3 , BCl 3 , BBr 3 、TiCl 4 、ZnBr 2 , LiBF 4 ) is hydrolyzed to obtain compound 3; Step 4. The unsaturated sulfenamide is dehydrogenated with a base such as n-butyl lithium at low temperature (e.g., -45°C to -78°C), and then reacts with compound 3 in the presence of a Lewis acid to obtain compound 4; Lewis acids include hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, pyridine p-toluenesulfonate, AlCl 3 、FeCl 3 、ZnCl 2 , BF 3 , BCl 3 , BBr 3 、TiCl 4 、ZnBr 2 , LiBF 4 ; Step 5. Compound 4 is reacted with a reducing agent (such as NaBH) at low temperature (such as -45°C to -78°C). 4 , LiBH 4、 Na(OAc) 3 BH、Na(CN)BH 3 etc.) and selectively reduced, and by adding a Lewis acid (such as Ti(Oet)4), its stereochemistry is controlled to obtain compound 5; Step 6. Compound 5 is dissolved in a solvent (such as methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile), and the tert-butylsulfinyl group is removed by an acid such as hydrochloric acid, sulfuric acid and phosphoric acid to obtain compound 6; Step 7. Dissolve compound 6 and hydrazoic acid in a solvent (such as n-heptane, tetrahydrofuran, dichloromethane, N,N-dimethylformamide), and carry out a condensation reaction in the presence of a condensation reagent (such as DIC / HOBt, DCC / HOBt, EDC / HOBt, HATU, BOP, T3P, BrOP) or through a condensation reaction route to obtain compound 7; Alternatively, hydrazoic acid and isobutyl chloroformate react in the presence of an organic base (such as triethylamine, diisopropylethylamine, N-methylmorpholine, etc.) in THF to obtain a mixed anhydride, which is then condensed with the hydrochloride of compound 6 to obtain compound 7; Alternatively, hydrazoic acid reacts with oxalyl chloride, triethylamine and a catalytic amount of DMF in a solvent (such as n-heptane, n-hexane, dichloromethane, tetrahydrofuran) to be converted into an acyl chloride, which is then condensed with the hydrochloride of compound 6 to obtain compound 7; Step 8. In a solvent (such as dichloromethane, tetrahydrofuran, acetonitrile), the hydroxyl group on compound 7 is treated with a hydroxyl protecting agent (such as TESCl) in the presence of an organic base (such as imidazole, triethylamine, pyridine) to obtain compound 8; Step 9. Compound 8 is dissolved in a solvent (such as tetrahydrofuran, dichloromethane, acetonitrile), and the amide is deprotonated under the action of a base (such as KHMDS, LiHMDS, NaHMDS, KOtBu, NaH, KH), and then alkylated with iodomethane, bromomethane, dimethyl sulfate, methyl trifluoromethanesulfonate or iodoethane to obtain compound 9; Step 10. Compound 9 is dissolved in a solvent (such as tetrahydrofuran, dichloromethane, ethyl acetate), and the azide group thereof is reduced to an amino group under certain conditions, such as in the presence of hydrogen and palladium carbon catalyst, triphenylphosphine and water (Staudinger reaction), and then condensed with an acid or a reactive acid derivative to obtain compound 10; Step 11. Hydroxyl protecting group PG in compound 10 1 Under appropriate conditions (e.g., TES protecting groups can be protected in hydrochloric acid, THF / MeOH / AcOH, nBu 4 NF or pyridine hydrofluoride in THF solution) to give compound 11; Step 12. The ester group in compound 11 is converted into acid compound 12 under the action of a base (such as LiOH, NaOH, KOH) or other appropriate conditions (such as methyl ester can be converted into carboxylic acid under the action of reagents such as LiCl, LiI, Me3SiOK, etc.); Step 13. In the presence of a base (such as triethylamine, N,N-diisopropylethylamine, pyridine) and a catalyst (such as DMAP), at a certain temperature condition (such as 0°C to 23°C), compound 12 reacts with an acid anhydride such as acetic anhydride, propionic anhydride, isopropionic anhydride, acetyl halide, propionic acid halide, propionic acid halide, formamide halide, isoamide halide, diformamide halide, and the like to obtain compound 13. This reaction may also be carried out without using a base or a catalyst; Step 14. Compound 13 reacts with a suitable hydroxyl-containing compound such as pentafluorophenol or N-hydroxysuccinimide in the presence of a condensation reagent (such as EDC, DIC, DCC, HATU, HBTU) to obtain an active ester compound 14; Step 15. Compound 15 and compound 14 are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 16; the reaction may also be performed without using any base, provided that a certain reaction temperature (such as 0°C to 23°C) and reaction time (such as 30 minutes to 18 hours) need to be controlled; Step 16. The nitro group in compound 16 is oxidized under reducing conditions, such as hydrogen gas and palladium carbon catalyst, hydrazine hydrate and FeCl 3 , iron powder and acetic acid, etc., is reduced to an amino group to obtain compound III; 14. The conjugate according to any one of claims 9 to 13, It is characterized in that The synthesis of the compound of formula (L') comprises one or more of the following steps:

15. The conjugate according to claim 14, It is characterized in that The synthesis of the compound of formula (L') comprises one or more of the following steps: Step 1. Compound 1-1 and compound 1-2 are condensed under the action of a condensation agent (such as EDC, HATU, DIC, DCC), or undergo a condensation reaction via a condensation reaction route (such as compound 1-2 undergoes a condensation reaction with pentafluorophenol, nitrophenol or N-hydroxysuccinimide under the action of a condensation agent such as DIC and EDC to produce a corresponding active ester, which is then reacted with compound 1-1) to obtain compound 1a; Alternatively, compound 1-3 and compound 1-4 undergo condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC), or via a condensation reaction route, to obtain compound 1b; Step 2. Carboxyl protecting group PG in compound 1 2 Under the action of a deprotecting agent (such as the tert-butyl ester group under the action of an acid), it is removed to obtain compound 2; Step 3. The carboxyl-containing compound 2 and the amino-containing compound 3 undergo a condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC) or via a condensation reaction route to obtain a compound 4; Step 4. Amino protecting group PG on compound 4 1 It can be removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 5; Step 5. The carboxyl-containing compound 6 and the amino-containing compound 5 undergo a condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC) or via a condensation reaction route to obtain compound 7; Step 6. Protecting group PG of the carboxyl group of compound 7 3 It is removed under deprotection conditions (for example, the tert-butyl ester protecting group on the carboxyl group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.) to obtain compound 8; Step 7. Compound 8 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, DCC) to obtain an ester compound having a reaction activity, or reacts with other acid-activated groups to obtain a compound L' having a condensation reaction activity; 16. The conjugate according to any one of claims 9 to 13, It is characterized in that The synthesis of the compound of formula (L') comprises one or more of the following steps:

17. The conjugate according to claim 16, It is characterized in that The synthesis of the compound of formula (L') comprises one or more of the following steps: Step 1. Amino protecting group PG on compound 1 1 It can be removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 2; Step 2. Compound 2 containing an amino group and compound 3 containing a carboxyl group undergo a condensation reaction under the action of a condensation reagent (such as EDC, HATU, DIC, DCC) or via a condensation reaction route to obtain compound 4; Step 3. Carboxyl protecting group PG on compound 4 2 It can be removed under deprotection conditions, such as the tert-butyl ester protecting group on the carboxyl group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc. to obtain compound 5; Step 4. The carboxyl-containing compound 5 and the amino-containing compound 6 undergo a condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC) or via a condensation reaction route to obtain compound 7; Step 5. Protecting group PG of the carboxyl group of compound 7 3 It can be removed under deprotection conditions, such as the tert-butyl ester protecting group on the carboxyl group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc. to obtain compound 8; Step 6. Compound 8 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation agent to obtain an ester compound having a reaction activity, or reacts with other acid-activated groups to obtain compound 9 having a condensation reaction activity; 18. The conjugate according to any one of claims 3 to 11, It is characterized in that The synthesis of the compound of structural formula (II) is obtained by condensation reaction of structural formula (IV) and structural formula (V): The definition of X and the condensation reaction conditions are as described in any one of claims 9 to 11.

19. The conjugate according to claim 18, It is characterized in that NH in the structural formula (V) 2 The group is condensed in the form of trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate.

20. The conjugate according to any one of claims 18 to 19, It is characterized in that The synthesis of structural formula (IV) comprises one or more of the following steps:

21. The conjugate according to claim 20, It is characterized in that The synthesis of structural formula (IV) comprises any of the following steps: The carboxylic acid compound 1 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, DIC, DCC, HATU, HBTU) to obtain an ester having a reactive activity; Alternatively, the carboxylic acid compound 1 reacts with ethyl chloroformate, isobutyl chloroformate, etc. in the presence of an organic base (such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc.) to obtain a mixed acid anhydride having a reaction activity; Alternatively, the carboxylic acid compound 1 reacts with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount (eg, 0.01 equivalent to 0.5 equivalent) of DMF to obtain an acid chloride.

22. The conjugate according to any one of claims 18 to 21, It is characterized in that The synthesis of structural formula (V) comprises one or more of the following steps:

23. The conjugate according to claim 22, It is characterized in that The synthesis of structural formula (V) comprises one or more of the following steps: Step 1. Compound 1 and compound 2 are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 3; the reaction may also be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled; Step 2. Amino protecting group PG on compound 3 4 It can be removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound V; 24. The conjugate according to any one of claims 22 to 23, It is characterized in that The synthesis of compound 2 includes one or more of the following steps: Among them, the compound 8 (compound XIVa) obtained in this synthesis step is the target compound 2.

25. The conjugate according to claim 24, It is characterized in that The synthesis of compound 2 includes one or more of the following steps: Step 1. Dissolve the L-tyrosine ester derivative 1 in a suitable solvent, such as acetone, tetrahydrofuran, acetonitrile, dichloromethane, etc., or a mixed solvent of these solvents and water, and react with benzyl chloride, benzyl bromide or other benzyl compounds at 0 to 60° C., and add a suitable organic or inorganic base to the reaction system, such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, DBU, sodium hydride, etc., and add a suitable additive such as sodium iodide or a phase transfer catalyst, such as benzyltriethylammonium chloride (TEBA), tetrabutylammonium bromide (TBAB), tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, etc., to obtain compound 2; Step 2. Dissolve compound 2 in an organic solvent such as dichloromethane, tetrahydrofuran, methanol, ethanol, ether, etc., and reduce it with a reducing agent such as lithium aluminum hydride, DIBAL, sodium borohydride, lithium borohydride, sodium dihydrobis(2-methoxyethoxy)aluminate (Red-Al), diborane, etc. Additives such as I may be added to the reaction system. 2 , ferric chloride, zinc chloride, magnesium chloride, lithium chloride, calcium chloride, etc. to control the activity of the reducing agent to obtain compound 3; Step 3. Alcohol compound 3 is oxidized to aldehyde 4 under appropriate oxidation conditions, such as Swern oxidation (oxalyl chloride, DMSO, triethylamine), Parikh-Doering oxidation (sulfur trioxide pyridine oxidation), Dess-Martin oxidation, etc.; Step 4. Aldehyde 4 reacts with phosphate ester (Horner-Wadsworth-Emmons reaction) or phosphorus ylide reaction (Wittig reaction) to extend the carbon chain to obtain compound 5; Step 5. The double bond in compound 5 is hydrogenated and reduced under the action of a homogeneous or biphasic catalyst, and the benzyl group is also removed at the same time to obtain a chiral compound with a single stereostructure or a mixture of two diastereomers; the catalyst includes Pd / C, Pd(OH) 2 / C、Pd / BaSO 4 , PtO 2 、Pt / Al 2 O 3 , Ru / C, Raney nickel and other two-phase catalysts, homogeneous asymmetric hydrogenation catalysts such as Crabtree catalyst, [Ru(II)-(BINAP)] type catalyst, [(Ph3P)CuH] 6 Catalysts, etc. Step 6. Dissolve compound 6 in an organic solvent, such as tetrahydrofuran, acetonitrile, or dichloromethane, and nitrate under nitration conditions. The nitration reagent includes nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, tert-butyl nitroso, nitric acid / trifluoroacetic anhydride, NO 2 BF 4 , nitropyridinium salts, etc.; Step 7. The nitro group in compound 7 was reduced to an amino group under the following conditions: 2 / Pd / C, Fe or Zn / HOAc, SnCl 2 / HCl.

26. The conjugate according to any one of claims 22 to 23, It is characterized in that The synthesis of compound 2 includes one or more of the following steps: Among them, the compound 8 (compound XIVb) obtained in this synthesis step is the target compound 2.

27. The conjugate according to claim 26, It is characterized in that The synthesis of compound 2 includes one or more of the following steps: Step 1. Compound 1 is reacted with an Evans chiral N-acyl oxazolidinone or thione 2, wherein X=O or S, R 16 =H, methyl, phenyl, R 17 =H, methyl, isopropyl, phenyl, benzyl, etc., undergo Aldol reaction to obtain compound 3 with a single stereo configuration; Step 2. The hydroxyl group on compound 3 was removed under the following conditions, which included a Barton–McCombie deoxygenation reaction, i.e., the alcohol was first converted to a thioacyl derivative, such as alkyl xanthate, phenyl chlorothioformate, or thiocarbonyl imidazole ester, and then reacted with Bu 3 SnH treatment, free radical cleavage occurs to obtain dehydroxylated products; free radical bond breaking conditions include: n-Bu 3 SnH / AIBN, n-Bu 3 SnH / AIBN / n-BuOH / PMHS, (Bu 4 N) 2 S 2 O 8 / HCO 2 Na; Step 3. Compound 4 was dissolved in tetrahydrofuran and the Evans chiral auxiliary group was reacted with LiOH / H 2 O 2 The corresponding acid 5 was cleaved under the conditions of Step 4. Compound 5 is dissolved in an organic solvent, such as ethyl acetate, methanol, dichloromethane, ethanol or acetic acid, and the benzyl group thereof is catalytically hydrogenated in the presence of a palladium-carbon catalyst to obtain compound 6; Step 5. Dissolve compound 6 in an organic solvent, such as tetrahydrofuran, acetonitrile, or dichloromethane, and nitrate under nitration conditions. The nitration reagent includes nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, tert-butyl nitroso, nitric acid / trifluoroacetic anhydride, NO 2 BF 4 , nitropyridinium salts, etc.; Step 6. The nitro group in compound 7 was reduced to an amino group under the following conditions: 2 / Pd / C, Fe or Zn / HOAc, SnCl 2 / HCl, etc., to obtain chiral compound 8 with a single stereo configuration.

28. The conjugate according to any one of claims 3 to 11, It is characterized in that The synthesis of structural formula (II) is obtained by condensation reaction of structural formula (VI) and structural formula (VII): The definition of X and the condensation reaction conditions are as described in any one of claims 9 to 11.

29. The conjugate according to claim 28, It is characterized in that The synthesis of structural formula (VI) comprises one or more of the following steps:

30. The conjugate according to claim 29, It is characterized in that The synthesis of structural formula (VI) comprises one or more of the following steps: Step 1. Compound 1 reacts with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent to obtain a reactive acid derivative compound 2; Step 2. Compound 2 and compound 3 are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 4; the reaction may also be carried out without using any base, but it is necessary to control the appropriate reaction temperature and reaction time; Step 3. Amino protecting group PG on compound 4 4 It can be selectively removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 5; Step 4. Compound 5 and structural formula (IV) are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 6; the reaction may also be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled; Step 5. Amino protecting group PG on compound 6 1 It can be removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound VI; 31. The conjugate according to any one of claims 28 to 30, It is characterized in that The synthesis of structural formula (VII) comprises one or more of the following steps:

32. The conjugate according to claim 31, It is characterized in that The synthesis of structural formula (VII) comprises one or more of the following steps: Step 1. Carboxylic acid compound 1 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, DCC) to obtain an ester having a reactive activity; Alternatively, the carboxylic acid compound 1 reacts with ethyl chloroformate, isobutyl chloroformate, etc. in the presence of an organic base (such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc.) to obtain a mixed acid anhydride having a reaction activity; Alternatively, the carboxylic acid compound 1 reacts with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount (e.g., 0.01 equivalent to 0.5 equivalent) of DMF to obtain an acyl chloride; 33. The conjugate according to any one of claims 3 to 11, It is characterized in that The synthesis of structural formula (II) is obtained by condensation reaction of structural formula (VIII) and structural formula (IX): The definition of X and the condensation reaction conditions are as described in any one of claims 9 to 11.

34. The conjugate according to claim 33, It is characterized in that NH in the structural formula (VIII) 2 The group is condensed in the form of trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate.

35. A conjugate as described in any one of claims 33-34, It is characterized in that The synthesis of structural formula (VIII) comprises one or more of the following steps:

36. The conjugate according to claim 35, It is characterized in that The synthesis of structural formula (VIII) comprises one or more of the following steps: Step 1. Carboxyl protecting group PG on compound 1 3 It is removed under deprotection conditions (for example, the tert-butyl ester protecting group on the carboxyl group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.) to obtain compound 2; Step 2. Compound 2 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, DCC) to obtain an ester compound 3 having a reaction activity; Step 3. Compound 3 and compound 4 are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 5; the reaction may also be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled; Step 4. Amino protecting group PG on compound 5 3 The protecting group of the amino group can be removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 6; Step 5. Compound 6 and structural formula (IV) (i.e. structural formula (IV) according to any one of claims 18 to 21) are reacted in an aqueous phase under a certain pH condition (e.g. pH = 5.0-8.0) or in an organic phase in the presence of an organic base (e.g. TEA, DBU, DIPEA) or an inorganic base (e.g. Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 7; the reaction may also be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled; Step 6. Amino protecting group PG on compound 7 1 It can be removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound VIII; 37. A conjugate as described in any one of claims 33 to 36, It is characterized in that The synthesis of structural formula (IX) comprises one or more of the following steps: The carboxylic acid compound 1 reacts with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation agent to obtain a reactive ester IX; Alternatively, the carboxylic acid compound 1 reacts with ethyl chloroformate, isobutyl chloroformate, etc. in the presence of an organic base (such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc.) to obtain a reactive mixed acid anhydride IX; Alternatively, the carboxylic acid compound 1 reacts with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount of DMF to obtain the acyl chloride IX; 38. A conjugate as described in any one of claims 3 to 11, It is characterized in that The synthesis of structural formula (II) is obtained by condensation reaction of structural formula (X) and structural formula (XI): where Y 1 and Y 2 The groups are condensed to form a Y group; 1 and Y 2 NH 2 、- + NH 3 , COOH, COX, SO 2 Cl、P(O)Cl 2 、NHCOX、NHSO 2 Cl、NHP(O)Cl 2 、NHP(O)(OH)Cl、 39. The conjugate according to claim 38, It is characterized in that The synthesis of structural formula (X) comprises one or more of the following steps:

40. The conjugate according to claim 39, It is characterized in that The synthesis of structural formula (X) comprises one or more of the following steps: Step 1. Compound 1 containing a carboxyl group and compound VI undergo a condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC), or through a condensation reaction route, to obtain compound 2; wherein Z 1 Y 1 Precursors of, such as amino, carboxyl, amide, phosphoramide and sulfonamide groups, carboxylates, phosphates, phosphonates, etc., which are protected by appropriate groups; Step 2. Amino Protecting Group PG on Compound 2 1 It can be removed under deprotection conditions, such as the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium carbon catalyst, and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 3; Step 3. The carboxyl-containing compound 4 and the amino-containing compound 3 undergo a condensation reaction under the action of a condensation agent or via a condensation reaction route to obtain a compound 5; Step 4. Functional group Z in compound 5 1 After appropriate chemical transformation, such as deprotection of carboxyl and amino groups, functional group Y is generated. 1 , to obtain compound X; 41. A conjugate as described in any one of claims 38 to 40, It is characterized in that The synthesis of structural formula (XI) comprises one or more of the following steps:

42. The conjugate of claim 41, It is characterized in that The synthesis of structural formula (XI) comprises one or more of the following steps: Step 1. Dissolve compound 1 in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, extract hydrogen with a base such as sodium hydride, sodium, sodium hydroxide, etc., and then react with compound 2 (wherein X is a halogen such as chlorine, bromine, iodine, etc. or other leaving groups) at a certain temperature to obtain compound 3; Step 2. The carboxyl protecting group PG1 on compound 3 is removed under deprotection conditions, such as the tert-butyl ester protecting group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc., to obtain compound XIa-1; Step 3. Dissolve compound 1 in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, extract hydrogen with a base such as sodium hydride, sodium, sodium hydroxide, etc., and then react with compound 4 (stirred at a certain temperature to obtain compound 5; Step 4. Protecting group PG of the carboxyl group of compound 5 1 It can be removed under deprotection conditions, such as the tert-butyl ester protecting group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc. to obtain compound XIa-2; Step 5. Dissolve compound 6 in an organic solvent, such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc., add a suitable organic base, such as triethylamine, N,N-diisopropylethylamine, pyridine, etc., and react with methanesulfonyl chloride, 4-toluenesulfonyl chloride, etc. at 0-5°C to obtain compound 7; Step 6. Compound 7 is reacted with ammonia in an aqueous phase or an organic solvent, such as methanol, ethanol, acetonitrile, tetrahydrofuran, hexacyclic epoxy, etc. The reaction may be heated appropriately to obtain compound XIb. Step 7. Compound 7 reacts with sodium azide in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc. to obtain compound 8; Step 8. Azide compound 8 is hydrogenated and reduced in the presence of a palladium-carbon catalyst, or reduced under the action of triphenylphosphine and water to obtain compound XIb; Step 9. Compound 7 reacts with dibenzylamine in an organic solvent, such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc., preferably N,N-dimethylformamide, at 100° C. to obtain compound 9; Step 10. Dissolve compound 9 in a solvent such as ethyl acetate, methanol, ethanol, acetic acid, tetrahydrofuran, etc., and reduce it on a palladium carbon catalyst under a certain hydrogen pressure. The reaction can be appropriately heated to 45° C. to obtain compound XIb.

43. The conjugate according to any one of claims 3 to 11, It is characterized in that The synthesis of structural formula (II) is obtained by condensation reaction of structural formula (XII) and structural formula (XIII): The definition of X and the condensation reaction conditions are as described in any one of claims 9 to 11.

44. The conjugate according to claim 43, It is characterized in that NH in the structural formula (XII) 2 The group is condensed in the form of trifluoroacetate, hydrochloride, formate, acetate, sulfate, phosphate, nitrate, citrate, succinate, benzoate or sulfonate.

45. A conjugate as described in any one of claims 43-44, It is characterized in that The synthesis of structural formula (XII) comprises one or more of the following steps:

46. ​​The conjugate according to claim 45, It is characterized in that The synthesis of structural formula (XII) comprises one or more of the following steps: Step 1. Compound 1 reacts with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent to obtain a reactive acid derivative compound 2; Step 2. Compound 2 and compound 3 are reacted in an aqueous phase under a certain pH condition (e.g., pH = 5.0-8.0), or in an organic phase in the presence of an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 4; the reaction may also be carried out without using any base, but it is necessary to control the appropriate reaction temperature and reaction time; Step 3. Amino protecting group PG on compound 4 4 It can be selectively removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound 5; Step 4. Compound 5 and structural formula (IV) (i.e. structural formula (IV) according to any one of claims 18 to 21) are reacted in an aqueous phase under a certain pH condition (e.g. pH = 5.0-8.0) or in an organic phase in the presence of an organic base (e.g. TEA, DBU, DIPEA) or an inorganic base (e.g. Na 2 CO 3 , Cs 2 CO 3 , K 2 CO 3 、NaHCO 3 ), a condensation reaction occurs to obtain compound 6; the reaction may also be carried out without using any base, but the appropriate reaction temperature and reaction time need to be controlled; Step 5. The amino protecting group PG1 on compound 6 is removed under deprotection conditions (e.g., the Cbz protecting group on the amino group can be removed under the action of hydrogen and palladium-carbon catalyst), and the Boc protecting group on the amino group can be removed under acidic conditions to obtain compound XII; 47. A conjugate as described in any one of claims 43 to 46, It is characterized in that The synthesis of structural formula (XIII) comprises one or more of the following steps:

48. The conjugate according to claim 47, It is characterized in that The synthesis of structural formula (XIII) comprises one or more of the following steps: Step 1. A carboxyl group-containing compound 1 and an amino group-containing compound 2 undergo a condensation reaction under the action of a condensation agent (such as EDC, HATU, DIC, DCC) or via a condensation reaction route to obtain a compound 3; Step 2. Carboxyl protecting group PG on compound 3 1 It is removed under deprotection conditions (for example, the tert-butyl ester protecting group on the carboxyl group can be removed under the action of formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.) to obtain compound 4; Step 3. Carboxylic acid compound 4 reacts with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation agent to obtain an ester having a reactive structure (XIII); Alternatively, the carboxylic acid compound 4 reacts with ethyl chloroformate, isobutyl chloroformate, etc., in the presence of an organic base such as N-methylmorpholine, triethylamine, diisopropylethylamine, etc. to obtain a reactive mixed acid anhydride of formula (XIII); Alternatively, the carboxylic acid compound 4 reacts with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount of DMF to obtain an acyl chloride having the structural formula (XIII).

49. The conjugate according to any one of claims 1 to 48, It is characterized in that The structure of the compound of formula (I) is as follows:

50. A compound, It is characterized in that The compound of formula (II) has the following structure:

51. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 49 or a conjugate composed of the compound according to claim 50, and a pharmaceutically acceptable excipient.

52. Use of the conjugate according to any one of claims 1 to 49 in the preparation of a medicament for treating cancer, infection or autoimmune disease.

53. Use of the compound of claim 50 in the preparation of a medicament for treating cancer, infection or autoimmune disease.

54. Use of the pharmaceutical composition of claim 51 in the preparation of a medicament for treating cancer, infection or autoimmune disease.