Cell-binding molecule-tubulosin derivative conjugate and method for preparing the same

Abstract

The present invention involves the conjugation of tubulysin derivatives (analogs) to cell-binding molecules via branched (side chain) linkers, with the resulting conjugates having better pharmacokinetic properties and thus enabling more precise targeting and killing of diseased cells. The present invention also relates to methods for synthesizing the conjugates of tubulysin analogs to cell-binding agents and the molecules involved therein, as well as targeted treatment of cancer, infectious diseases, and autoimmune diseases using the conjugates. Conjugates of tubulysin with long branched linkers extend half-life during targeted delivery and minimize exposure to non-target cells, tissues, or organs in the blood circulation, thereby reducing off-target toxicity.

Description

[Technical Field]

[0001] This invention relates to a conjugate of a tubulicin derivative (congener) and a cell-binding molecule using a branched (side-chain) linkage, wherein the resulting conjugate has superior pharmacokinetic properties and can more accurately target and kill abnormal cells. This invention also relates to a method for conjugating a tubulicin derivative (congener) with a cell-binding agent, a method for synthesizing the molecules contained therein, and a method for using the conjugate for targeted therapy of cancer, infectious diseases, and autoimmune diseases. [Background technology]

[0002] The clinical success of "Adcetris" for relapsed / refractory Hodgkin lymphoma (Non-Patent Documents 1 and 2) and "Kadcyla" for relapsed HER2-positive breast cancer (Non-Patent Documents 3 and 4) has made antibody-drug conjugates (ADCs) one of the most promising targeted therapies for cancer. All three components of an ADC—monoclonal antibody, cytotoxic agent, and conjugate—are crucial factors in the success of ADCs (Non-Patent Documents 5 and 6). Research into each component of ADCs has continued for 30 years. The conjugate must react with specific reactive functional groups on the drug, possess stability in the human blood circulation, and be able to readily release the drug after binding to the antigen on a cell and being taken up into the cell. It is also important that the conjugate-payload portion is off-target in the blood circulation and does not damage normal tissue, but existing conjugation technologies remain limited (Non-Patent Documents 7-10).

[0003] Early ADCs were primarily used for targeted therapy of liquid tumors, but the conjugates used were highly unstable, leading to the release of free drugs into the bloodstream and resulting in off-target toxicity (Non-Patent Literature 11). Today's generation of ADCs uses more stable conjugates and highly active cytotoxic agents (Non-Patent Literature 12). However, off-target toxicity remains one of the major challenges in ADC drug development (Non-Patent Literature 13). For example, T-DM1 (Kadcyla®), which uses a stable (uncleavable) MCC conjugate, showed significant benefits in clinical trials in patients with HER2-positive metastatic breast cancer (mBC) or patients who had already been treated for mBC or whose HER2 tumor recurred within 6 months of adjuvant therapy (Non-Patent Literature 14-16). However, clinical trials comparing the side effects and efficacy of T-DM1 as a first-line treatment for patients with HER2-positive, unresectable locally advanced or metastatic breast cancer, and as a second-line treatment for HER2-positive, advanced gastric cancer, were unsuccessful because the patient benefits were minimal (Non-Patent Documents 17-20).

[0004] To address the issue of off-target toxicity, research and development in the chemistry and design of ADCs is now expanding beyond mere payload potency to encompass the scope of conjugate-payload compartments and conjugation chemistry, particularly addressing the conjugate-payload activity of ADCs toward targeted / target diseases (Non-Patent Literature 21-22). Today, many drug developers and academic institutions are focusing on establishing reliable, specific, novel conjugated conjugates and site-specific ADC conjugation methods that are expected to have longer circulating half-lives, higher efficacy, reduced off-target toxicity, a narrower range of in vivo pharmacokinetic (PK) properties of ADCs, and improved batch-to-batch consistency in ADC production (Non-Patent Literature 23-27). These specific conjugation methods reported to date include: insertion of manipulated cysteine ​​(Non-Patent Documents 28-29, Patent Documents 1-5), selenocysteine ​​(Non-Patent Documents 30-31, Patent Document 6), cysteine-containing tags including perfluoroaromatic reagents (Non-Patent Document 27), thiolfucose (Non-Patent Document 33), unnatural amino acids (Non-Patent Documents 34-37, Patent Documents 7-17), binding to reduced intermolecular disulfide by re-crosslinking dibromomalemide (Non-Patent Document 38), bissulfone reagents (Non-Patent Document 39, Patent Documents 18-19), dibromopyridazinedione (Non-Patent Document 40), galactosyltransferase and sialyltransferase (Non-Patent Document 41, Patent Document 20), formylglycine-producing enzyme (FGE) (Non-Patent Document 42, Patent Documents 21-25), phosphopantetheinyltransferase (PPTase) (Non-Patent Document 43), saltase A (Non-Patent Document 44), and Streptoverticillium. This includes a genetically introduced glutamine tag using mobaraense transglutaminase (mTG) (Non-Patent Documents 45-46, Patent Document 26) or microbial transglutaminase (MTGase) (Non-Patent Documents 47-48, Patent Documents 27-28), and enzymes / bacteria that form isopeptide bond-peptide bonds on the outside of the protein backbone (Non-Patent Documents 49-51). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Publication No. 8,309,300 [Patent Document 2] U.S. Publication No. 7,855,275 [Patent Document 3] U.S. Publication No. 7,521,541 [Patent Document 4] U.S. Publication No. 7,723,485 [Patent Document 5] International Publication No. WO2008 / 141044 [Patent Document 6] U.S. Publication No. 8,916,159 [Patent Document 7] U.S. Publication No. 8,778,631 [Patent Document 8] U.S. Patent Application Publication No. 20100184135 [Patent Document 9] International Publication No. WO2010 / 081110 [Patent Document 10] International Publication No. WO2006 / 069246 [Patent Document 11] International Publication No. WO2007 / 059312 [Patent Document 12] U.S. Publication No. 7,332,571 [Patent Document 13] U.S. Publication No. 7,696,312 [Patent Document 14] U.S. Publication No. 7,638,299 [Patent Document 15] International Publication No. WO2007 / 130453 [Patent Document 16] U.S. Publication No. 7,632,492 [Patent Document 17] U.S. Publication No. 7,829,659 [Patent Document 18] International Publication No. WO2013 / 190272 [Patent Document 19] International Publication No. WO2014 / 064424 [Patent Document 20] U.S. Patent and Trademark Office Publication No. 20140294867 [Patent Document 21] U.S. Patent No. 7,985,783 [License 22] U.S. Patent No. 8,097,701 [License 23] U.S. Patent No. 8,349,910 [License 24] U.S. Patent and Trademark Office Publication No. 20140141025 [License 25] U.S. Patent and Trademark Publication No. 20100210543 [License 26] U.S. Patent No. 8,871,908 [License 27] U.S. Patent and Trademark Publication No. 20130189287 [License 28] U.S. Patent No. 7,893,019 [Non-licensed literature]

[0006] [Non-licensed Document 1] Okeley, N. et al., Hematol Oncol.Clin.North.Am,2014,28,13-25 [Non-licensed Document 2] Gopal,A.,et al., Blood 2015,125,1236-43 [Non-licensed Document 3] Peddi, P., Hurvitz, S., Ther. Adv. Med. Oncol. 2014, 6(5), 202-9 [Non-licensed Document 4] Lambert, J. and Chari, R., J. Med. Chem. 2014, 57, 6949-64 [Non-licensed Document 5] L. Ducry and B Stump, Bioconjugate Chem., 2010, 21, 5-13

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[0007] The applicant has disclosed several conjugation methods for recrosslinking reduced thiol pairs of interchain disulfide bonds in natural antibodies, including using bromomaleimide and dibromomaleimide conjugates (International Publication WO2014 / 009774), 2,3-disubstituted succinic acid / 2-monosubstituted / 2,3-disubstituted fumaric acid or maleic acid conjugates (International Publication WO2015 / 155753 and International Publication WO20160596228), acetylenedicarboxylic acid conjugates (International Publication WO2015 / 151080 and International Publication WO20160596228), or hydrazine conjugates (International Publication WO2015 / 151081). ADCs prepared using these conjugates and methods exhibit superior therapeutic index compared to conventional non-selective conjugation via cysteine ​​or lysine residues of antibodies. Herein, the applicant discloses an invention of a tubulicin conjugate containing a long side chain linkage. The long side chain linkage prevents the antibody-drug conjugate from being hydrolyzed by hydrolytic enzymes such as proteinases or esterases, and the applicant has found that this makes the conjugate more stable during target delivery, thus completing the present invention.

[0008] Tubricins, a class of highly effective cytotoxic agents, are well known in this art and can be extracted from natural products based on known methods or synthesized by known organic synthesis methods (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., etal. J. Am. Chem. Soc., 2011, 133, 7692-5; Reddy, JA, et al. Mol. Pharmaceutics, 2009, 6, 1518-25; Raghavan, B., et al. J. Med. Chem., 2008, 51, 1530-33; Patterson, AW, etal. 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, GK, Org. Lett., 2004, 6, 3249-52; Peltier, HM, e tal. J. Am. Chem. Soc., 2006, 128, 16018-9; Chanrasekhar, S., et al J. Org. Chem., 2009, 74, 9531-4; Liu, Y., etal. Mol. Pharmaceutics, 2012, 9, 168-75; Friestad, GK, etal. Org. Lett., 2009, 11, 1095-8; Kubicek, K., et al., Angew Chem Int Ed Engl, 2010.49: 4809-12; Chai, Y., etal., Chem Biol, 2010, 17: 296-309; Ullrich, A., et al., Angew Chem Int Ed Engl, 2009, 48, 4422-5; Sani, M., etal.Angio 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), WO2010034724; Leamon,C.et al, WO2010033733, WO2009002993; Ellman , J. , et al , PCT WO2009134279 ; WO 2009012958; NUMBER SYSTEM NUMBER20110263650, 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. ; etal, National Vehicle Issue 20070041901, WO2006096754; Matschiner , G. , et al , WO2006056464 ; Vaghefi, F., et al, WO2006033913; Doemling , A. , Ger. Offen. DE102004030227, WO2004005327, WO2004005326, WO2004005269; Stanton, M., et al, Documentation 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. (1999).U.S. Patent Application 20110027274. We previously disclosed the construction of a tubulicin conjugate for targeted therapy of cancer, infectious diseases, and autoimmune diseases (PCT / IB2012 / 053554). The tubulicin conjugate comprising the long-chain branched (side-chain) linkage in this invention extends the half-life during targeted delivery, minimizing exposure to non-target cells, tissues, or organs in the bloodstream, and consequently reducing off-target toxicity.

[0009] The present invention relates to the conjugation of a cell-binding molecule having branched (side-chain) links with a tubulicin analog, and to a conjugate having better pharmacokinetic properties and therefore being able to more accurately target and kill abnormal cells. The present invention also provides a method for conjugating a tubulicin analog with a cell-binding agent, a method for synthesizing the tubulicin molecule used therein, and a method for targeted therapy of cancer, infectious diseases, and autoimmune diseases using the conjugate. [Means for solving the problem]

[0010] In one embodiment, the present invention relates to an antibody-tubulisin B derivative conjugate, wherein the conjugate has the structure of formula (I) below, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or a polymorphic crystalline structure represented by formula (I); or an optical isomer of the structure represented by formula (I); or one or more hydrogens ( 1 H) Atoms containing 1 or more deuterium ( 2 Substituted with H atoms, or one or more 12 C atom 1 or more 13 These are derivatives of those substituted with a carbon atom: [ka]

[0011] In the formula, P 1 is H, COCH3, COH, PO(OH)2, CH2OPO(OH)2, CONHCH3, CON(CH3)2, CON(CH2CH2)2NCH3, CON(CH2CH3)2, or CON(CH2CH2)2CHN(CH2CH2)2CH2;

[0012] R1, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkyl ether group (R1OR2), C1-C6 alkyl carbonyl (R1COR2), C1-C6 alkyl ester (R1COOR2), C1-C6 alkyl carboxy (R1COOH), or C1-C6 alkylamide group (R1CONHR2);

[0013] Alternatively, R1 and R2, R1 and R3, R2 and R3, or R3 and R4 together form a C2-C7 heterocyclyl or C2-C7 cycloalkyl structure;

[0014] R 5 These are H, O-C1~C6 alkyl groups, C(O)-H, C(O)-C1~C6 (linear or branched) alkyl groups, C(O)-NH-C1~C6 (linear or branched) alkyl groups, or C(O)-N(C1~C6 (linear or branched) alkyl) 2 groups;

[0015] R6, R7, and R8 are each independently H, a C1-C6 alkyl group, a C1-C6 alkyl ether group (R1OR2), a C1-C6 alkylcarbonyl group (R1COR2), a C1-C6 alkyl ester group (R1COOR2), a C1-C6 alkylcarboxyl group (R1COOH), or a C1-C6 alkylamide group (R1CONHR2); preferably R6, R7, and R8 are each independently H or CH3;

[0016] mAb refers to an antibody, antibody fragment, monoclonal antibody, polyclonal antibody, nanobody, prodrug antibody (probody), or antibody and antibody fragment modified with a synthetic molecule or protein;

[0017] L is a connective including hydrophilic branches, and its main frame is C2-C 100 The peptide unit (1-12 natural or unnatural amino acids), hydrazone, disulfide, ester, oxime, amide, or thioether linkage;

[0018] In one aspect, in the conjugate of the present invention, the structure of L is as follows:

Chemical formula

[0019] Wherein, Aa is an L- or D- natural or non-natural amino acid;

[0020] r is an integer from 0 to 12; when r is not 0, (Aa) r is a peptide unit composed of the same or different amino acids;

[0021] m1 = an integer from 1 to 18; m2 = an integer from 1 to 100; m3 = an integer from 1 to 8; m4 = an integer from 0 to 8; m5 = an integer from 1 to 8;

[0022] Y is NHC(=O), NHS(O2), NH(SO), NHS(O2)NH, NHP(O)(OH)NH, or C(O)NH;

[0023] R9 is H, (O=)CR1, (O=)CNHR1, R1COOH, R1(COCH2NH) m2 H, R1(Aa) r or R1(COCH2NCH3) m2 H;

[0024] R1, m2, and (Aa) r are as described in claims 1 and the above definitions.

[0025] Furthermore, the cell surface receptor-binding molecule mAb can take the form of any cell-binding structure and includes a cell-binding agent / molecule selected from the group consisting of: peptides or peptide-like structures; antibodies; single-chain antibodies; antibody fragments that bind to target cells; monoclonal antibodies; single-chain monoclonal antibodies; or monoclonal antibody fragments that bind to target cells; chimeric antibodies; chimeric antibody fragments that bind to target cells; domain antibodies; domain antibody cross-sections that bind to target cells; antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules; transferrin; binding peptides or proteins, or small molecules, polymers, dendrimers, liposomes, nanoparticles, vesicles, or (viral) capsids bound to antibodies or albumin.

[0026] Method for producing cell-binding molecule-tubulisin B derivative conjugates

[0027] In certain embodiments, the synthesis of a cell-binding molecule-tubulosine B derivative conjugate comprises one or more of the following steps: [ka]

[0028] In formula (II), P 1 , [ka] R1, R2, R3, R4, R5, R6, R7, and R8, as well as mAb, are as shown in formula (I);

[0029] The structure of L' is (II-0) and (II-00): [ka]

[0030] In the formula, m1, m2, m3, m4, m5, Aa, r, Y, and R9 are as described in formula (I).

[0031] Specifically, the ideal structure of L' is as follows: [ka]

[0032] In the formula, m1, m2, m3, m4, m5, Aa, r, and R9 are as described in claim 1 or 2.

[0033] In other specific embodiments, the preparation of mAb-SH in the process of producing the conjugate includes any of the following a) to c):

[0034] a) Reduction of disulfide bonds between heavy chains and light chains or between heavy chains, or intramolecular disulfide bonds of antibodies, antibody fragments, monoclonal antibodies, polyclonal antibodies, nanobodies, probodies or antibodies, and antibody fragments modified by 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));

[0035] b) Generation of thiol groups by reaction of Traut reagent or thiolactone with the amino group of the antibody molecule; [ka]

[0036] c) In a buffer system, introduction of readily reducible disulfide bonds into the antibody by biochemical reaction, followed by reduction with TCEP, DTT, GSH, β-MEA, or β-ME; [ka]

[0037] In another specific embodiment, the step of preparing the above-described conjugate is characterized in that the buffer system used in the synthesis of the conjugate is a pH 5.0 to 9.5, 1 mM to 1000 mM buffer of phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (trimethylolaminomethane), benzoic acid, or triethanolamine, or a mixture thereof, containing 0% to 35% of a water-soluble organic solvent which is methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA, or DMSO; the reaction temperature is 0°C to 45°C; and the reaction time is 5 minutes to 96 hours.

[0038] In other specific embodiments, the step of preparing the conjugate involves obtaining the conjugate of formula (I) by ultrafiltration or column chromatography after the coupling reaction is complete. The purification column is typically a molecular sieve column, a cation column, an anion column, a hydrophobic (HIC) column, a reversed-phase column, or a protein A or G affinity column.

[0039] In another specific embodiment, in the process of preparing the above conjugate, the compound of formula (II) is obtained by condensation of the tubulicine B derivative of formula (III) and the compound of formula (L'): [ka]

[0040] In the formula, 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, [ka] An anhydride formed from itself or other anhydrides such as acetic anhydride and formic anhydride; or an intermediate in a peptide condensation reaction or a Mitsunobu reaction;

[0041] The condensation reaction is carried out at -20°C to -150°C for 5 minutes to 120 hours in a solvent containing 1% to 100% by volume of pyridine, triethylamine, or diisopropylethylamine, in a solvent such as dichloroethane, DMF, DMA, tetrahydrofuran (THF), DMSO, acetone, isopropanol n-butanol, or acetonitrile, or a mixture of two or three of these, with or without protection from an inert gas (nitrogen, argon, helium).

[0042] Alternatively, the condensation reaction may be carried out in the following buffer system under the following conditions: in a buffer system containing a water-soluble organic solvent (methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA, or DMSO) with a pH of 5.0 to 9.5 and a volume ratio of 0% to 35%, and containing phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, tris(tris-hydroxymethylaminomethane), benzoic acid, or triethanolamine, or a mixture thereof, at a concentration of 1 mM to 1000 mM, the reaction temperature may be 0°C to 45°C, and the reaction time may be 5 minutes to 96 hours.

[0043] Furthermore, the NH2 group of formula (III) participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

[0044] If X is an OH group, a condensation reagent is required for the above condensation reaction. Typically, the condensation reagent is selected from the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide, meso-p-toluenesulfonate (CMC or CME-CDI), carbonyldiimidazole (CDI), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoro Borate (TBTU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole hexafluorophosphate-1-yloxytripyrrolidinyl phosphate (PyBOP), diethyl pyrocarbonate (DEPC), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate, 2-( 7-Oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-[(dimethylamine)(morpholino)methylene]-1[1,2,3]triazolo[4,5-b]1-pyridine-3-oxohexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), chlorotripyrrolidinylphosphonium hexafluorophosphate (PyCloP), bis(teto Lamethylene)fluoroformamide (BTFFH), N,N,N',N'-tetramethyl-thio-(1-oxo-2-pyridyl)thiouronium hexafluorophosphate, 2-(2-pyridon-1-yl)-1,1,3,3-tetramethylureatetrafluoroborate (TPTU), sulf-(1-oxo-2-pyridyl)-N,N,N',N'-tetramethylthiourea hexafluorophosphate, O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-Tetramethylthiourea hexafluorophosphate fluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine-carbonium hexafluorophosphate (COMU), (benzenetriazol-1-yloxy)dipyrrolidinecarbohexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (or on a solid support), dipyrrolidinyl (N-succinimidyloxy) hexafluorophosphate carbonyl (HSPyU) ), 1-(chloro-1-pyrrolidinylmethylene)pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazole tetrafluoroborate (CIB), (benzotriazole-1-yloxy)dipiperidine carbohexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluratetrafluoroborate (TCTU), tris(dimethylamino)phosphine hexafluorophosphate (BrOP), 1-n-propyl phosphate anhydride (PPACA), T3P ( (Registered trademark)), 2-isocyanoethylmorpholine (MEI), N,N,N',N'-tetramethylurea-oxy-(N-succinimidyl)hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinetetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthioureatetrafluoroborate (TOTU), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl Lu-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)urea tetrafluoroborate (TDBTU), azodicarboxydipiperidine (ADD), bis(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butylazodicarboxylate (DBAD), diisopropylazodicarboxylate (DIAD), or diethylazodicarboxylate (DEAD).

[0045] In another specific embodiment, the method for preparing the above conjugate comprises one or more of the following steps: [ka]

[0046] In the formula, R 5’ The group is H, a C1-C6 alkyl group, a C1-C6 alkenyl group, or a C1-C6 linear or branched aminoalkyl group, and the other groups are as defined above.

[0047] A method for synthesizing the tubulosine B derivative of formula (III) under ideal conditions includes one or more of the following steps:

[0048] Step 1: Stir the aqueous solutions of diethoxyacetonitrile and ammonium sulfide at room temperature to obtain compound 1 (2,2-diethoxythioacetamide); [ka]

[0049] Step 2: Compound 1 and bromopyruvate are heated and condensed in an anhydrous solvent (anhydrous tetrahydrofuran, dichloromethane, acetonitrile, N,N-dimethylformamide, methanol, isopropanol, etc.) to obtain compound 2; [ka]

[0050] Step 3: Compound 3 is dissolved in a solvent (tetrahydrofuran, dichloromethane, ethyl acetate, n-heptane, dioxane, acetonitrile, etc.) and hydrolyzed in the presence of a Lewis acid or protic acid (hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, AlCl3, FeCl3, ZnCl2, BF3, BCl3, BBr3, TiCl4, ZnBr2, LiBF4, etc.) to obtain compound 3; [ka]

[0051] Step 4: Under low temperature conditions (-45°C to -78°C, etc.), the sulfinamide is dehydrogenated with n-butyllithium, and then compound 4 is obtained by condensing it with compound 3 in the presence of a Lewis acid; [ka] The Lewis acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, pyridine p-toluenesulfonate, AlCl3, FeCl3, ZnCl2, BF3, BCl3, BBr3, TiCl4, ZnBr2, and LiBF4;

[0052] Step 5: Under low temperature conditions (e.g., -45°C to -78°C), compound 4 is selectively reduced with a reducing agent (NaBH4, LiBH4, Na(OAc)3BH, Na(CN)BH3, etc.) to obtain compound 5. During the reaction, a Lewis acid (Ti(Oet)4, etc.) is added to control its stereochemistry; [ka]

[0053] Step 6: Compound 5 is dissolved in a solvent (methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, etc.), and the tert-butylsulfinyl group is removed with an acid such as hydrochloric acid, sulfuric acid, or phosphoric acid to obtain compound 6; [ka]

[0054] Step 7: Compound 6 and azidic acid are condensed in a solvent (n-heptane, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, etc.) in the presence of a condensation reagent (DIC / HOBt, DCC / HOBt, EDC / HOBt, HATU, BOP, T3P, BrOP, etc.) to obtain compound 7; or, azidic acid and isobutyl chloroformate are reacted in THF in the presence of an organic base (triethylamine, diisopropylethylamine, N-methylmorpholine, etc.) to obtain a mixture of anhydrides, which is then condensed with the hydrochloride salt of compound 6 to obtain compound 7; or, azidic acid and oxalyl chloride are reacted in a solvent (n-heptane, n-hexane, dichloromethane, tetrahydrofuran, etc.) in the presence of triethylamine and a catalytic amount of DMF to obtain an acid chloride, which is then condensed with the hydrochloride salt of compound 6 to obtain compound 7; [ka]

[0055] Step 8: In the presence of an organic base (imidazole, triethylamine, or pyridine, etc.), the hydroxyl group of compound 7 is reacted with a hydroxyl protecting reagent (TESCl, etc.) in a solvent (dichloromethane, tetrahydrofuran, or acetonitrile, etc.) to obtain compound 8; [ka]

[0056] Step 9: Add a base (such as KHMDS, LiHMDS, NaHMDS, KOtBu, NaH, KH, etc.) to Compound 8 in a solvent (such as tetrahydrofuran, dichloromethane, or acetonitrile, etc.) for deprotection, and then alkylate with methyl iodide, methyl bromide, dimethyl sulfate, methyl trifluoromethanesulfonate, or ethyl iodide, etc. to obtain Compound 9;

Chem.

[0057] Step 10: Dissolve Compound 9 in a solvent (such as tetrahydrofuran, dichloromethane, ethyl acetate, etc.), reduce the azide group to an amino group under specific conditions of hydrogen and palladium-carbon catalyst, triphenylphosphine, and water, etc. (Staudinger reaction), and then condense with an acid or an acid derivative having similar reactivity to obtain Compound 10;

Chem.

[0058] Step 11: Under appropriate conditions (for example, the TES protecting group can be deprotected with HCl, THF / MeOH / AcOH, n Bu4NF, or HF-pyridine in THF), deprotect the hydroxyl protecting group PG1 of Compound 10 to obtain Compound 11;

Chem.

[0059] Step 12; Convert the ester Compound 11 to the acid Compound 12 under the action of an alkali (such as LiOH, NaOH, KOH, etc.) or other appropriate conditions (for example, a methyl ester can be converted to a carboxylic acid with LiCl, LiI, Me3SiOK, etc.);

Chem.

[0060] Step 13: In the presence of a base (such as triethylamine, N,N - diisopropylethylamine, pyridine, etc.) and a catalyst (such as DMAP, etc.), under specific temperature conditions (such as 0°C to 23°C, etc.), react Compound 12 with an acid anhydride (such as acetic anhydride, propionic anhydride, isopropionic anhydride, and other acid anhydrides) or a carboxylic acid chloride (such as acetyl chloride, propionyl chloride, carbamoyl chloride, methylcarbamoyl chloride, ethylcarbamoyl chloride, dimethylcarbamoyl chloride, and other acid chlorides) to obtain Compound 13. Here, the reaction may also be carried out without a base or a catalyst;

Chemical formula

[0061] Step 14: In the presence of a condensation reagent (such as EDC, DIC, DCC, HATU, HBTU, etc.), condense Compound 13 with a suitable hydroxyl - containing compound such as pentafluorophenol or N - hydroxysuccinimide to obtain a reactive ester compound 14;

Chemical formula

[0062] Step 15: In an aqueous phase under specific pH conditions (such as pH = 5.0 - 8.0), or in an organic phase containing an organic base (such as TEA, DBU, or DIPEA, etc.) or an inorganic base (such as Na2CO3, Cs2CO3, K2CO3, or NaHCO3, etc.), condense Compound 15 and Compound 14 to obtain Compound 16. Here, optionally, it is not always necessary to use a base to promote the reaction, but it is necessary to appropriately control the reaction temperature (such as 0°C to 23°C, etc.) and the reaction time (such as 30 minutes to 18 hours, etc.);

Chemical formula

[0063] Step 16: Compound III is obtained by reducing the nitro group of compound 16 to an amino group under reducing conditions such as hydrogen and a palladium-carbon catalyst, hydrazine hydrate and FeCl3, iron powder and acetic acid. [ka]

[0064] In other specific embodiments, the method for preparing the conjugate includes one or more of the following steps: [ka]

[0065] The synthesis of the compound of formula (L') includes one or more of the following steps:

[0066] Step 1: Compound 1a is obtained by directly condensing compound 1-1 and compound 1-2 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.), or by reacting compound 1-2 with pentafluorophenol, nitrophenol, or N-hydroxysuccinimide in the presence of a condensing agent such as DIC or EDC, and then reacting it with compound 1-1; or by directly condensing compound 1-3 and compound 1-4 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.), or by condensing compound 1-3 and compound 1-4 by other indirect condensation reaction pathways; [ka]

[0067] Step 2: Remove the carboxyl protecting group PG2 from compound 1 using a deprotection reagent (e.g., an acid to remove the tert-butyl ester group) to obtain compound 2; [ka]

[0068] Step 3: Condense the carboxyl-containing compound 2 and the amino-containing compound 3 in the presence of a condensing agent (such as EDC, HATU, DIC, or DCC, etc.) or through other indirect condensation reaction pathways to obtain compound 4;

Chemical formula

[0069] Step 4: Remove the amino protecting group PG1 of compound 4 under deprotection conditions (for example, hydrogen and palladium-carbon catalyst for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound 5;

Chemical formula

[0070] Step 5: Condense the carboxylic acid 6 and the amine 5 in the presence of a condensing agent (such as EDC, HATU, DIC, or DCC, etc.) or through other indirect condensation reaction pathways to obtain compound 7;

Chemical formula

[0071] Step 6: Remove the carboxyl protecting group PG3 of compound 7 under deprotection conditions (for example, 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 8;

Chemical formula

[0072] Step 7: React compound 8 with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing agent (such as EDC, HATU, DIC, or DCC) to obtain a reactive ester compound L'; [ka]

[0073] 16. The conjugate according to any one of claims 9 to 13, wherein the synthesis of the compound of formula (L') comprises one or more of the following steps: [ka]

[0074] Under ideal conditions, the synthesis of the compound of formula (L') comprises one or more of the following steps:

[0075] Step 1: Remove the amino protecting group PG1 from compound 1 under deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for a Cbz protecting group on an amino group, or acidic conditions for a Boc protecting group) to obtain compound 2; [ka]

[0076] Step 2: Condensing amine compound 2 and carboxylic acid 3 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.) or via other indirect condensation reaction pathways to obtain compound 4; [ka]

[0077] Step 3: Remove the carboxyl protecting group PG2 of compound 4 under deprotection conditions (for example, 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 5; [ka]

[0078] Step 4: Condensing carboxylic acid 5 and amine 6 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.) or via other indirect condensation reaction pathways to obtain compound 7; [ka]

[0079] Step 5: Remove the carboxyl protecting group PG3 of compound 7 under deprotection conditions (for example, 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 8; [ka]

[0080] Step 6: Compound 8 is reacted with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensing agent (such as EDC, HATU, DIC, or DCC) to obtain a reactive ester compound 9, or compound 8 is reacted with another carboxylic acid activating compound. [ka]

[0081] In another specific embodiment, the compound of formula (II) is obtained by a condensation reaction between the compound of formula (IV) and the compound of formula (V) in the process of preparing the above conjugate: [ka]

[0082] Here, the definition of X and the condensation reaction conditions are as described above.

[0083] The NH2 group of formula (V) is preferably involved in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

[0084] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of the compound of formula (IV) includes one or more of the following steps: [ka]

[0085] Under ideal conditions, the synthesis of equation (IV) involves one of the following steps:

[0086] A reactive ester is obtained by reacting carboxylic acid compound 1 with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, DIC, DCC, HATU, or HBTU);

[0087] Alternatively, reactive mixed acid anhydrides are obtained by reacting carboxylic acid compound 1 with ethyl chloroformate, isobutyl chloroformate, etc., in the presence of an organic base (such as N-methylmorpholine, triethylamine, or diisopropylethylamine);

[0088] Alternatively, acid chlorides are obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount (e.g., 0.01 to 0.5 equivalents) of DMF.

[0089] In another specific embodiment, the method for preparing the above-mentioned conjugate is characterized in that the synthesis of formula (V) includes one or more of the following steps: [ka]

[0090] Under ideal conditions, the synthesis of equation (V) involves one or more of the following steps:

[0091] Step 1: Compound 1 and Compound 2 are condensed in an aqueous phase under specific pH conditions (e.g., pH = 5.0 to 8.0) or in an organic phase containing an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain Compound 3. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled. [ka]

[0092] Step 2: Remove the amino protecting group PG4 of compound 3 under appropriate deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound V; [ka]

[0093] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of compound 2 includes one or more of the following steps: [ka]

[0094] Here, compound 8 (compound XIVa) obtained in this synthesis step is target compound 2, and PG4 is the amino protecting group.

[0095] Under ideal conditions, the synthesis of compound 2 involves one or more of the following steps:

[0096] Step 1: At 0-60°C, benzyl chloride, benzyl bromide, or other benzyl compounds are added to an L-tyrosine ester derivative (1) in a suitable solvent (acetone, tetrahydrofuran, acetonitrile, dichloromethane, etc., or a mixed solvent of these solvents and water). Then, an organic or inorganic base such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, DBU, or sodium hydride is added. Optionally, a suitable additive, such as sodium iodide or a phase transfer catalyst (e.g., benzyltriethylammonium chloride (TEBA), tetrabutylammonium bromide (TBAB), tetrabutylammonium dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, etc.) is added to obtain compound 2;

[0097] Step 2: Compound 2 is dissolved in an organic solvent (dichloromethane, tetrahydrofuran, methanol, ethanol, diethyl ether, etc.), and then reacted with a reducing agent such as lithium aluminum hydride, DIBAL, sodium borohydride, lithium borohydride, sodium bis(2-methoxyethoxy)aluminum hydride (Red-Al), or diborane, optionally in the presence of an additive (I2, ferric chloride, zinc chloride, magnesium chloride, lithium chloride, or calcium chloride, etc.) to adjust the activity of the reducing agent, to obtain compound 3;

[0098] Step 3: Aldehyde 4 is obtained by oxidizing alcohol compound 3 under oxidative conditions such as Swarn oxidation (oxalyl chloride, DMSO, triethylamine), Parik-Dering oxidation (sulfur trioxide), or Dess-Martin oxidation;

[0099] Step 4: Compound 5 is obtained by extending the carbon chain by reacting aldehyde 4 with a phosphate ester (Horner-Wadsworth-Emmons reaction) or a phosphate ylide (Wittig reaction);

[0100] Step 5: In the presence of a homogeneous or heterogeneous catalyst, the double bond of compound 5 is reduced, thereby removing the benzyl group and obtaining a stereochemically pure compound or two diastereomers; the heterogeneous catalyst includes Pd / C, Pd(OH)2 / C, Pd / BaSO4, PtO2, Pt / Al2O3, Ru / C, Raney nickel, etc., and the homogeneous asymmetric hydrogenation catalyst includes Crabtree catalyst, [Ru(II)-(BINAP)] system catalyst, [(Ph3P)CuH]6, etc.;

[0101] Step 6; Dissolve compound 6 in an organic solvent (tetrahydrofuran, acetonitrile, dichloromethane, etc.), and nitrate it with a nitrating reagent containing nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, tert-butyl nitrite, nitric acid / trifluoroacetic anhydride, NO2BF4, or nitropyridinium salt, etc.;

[0102] Step 7: The nitro group of compound 7 is reduced to an amino group under conditions containing H2 / Pd / C, Fe or Zn / HOAc, or SnCl2 / HCl.

[0103] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of compound 2 comprises one or more of the following steps: [ka]

[0104] In the formula, compound 8 (compound XIVb) is target compound 2.

[0105] Under ideal conditions, the synthesis of compound 2 involves one or more of the following steps:

[0106] Step 1: At -78°C to -45°C, an aldol reaction is carried out between Evans asymmetric N-acyloxazolidinone or thion 2 and compound 1 to obtain stereochemically pure compound 3, where X=O or S;R 16 =H, methyl, phenyl; R 17 =H, methyl, isopropyl, phenyl, benzyl, etc.

[0107] Step 2: The hydroxyl group of compound 3 is deoxygenated under Burton-McCombee deoxygenation conditions, i.e., the alcohol is first converted to a thiocarbonyl derivative (alkyl xanthogenic acid, phenylcarbothioate, imidazolecarbothioate, etc.), and then treated with Bu3SnH to perform radical cleavage and obtain the dehydroxylated product. Here, the conditions for radical cleavage include n-Bu3SnH / AIBN, n-Bu3SnH / AIBN / n-BuOH / PMHS, and (Bu4N)2S2O8 / HCO2Na;

[0108] Step 3: Compound 4 is dissolved in tetrahydrofuran, and the Evans chiral auxiliary is cleaved under LiOH / H2O2 conditions to obtain the corresponding acid 5;

[0109] Step 4: Compound 5 is dissolved in an organic solvent (ethyl acetate, methanol, dichloromethane, ethanol, or acetic acid, etc.) and hydrogenated in the presence of a palladium-carbon catalyst. Here, the benzyl group is also removed to obtain compound 6;

[0110] Step 5; Dissolve compound 6 in an organic solvent (tetrahydrofuran, acetonitrile, dichloromethane, etc.), and nitrate it with a nitrating reagent containing nitric acid, nitric acid / acetic acid, potassium nitrate / sulfuric acid, tert-butyl nitrite, nitric acid / trifluoroacetic anhydride, NO2BF4, or nitropyridinium salt, etc.

[0111] Step 6: Under conditions containing H2 / Pd / C, Fe or Zn / HOAc, or SnCl2 / HCl, the nitro group of compound 7 is reduced to an amino group to obtain a stereochemically pure compound 8.

[0112] In another specific embodiment, the method for preparing the above conjugate is characterized in that the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (VI) and the compound of formula (VII): [ka]

[0113] In the formula, the definition of X and the conditions for the condensation reaction are as described in any one of claims 9 to 11.

[0114] In another specific embodiment, the synthesis of the compound of formula (VI) comprises one or more of the following steps: [ka]

[0115] Under ideal conditions, the synthesis of equation (VI) involves one or more of the following steps:

[0116] Step 1: Compound 1 is condensed with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent to obtain reactive acid derivative compound 2; [ka]

[0117] Step 2: Compounds 2 and 3 are condensed in an aqueous phase under specific pH conditions (e.g., pH = 5.0 to 8.0) or in an organic phase containing an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain compound 4. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled. [ka]

[0118] Step 3: Remove the amino protecting group PG4 of compound 4 under appropriate deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound 5; [ka]

[0119] Step 4: Compound 5 and the compound of formula (IV) are condensed in an aqueous phase under specific pH conditions (e.g., pH = 5.0 to 8.0), or in an organic phase containing an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain compound 6. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled. [ka]

[0120] Step 5: Remove the amino protecting group PG1 from compound 6 under appropriate deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound VI; [ka]

[0121] In another specific embodiment, the method for preparing the above conjugate is characterized in that the synthesis of the compound of formula (VII) comprises one or more of the following steps: [ka]

[0122] Under ideal conditions, the synthesis of the compound of formula (VII) involves one or more of the following steps:

[0123] Step 1: Reactive esters are obtained by reacting carboxylic acid 1 with a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, or DCC);

[0124] Alternatively, in the presence of an organic base (such as N-methylmorpholine, triethylamine, or diisopropylethylamine), carboxylic acid compound 1 is reacted with ethyl chloroformate, isobutyl chloroformate, etc. to obtain a reactive mixed anhydride;

[0125] Alternatively, acid chlorides are obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount of DMF (e.g., 0.01 to 0.5 equivalents); [ka]

[0126] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (VIII) and the compound of formula (IX): [ka]

[0127] In the formula, the definition of X and the conditions for the condensation reaction are as described above.

[0128] The NH2 group of formula (VIII) ideally participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

[0129] Under specific operating conditions, the synthesis of formula (VIII) includes one or more of the following steps: [ka]

[0130] Under ideal conditions, the synthesis of the compound of formula (VIII) involves one or more of the following steps:

[0131] Step 1: Remove the carboxyl protecting group PG3 from compound 1 under deprotection conditions (for example, 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 2; [ka]

[0132] Step 2: Reactive ester compound 3 is obtained by reacting compound 2 and a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent (such as EDC, HATU, DIC, or DCC); [ka]

[0133] Step 3: Compounds 3 and 4 are condensed in an aqueous phase under appropriate pH conditions (e.g., pH = 5.0 to 8.0) or in an organic phase containing an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain compound 5. While it is not strictly necessary to use a base to advance the reaction, the reaction temperature and reaction time must be appropriately controlled. [ka]

[0134] Step 4: Remove the amino protecting group PG3 from compound 5 under deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound 6; [ka]

[0135] Step 5: Compound 6 and the compound of formula (IV) described in claims 18-21 are condensed in an aqueous phase under appropriate pH conditions (e.g., pH = 5.0-8.0) or in an organic phase containing an organic base (e.g., TEA, DBU, DIPEA) or an inorganic base (e.g., Na2CO3, Cs2CO3, K2CO3, NaHCO3) to obtain compound 7. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled; [ka]

[0136] Step 6: Remove the amino protecting group PG1 from compound 7 under deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound VIII. [ka]

[0137] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of formula (IX) includes one or more of the following steps:

[0138] Reactive ester IX is obtained by condensing carboxylic acid compound 1 and a suitable hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) in the presence of a condensation reagent;

[0139] Alternatively, in the presence of an organic base (such as N-methylmorpholine, triethylamine, or diisopropylethylamine), carboxylic acid compound 1 is reacted with ethyl chloroformate, isobutyl chloroformate, etc. to obtain reactive mixed anhydride IX;

[0140] Alternatively, acid chloride IX is obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base such as triethylamine and a catalytic amount of DMF; [ka]

[0141] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the compound of formula (II) is obtained by a condensation reaction between the compound of formula (X) and the compound of formula (XI): [ka]

[0142] In the formula, Y 1 and Y 2 They condense to form a Y group; Y 1 and Y 2 These are NH2, - + NH3, COOH, COX, SO2Cl, P(O)Cl2, NHCOX, NHSO2Cl, NHP(O)Cl2, NHP(O)(OH)Cl, [ka]

[0143] In another specific embodiment, the method for preparing the above-described conjugate includes one or more of the following steps in the synthesis of the compound of formula (X): [ka]

[0144] Under ideal conditions, the synthesis of equation (X) involves one or more of the following steps:

[0145] Step 1: Compound 2 is obtained by condensing compound 1 containing a carboxyl group with compound VI in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.) or by other indirect condensation reaction pathways, where Z 1 This includes protected amino acids, protected carboxyls, amides, phosphoramides and sulfonamides, carboxylates, phosphates, phosphonates, etc. 1 It is a precursor of; [ka]

[0146] Step 2: Remove the amino protecting group PG1 from compound 2 under deprotection conditions (e.g., hydrogen and palladium-carbon catalysts for the Cbz protecting group on the amino group, or acidic conditions for the Boc protecting group) to obtain compound 3; [ka]

[0147] Step 3: Compound 5 is obtained by condensing carboxylic acid 4 and amine 3 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.) or by other indirect condensation reaction pathways; [ka]

[0148] Step 4: By appropriate chemical operations such as deprotection of the carboxyl group and amino group, the functional group Z of compound 5 is removed. 1 functional group Y 1 This is converted to obtain compound X. [ka]

[0149] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of the compound of formula (XI) comprises one or more of the following steps: [ka]

[0150] Under ideal conditions, the synthesis of equation (XI) involves one or more of the following steps:

[0151] Step 1: Compound 1 is dissolved in an organic solvent such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, or dimethyl sulfoxide, then dehydrogenated with a base such as sodium hydride, sodium hydroxide, or sodium hydroxide, and then reacted with compound 2 (where X is a halogen such as chlorine, bromine, or iodine, or another leaving group) while stirring at an appropriate temperature to obtain compound 3;

[0152] Step 2: Remove the carboxyl protecting group PG1 of compound 3 under deprotection conditions (for example, 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;

[0153] Step 3: Compound 1 is dissolved in an organic solvent (tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc.), then dehydrogenated with a base such as sodium hydride, sodium hydroxide, etc., and compound 5 is obtained by stirring with compound 4 at an appropriate temperature;

[0154] Step 4: Remove the carboxyl protecting group PG1 of compound 5 under deprotection conditions (for example, 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;

[0155] Step 5: Compound 6 is dissolved in an organic solvent (tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc.), and then reacted with methylsulfonyl chloride, 4-toluenesulfonyl chloride, etc., at 0-5°C in the presence of an organic base such as triethylamine, N,N-diisopropylethylamine, or pyridine to obtain compound 7;

[0156] Step 6: React compound 7 and ammonia solution in water or an organic solvent (methanol, ethanol, acetonitrile, tetrahydrofuran, or dioxane, etc.), and optionally heat to obtain compound XIb;

[0157] Step 7; Compound 7 and sodium azide are reacted in an organic solvent (such as tetrahydrofuran, dichloromethane, N,N-dimethylformamide, or dimethyl sulfoxide) to obtain compound 8;

[0158] Step 8; Compound XIb is obtained by hydrogenation in the presence of a palladium-carbon catalyst, or by reduction of azide compound 8 under the conditions of triphenylphosphine and water;

[0159] Step 9; Compound 9 is obtained by heating compound 7 and dibenzylamine in an organic solvent (tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, etc., preferably N,N-dimethylformamide) to 100°C;

[0160] Step 10: Compound 9 is dissolved in a solvent (ethyl acetate, methanol, ethanol, acetic acid, tetrahydrofuran, etc.), hydrogenated in a hydrogen atmosphere in the presence of a palladium-carbon catalyst, and optionally heated to 45°C to obtain compound XIb.

[0161] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (XII) and the compound of formula (XIII): [ka]

[0162] In the formula, the definition of X and the conditions for the condensation reaction are as described above.

[0163] Under ideal conditions, the NH2 group of formula (XII) participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

[0164] In another specific embodiment, the method for preparing the above conjugate is characterized in that the synthesis of the compound of formula (XII) comprises one or more of the following steps: [ka]

[0165] Under ideal conditions, the synthesis of the compound of formula (XII) involves one or more of the following steps:

[0166] Step 1: In the presence of a condensation reagent, compound 1 is reacted with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) to obtain a reactive carboxylic acid derivative compound 2; [ka]

[0167] Step 2: Compound 2 and compound 3 are condensed in an aqueous phase under specific pH conditions (e.g., pH = 5.0 to 8.0), or in an organic phase containing an organic base (TEA, DBU, DIPEA, etc.) or an inorganic base (Na2CO3, Cs2CO3, K2CO3, NaHCO3, etc.) to obtain compound 4. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled. [ka]

[0168] Step 3: The amino protecting group PG4 of compound 4 can be selectively removed under appropriate deprotection conditions (for example, the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium-carbon catalysts), and the Boc protecting group on the amino group can be cleaved under acidic conditions; [ka]

[0169] Step 4: Compound 5 is condensed with the compound of formula (IV) (i.e., formula (IV) as described in any one of claims 18 to 21) in an aqueous phase under specific pH conditions (e.g., pH = 5.0 to 8.0), or in an organic phase containing an organic base (TEA, DBU, DIPEA, etc.) or an inorganic base (Na2CO3, Cs2CO3, K2CO3, NaHCO3, etc.) to obtain compound 6. Here, it is not necessary to use a base to carry out the reaction, but the reaction temperature and reaction time must be appropriately controlled; [ka]

[0170] Step 5: The amino protecting group PG1 of compound 6 can be selectively removed under appropriate deprotection conditions (for example, the Cbz protecting group on the amino group can be cleaved under the action of hydrogen and palladium-carbon catalysts), and the Boc protecting group on the amino group can be cleaved under acidic conditions to obtain compound XII. [ka]

[0171] In another specific embodiment, the method for preparing the above-described conjugate is characterized in that the synthesis of the compound of formula (XIII) includes one or more of the following steps: [ka]

[0172] Under favorable conditions, the synthesis of formula (XIII) includes one or more of the following steps:

[0173] Step 1: Compound 3 is obtained by condensing carboxylic acid 1 and amine 2 in the presence of a condensing agent (EDC, HATU, DIC, or DCC, etc.) or by other indirect condensation reaction pathways; [ka]

[0174] Step 2: Under deprotection conditions such as an acid (formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, phosphoric acid, etc.), the carboxyl protecting group PG1 of compound 3 is removed by cleavage of the tert-butyl ester protecting group to obtain compound 4; [ka]

[0175] Step 3: In the presence of a condensing agent, carboxylic acid compound 4 is reacted with a hydroxyl-containing compound (such as pentafluorophenol or N-hydroxysuccinimide) to obtain a reactive ester compound of formula (XIII);

[0176] Alternatively, in the presence of an organic base (such as N-methylmorpholine, triethylamine, or diisopropylethylamine), carboxylic acid compound 4 is reacted with ethyl chloroformate, isobutyl chloroformate, etc. to obtain the reactive mixed acid anhydride of formula (XIII);

[0177] Alternatively, carboxylic acid compound 4 and oxalyl chloride are reacted in the presence of an organic base such as triethylamine and a catalytic amount of DMF to obtain the acid chloride of formula (XIII); [ka]

[0178] The preferred structure of the compound of formula (II) is as follows: [ka]

[0179] In another specific embodiment, the pharmaceutical composition comprises a conjugate formed by reacting a compound containing any of the above-described conjugates or the above-described conjugates with a cell-binding molecule, and a pharmacochemically acceptable excipient. Any of the above-described conjugates can be used in the preparation of pharmaceuticals for the treatment of cancer, infectious diseases, or autoimmune diseases. [Brief explanation of the drawing]

[0180] [Figure 1] The synthesis of tubulosine derivative fragments 13 and 18 is shown. [Figure 2] The synthesis of tubulicine derivative fragment 34 is shown. [Figure 3] The synthesis of tubulosine derivative fragments 37, 38, and 45 is shown. [Figure 4] The synthesis of tubulosine derivative fragment 57 is shown. [Figure 5] The synthesis of tubulosine derivative fragment 71 is shown. [Figure 6] The synthesis of 72 tubulosine derivatives that can be bound is shown. [Figure 7] The conjugate exhibits in vivo antitumor activity against BALB / c nude mice with NCI-N87 xenograft tumors. [Figure 8] Toxicity tests of the tubulicin derivative Her2 antibody conjugate are shown (compared to T-DM1). [Modes for carrying out the invention]

[0181] Explanation of related terms:

[0182] "Alkyl" refers to an aliphatic hydrocarbon group or monovalent group derived from an alkane by removing one or two hydrogen atoms from a carbon atom. It may be linear or branched, having C1-C8 (1-8 carbon atoms) in the chain. "Branched" refers to a linear alkyl group to which one or more lower C-number alkyl groups, such as methyl, ethyl, or propyl groups, are bonded. Specific examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, 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 isooctyl. C1-C8 alkyl groups may be unsubstituted or substituted with one or more substituents (but not limited to the following substituents). Examples of the substituents include -C1-C8alkyl, -O-(C1~C8alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where R' is independently selected from C1~C8alkyl and aryl.

[0183] A C3-C8 carbocycle refers to a saturated or unsaturated non-aromatic carboncyclic compound with 3, 4, 5, 6, 7, or 8 members. The C3-C8 carbocycle may be unsubstituted or substituted with one or more substituents. These substituents include, but are not limited to, -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where R' is independently selected from C1-C8 alkyl and aryl.

[0184] A "C3-C8 carbon ring group" is a group formed when a hydrogen atom in the above-mentioned C3-C8 carbon ring is replaced by a chemical bond.

[0185] "Alkenyl" refers to an aliphatic hydrocarbon group that has 2 to 8 carbon atoms in its chain and contains a carbon-carbon double bond, and may be linear or branched. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbuto-2-enyl, n-pentenyl, hexylenyl, heptenyl, and octenyl.

[0186] "Alkynyl" refers to an aliphatic hydrocarbon group that has 2 to 8 carbon atoms in its chain and contains a carbon-carbon triple bond, and may be linear or branched. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n-pentynyl, hexylinyl, heptynyl, and octynyl.

[0187] A "heteroalkyl" refers to a C2-C8 alkyl group in which 1 to 4 carbon atoms are independently substituted with heteroatoms selected from the group consisting of O, S, and N.

[0188] The term "aryl" or "Ar" refers to an aromatic or heteroaromatic group consisting of one or more rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. The term "heteroaromatic group" refers to an aromatic group in which some carbon atoms, preferably 1, 2, 3, or 4 carbon atoms, are replaced with O, N, Si, Se, P, or S, preferably O, S, and N. The term aryl or Ar also refers to a group in which one or more H atoms are independently R 13 F, Cl, Br, I, OR 13 , SR 13 , NR 13 R 14 N=NR 13 N=R 13 , NR 13 R 14 NO2, SOR 13 R 14 SO2R 13 SO3OR 13 OSO3OR 13 PR 13 R 14 , POR 13 R 14 PO2R 13 Ure 14 OPO3R 13 R 14 , or PO3R 13 R 14 This also refers to those replaced by the aforementioned R 13 , R 14 These are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt.

[0189] "Halogen" refers to fluorine, chlorine, bromine, or iodine atoms, with fluorine and chlorine atoms being preferred.

[0190] A "heterocycle" is an aromatic or non-aromatic ring system containing 2 to 8 carbon atoms, in which 1 to 4 ring carbon atoms are independently substituted with heteroatoms from the group O, N, S, Se, B, Si, and P. Preferred heteroatoms are O, N, and S. Heterocycles are described in The Handbook of Chemistry and Physics, 78th edition, CRC Press, Inc., 1997-1998, pp. 225-226, the disclosure of which is incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to, epoxy, azilidinyl, tyranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxylanyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolidinyl, pyrazolinyl, thiazolidinyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, and condensation systems resulting from condensation with phenyl groups.

[0191] The term "heteroaryl" or aromatic heterocyclic refers to an aromatic hetero, monocyclic, bicyclic, or polycyclic ring with 3 to 14 members, preferably 5 to 10 members. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanil, benzofuranil, 1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and condensation systems resulting from condensation with a phenyl group.

[0192] The terms "alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," and "heterocyclic" also refer to the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," and "heterocyclene" groups, which are formed by the removal of two hydrogen atoms.

[0193] "Arylalkyl" refers to carbon atoms, typically terminal or sp 3 This refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, and 2-naphthophenylethane-1-yl.

[0194] "Heteroarylalkyl" refers to carbon atoms, typically terminal or sp 3 This refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group. Typical heteroarylalkyl groups include 2-benzimidazolylmethyl and 2-furylethyl.

[0195] Examples of "hydroxy protecting groups" include methoxymethyl ether (MOM), 2-methoxyethoxymethyl ether (2-MOEOM), tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate esters, substituted acetate esters, pivaloates, adamantanoates, mesitoates, methanesulfonates, tosylates, and p-toluenesulfonates.

[0196] "Amino acids" may be natural and / or non-natural amino acids, preferably α-amino acids. Natural amino acids are those encoded by the genetic code and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. Non-natural amino acids are derivatives of protein-forming amino acids. Examples include hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (neurotransmitter), ornithine, citrulline, β-alanine (3-aminopropanoic acid), γ-carboxyglutamate, selenocysteine ​​(present in most eukaryotes but not directly encoded by DNA), pyrrolidine (found in some archaea and only one bacterium), N-formylmethionine (often the first amino acid in bacterial, mitochondrial, and chloroplast proteins), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoserine. The term amino acid also includes amino acid analogs and mimics. Analogues are compounds that have the same common H2N(R)CHCO2H structure as natural amino acids, except that the R group is not found in natural amino acids. Examples of analogues include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Preferably, amino acid mimetic compounds are compounds that have a different structure from the general chemical structure of α-amino acids but function similarly. The term "non-natural amino acid" is intended to represent the stereochemical form "D," while natural amino acids are in the "L" form. When 1 to 12 amino acids are used in this application, the amino acid sequence is preferably a protease cleavage recognition sequence.Many cleavage recognition sequences are known in the art; see, for example, 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); their disclosures are incorporated herein by reference. In particular, the sequence is selected from the group consisting of Val-Cit, Ala-Val, Ala-Ala, Val-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.

[0197] A "peptide" is formed by two or more amino acids connected by a peptide bond (i.e., an amide bond) between an amino group and the carboxyl group of another amino acid. Compounds in which two amino acids are linked by peptide bonds are called dipeptides, and compounds in which three amino acids are linked by peptide bonds are called tripeptides. Peptides composed only of naturally occurring α-amino acids are natural peptides (natural proteins), while peptides containing one or more unnatural amino acids or amino acid analogs are unnatural peptides (peptoid compounds). A peptide consisting of two or more amino acids is called a peptide unit.

[0198] A "glycoside" is a molecule in which a sugar group is linked to another group via a glycosidic bond through its anomeric carbon. Glycosides can be linked by O-(O-glycoside), N-(glycosylamine), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds. The core empirical formula is C m(H2O) n (where m is different from n, and both m and n are < 36), and here glycosides include glucose (dextrose), fructose (levrose), allose, altrose, mannose, gross, iodose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol, sucrose, lactose, maltose, trehalose, maltodextrin, raffinose, glucuronic acid (glucuronide), and stachyose. It may be a D-type or L-type, a pentatomic cyclic furanose type, a hexatomic cyclic pyranose type, or an acyclic type, an α-isomer (anomer carbon -OH below the plane of carbon atoms in the Haworth projection), or a β-isomer (anomer carbon -OH above the plane of carbon atoms in the Haworth projection). It is used herein as a monosaccharide, disaccharide, polyol, or oligosaccharide containing 3 to 6 sugar units.

[0199] The term "antibody" as used here is used in its broadest sense, covering complete monoclonal antibodies, polyclonal antibodies, specific antibodies, and multispecific antibodies (e.g., bispecific antibodies and antibody fragments), with the required number of drug-binding sites linked to the biological activity of an ideal antibody fragment. The native form of an antibody is a tetramer consisting of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the variable regions (VL and VH) of the light and heavy chains jointly participate in binding to the antigen. The light and heavy chain variable regions are interrupted by a framework region and three hypervariable regions. These are also called "complementarity-determining regions" or "services." Certain regions may be recognized as interacting with the immune system. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), major classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. Antibodies can be obtained from any suitable species. In some aspects, the antibodies are derived from humans or mice. The antibodies may be human, humanized antibodies, or chimeric antibodies.

[0200] The term "specific binding" means that an antibody or antibody derivative binds to a corresponding target antigen with high selectivity, rather than to many other antigens. Generally, an antibody or antibody derivative binds to at least about 1 × 10⁻¹⁶ antigens. -7 M, preferably 1 × 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It has a binding affinity of M. The binding affinity of a given antigen is at least twice as great as the binding affinity of a nonspecific antigen (such as bovine serum albumin or casein).

[0201] "Pharmacologically" or "pharmaceutically acceptable" means that the corresponding compound or compound composition is not harmful, allergic, or otherwise harmful when administered to animals or humans in an appropriate manner.

[0202] Pharmaceutically acceptable auxiliary materials include all carriers, diluents, adjuvants, or molding agents, such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersing media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption retarders. In the pharmaceutical field, adding these auxiliary materials to active drug components is a common practice. It can be said that adding auxiliary materials to drug components is appropriate unless the auxiliary material is incompatible with the drug-active component. Active auxiliary materials may be added to drug components to obtain favorable results.

[0203] In the present invention, "medicinal salt" refers to salt derivatives of the compound of the present invention. By appropriate modification, the compound of the present invention can be formed into a corresponding acid salt or alkali salt. Medicinal salts include commonly used non-toxic salts or quaternary ammonium compounds, which can be prepared with the compound of the present invention and a corresponding non-toxic inorganic or organic acid. For example, inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, while organic acids include acetic acid, propioic 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, and these acids can be used in pharmaceutically acceptable salts. Other salts include ammonium salts such as trometamol, meglumine, and pyrroleethanol, and metal salts such as sodium, potassium, calcium, zinc, and magnesium.

[0204] In the present invention, pharmaceutical salts can be prepared from parent compounds containing acidic or basic residues by conventional chemical methods. Generally, these salts can be obtained by reacting the free acidic or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Preferred non-aqueous reaction solvents are generally ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts is given in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, 1985, page 1418, which is incorporated by reference.

[0205] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a ligand-drug conjugate or conjugate-drug conjugate. A conjugate may contain at least one amino group and thus may form an acid addition salt with the amino group, such as nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamine salt, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis-(2-hydroxynaphthoate)). A pharmaceutically acceptable salt may contain additional molecules such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts may have multiple charged atoms in their structure. Embodiments in which multiple charged atoms are part of the pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0206] A "pharmaceutically acceptable solvate" or "solvate" refers to the association of one or more solvent molecules with a disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, butanol, tert-butanol, methanol, acetone, glycerol, DMSO, ethyl acetate, formic acid, acetic acid, triethanolamine, and ethanolamine.

[0207] A hydrate refers to a compound that contains water. Water can bond to other parts of a compound via coordinate bonds to form complexes such as metal ion hydration ligands, or to other parts of a compound via covalent bonds to form hydrated chloroacetaldehyde, etc. It can also refer to crystals or liquid molecules formed by water in a specific compound under specific temperature and pressure conditions. A certain amount of water is present in hydrates; for example, the composition of the hydrate of anhydrous sodium sulfate (Na2SO4) is Na2SO4·10H2O. There are several ways in which water can bond in hydrates. One is through ligands, which coordinate to metal ions and are known as coordinate water. Another is as anionic water, which bonds to anions. Although water cannot directly bond with cations or anions, it can be present in a specific proportion in crystals and occupy specific positions in the crystal lattice. This combined form of water is called lattice water and generally contains 12 water molecules. Some crystalline compounds also contain water, but not in a constant proportion. Hydrate salts refer to pharmaceutically acceptable salts formed based on hydrates.

[0208] Optical isomers, also called enantiomers, opposite-legged isomers, optical isomers, enantiomers, or chiral isomers, cannot perfectly overlap with the stereoisomers of each other. When a substance contains chiral carbon atoms, there are two optical isomers, which are also called enantiomers because they are physically and mirror images of each other. Enantiomers have the same optical rotation but rotate in opposite directions, and are very likely to have similar physical and chemical properties. A molecule containing two carbon atoms with the same properties will have three optical isomers. If a molecule contains several different chiral atoms, the number of its optical isomers will be 2 n Here, n is the number of different chiral atoms. When two substances that are equal amounts of optical isomers are uniformly mixed, their optical rotations cancel each other out, forming a racemate.

[0209] Examples of “patient” or “subject” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In exemplary embodiments, the patient or subject is human.

[0210] "Administration" refers to any manner in which a pharmaceutical or other drug is transferred, delivered, introduced, or transported. Such manners include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, lesional, nasal, subcutaneous, or intracavitary administration. Furthermore, the present invention aims to utilize devices or equipment for administering drugs. Such devices may utilize active or passive transport and may be slow-release or fast-release delivery devices.

[0211] The following abbreviations may be used herein, having the definitions set forth below: Boc, tert-butoxycarbonyl; BroP, bromotrispirolidinophosphonium hexafluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylic acid; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethylphosphoroanidiate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothrey Thor; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU, O-(7-azabenzotriazole-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.

[0212] Detailed description of the embodiment

[0213] Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While the drawings and the following embodiments illustrate specific examples of the present invention, it should be understood that the present invention can be carried out in various forms and should not be limited to the embodiments described herein. Rather, these embodiments are provided to enable a more complete understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0214] The disclosed subject matter can be embodied in many different forms and should not be construed as being limited to the embodiments described herein. Indeed, a person skilled in the art who is familiar with the subject matter of this disclosure will likely envision many modifications and other embodiments of the subject matter of this disclosure that will benefit from the teachings given in the description contained herein. Therefore, it should be understood that the disclosed subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to fall within the scope of the disclosed subject matter.

[0215] Certain terms are used herein, but these are used only in a general and descriptive sense and not for any limited purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom the subject matter described herein belongs.

[0216] Standard nomenclature is used to describe the compounds. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs.

[0217] The terms "one" or "one" ("a" or "one") do not imply a limitation of quantity, but rather that there is at least one of the referenced items. Enumeration of numerical ranges is intended solely as a simplified way of referring individually to the individual values ​​within the ranges, unless otherwise stated herein, and the individual values ​​are incorporated into the description as if they were individually cited herein. All range endpoints are included within the ranges and can be combined individually. All methods described herein may be performed in any order unless otherwise indicated herein or unless it is clearly inconsistent with the context. Unless otherwise specified, the use of example or illustrative language ("for example," etc.) is intended solely to better illustrate the invention and does not limit its scope.

[0218] The present invention includes compounds of formula (I) having at least one desired isotopic atom substitution in greater quantities than the natural abundance of the isotope (i.e., enriched), and the use of such compounds. Isotopes are atoms with the same atomic number but different mass numbers; that is, they have the same number of protons but different numbers of neutrons. Isotopic substitutions, such as deuterium substitution, may be partial or complete. Partial deuterium substitution means that at least one hydrogen atom is replaced by deuterium. In certain embodiments, the isotope is enriched to 90%, 95%, or 99% or more at any desired location. In one embodiment, deuterium is enriched to 90%, 95%, or 99% at a desired location.

[0219] It should be noted that the specification and claims use specific words to refer to specific components. Those skilled in the art should understand that different terminology may be used to refer to the same component. The specification and claims do not use differences in terminology as a way to distinguish components, but use differences in the function of the components as the criterion for distinction. Where “includes” or “contains” is an open term as referred to in the specification and claims, it should be interpreted as “includes, but not limited to.” The following description in the specification is a preferred embodiment for carrying out the invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the invention shall be as defined by the appended claims.

[0220] The term "C1-C6" refers to a group containing one to six carbon atoms.

[0221] The term "connection with hydrophilic branching" refers to a structure where the main skeleton is C2-C 100 This means that the peptide unit (1 to 12 natural or unnatural amino acids), hydrazone group, disulfide group, ester group, oxime group, amide group, or thioether group is present.

[0222] The term "pharmaceutically acceptable salt" means a salt of a compound suitable for use in pharmaceutical formulations. When a compound has one or more basic groups, the salt may be an acid addition salt such as sulfate, hydrobromide, tartrate, methanesulfonate, maleate, citrate, phosphate, acetate, pamoate, alginate, hydroiodide, nitrate, hydrochloride, lactate, methyl sulfate, fumarate, benzoate, succinate, methanesulfonate, lactobionate, sverate, or tosylate. When a compound has one or more acidic groups, the salt may be a salt such as calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, or tetramethylammonium salt. Polymorphic crystalline forms and solvates are also included in the scope of the present invention.

[0223] In the present invention, pharmaceutical salts can be prepared by conventional chemical methods. Generally, these salts can be obtained by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Preferred non-aqueous reaction solvents are generally ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts is given in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, 1985, page 1418, which is incorporated by reference.

[0224] Pharmaceutically acceptable auxiliary materials include all carriers, diluents, adjuvants, or molding agents, such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersing media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption retarders. In the pharmaceutical field, adding these auxiliary materials to active drug components is a common practice. It can be said that adding auxiliary materials to drug components is appropriate unless the auxiliary material is incompatible with the drug-active component. Active auxiliary materials may be added to drug components to obtain favorable results.

[0225] In this application, the formula (I) [ka] This represents a chiral carbon atom moiety and can be selected as pure R, pure S, or a mixture with different R / S ratios.

[0226] Unless a specific stereoisomer is explicitly indicated (for example, by thick or dashed lines indicating a bond at the center of the relevant stereochemistry in the structural formula, by indicating that the double bond in the structural formula has an E or Z configuration, or by stereochemically assigned nomenclature), all stereoisomers are included in the scope of the present invention as pure compounds and mixtures thereof. Unless otherwise specified, individual enantiomers, diastereomers, geometric isomers, and combinations and mixtures thereof are all included in the present invention.

[0227] Those skilled in the art will understand that compounds may have tautomers (e.g., keto and enol forms), resonance forms, and zwitterionic forms equivalent to those shown in the structural formulas used herein, and that the structural formulas include these tautomers, resonance forms, and zwitterionic forms.

[0228] Another aspect of the present invention relates to the production and preparation of antibodies. The manufacturing process includes in vivo, in vitro, or a combination thereof. Methods for preparing anti-receptor peptide polyclonal antibodies are well known, for example, as described in U.S. Patent No. 4,493,795 (Nestor et al.). A typical method for preparing monoclonal antibodies involves fusing mouse spleen cells isolated from a specific antigen-immunized mouse 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), and the contents of that document are incorporated herein by reference as forming part of this specification. In particular, monoclonal antibodies can be obtained by immunizing mice, rats, hamsters, or other mammals with the target antigen. Examples of target antigens include intact target cells, antigens isolated from target cells, whole viruses, weakened whole viruses, and viral proteins. Splenocytes and myeloma cells are fused using PEG6000. The hybridomas obtained after fusion are screened using their sensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomas that produce monoclonal antibodies useful for carrying out the present invention are identified by inducing an immune response with or suppression of receptor activity of specific target cell receptors.

[0229] The monoclonal antibodies used in the present invention can be obtained by initiating the culture of monoclonal hybridoma cells in a nutrient medium containing hybridoma cells that secrete antibodies with appropriate antigen specificity. In this culture, it is necessary to maintain sufficient time and conditions for the hybridoma cells to secrete antibodies into the culture medium. After collecting the supernatant of the antibody-containing medium, the antibodies can be isolated by well-known techniques, such as protein A affinity chromatography; anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, and molecular sieve chromatography (particularly affinity chromatography and molecular sieve chromatography using antigen-crosslinked protein A); centrifugation, precipitation, or standard methods for purifying other proteins.

[0230] Culture media useful for preparing these compositions are well known in the art and commercially available, and include synthetic media. An example of a synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) to which 4.5 gm / L glucose, 20 mM glutamine, 20% fetal bovine serum, and an antifoaming agent (e.g., polyoxyethylene-polyoxypropylene block copolymer) are added.

[0231] Furthermore, in addition to cell fusion technology, cell lines for antibody production can also be constructed by the following methods. For example, direct transformation of B lymphocytes with oncogenic DNA, or transfection with oncogenic viruses, such as Epstein-Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi's sarcoma-associated virus (KSHV) (see U.S. Patent Nos. 4341761;4399121;4427783;4444887;4451570;4466917;4472500;4491632;4493890 for details). Monoclonal antibodies can be prepared using antireceptor peptides or peptides containing terminal carboxyl groups based on known methods (see 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) for details). Typically, antireceptor polypeptides or polypeptide analogs can be used alone or conjugated to a crosslinked immunogenic carrier as immunogens for preparing antireceptor polypeptides for monoclonal antibodies.

[0232] There are many other well-known methods for producing monoclonal antibodies as binding molecules in the present invention. Among these, the method for producing fully human antibodies has attracted particular attention. Phage display technology allows for the acquisition of fully human antibodies that specifically bind to known antigens from a fully human antibody library through affinity selection. The literature contains detailed descriptions of phage display technology itself, vector construction, and library screening. For more information, see 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).

[0233] Monoclonal antibodies obtained from non-human species (e.g., mice) using hybridoma technology can be humanized to avoid human anti-mouse antibodies when administered to humans. Among the well-known methods for antibody humanization are the transplantation and remodeling of complementarity-determining regions. For details, see U.S. Patents 5,859,205 and 6,797,492; 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). The disclosures in the aforementioned literature are incorporated as references. Fully human antibodies can be prepared by antigen immunization against transgenic mice, rabbits, monkeys, and other mammals that possess most of the light and heavy chains of human immunoglobulins. Examples of such mice include Xenomouse (Abgenix, Inc.), HuMab-Mouse (Medarex / BMS), and VelociMouse (Regeneron). For details, see U.S. Patents 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181, 5,922,545, 5,661,016, 5,545,806, 5,436,149, and 5,569,825. In the course of human treatment, the immunogenicity produced in the human body by chimeric antibodies constructed by integrating mouse antibody variable region genes and human antibody constant region genes is far lower than that of mouse antibodies (Kipriyanov et al., Mol Biotechnol. 26:39-60 (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002)). The disclosures in the aforementioned literature are incorporated as references. Furthermore, antibody affinity and specificity can be improved by inducing specific mutagenesis in the antibody variable region (Brannigan et al., Nat Rev Mol Cell Biol. 3:964-70 (2002); Adams et al., J. Immunol Methods. 231:249-60 (1999)).By partially replacing the constant region of an antibody, its affinity for immunoeffector cells can be effectively enhanced, thereby increasing its cytotoxic effect.

[0234] Immunospecific antibodies against malignant cell antigens can be obtained through commercial channels or several commonly used technical methods, such as chemical synthesis or recombinant expression techniques. Similarly, nucleotide sequences encoding immunospecific antibodies against malignant cell antigens can be obtained through commercial channels such as the GenBank database or other similar databases, publicly available literature, or routine cloning and sequencing.

[0235] Besides antibodies, polypeptides or proteins similarly interact with corresponding receptors or epitopes on the target cell surface by binding, blocking, attacking, or other means, acting as binding molecules. These peptides or proteins do not need to belong to the immunoglobulin family, as long as they can specifically bind to the epitope or its corresponding receptor. These polypeptides are also isolated using techniques similar to those for phage display antibodies (Szardenings, J Recept Signal Transduct Res. 2003;23(4):307-49). Peptide fragments obtained from random peptide libraries have applications similar to those of antibodies and antibody fragments. Polypeptide or protein molecules maintain their antigen-binding specificity by linking to several macromolecules or media via binding molecules. These macromolecules include, but are not limited to, albumin, polymers, liposomes, nanoparticles, or dendrimers.

[0236] Antibodies used in conjugation to treat cancer, autoimmune diseases, and infectious diseases include, but are not limited to, the following: 3F8 (anti-GD2 antibody), avagovomab (anti-CA-125 antibody), absiximab (anti-CD41 antibody (integrin α-IIb)), adalimumab (anti-TNF-α antibody), adalimumab (anti-EpCAM antibody, CD326), aferimomab (anti-TNF-α); aftuzumab (anti-CD20 antibody), and alacizumab pegol. pegol (anti-VEGFR2 antibody), ALD518 (anti-IL-6 antibody), alemtuzumab (also known as Campus, MabCampus, anti-CD52 antibody), artumomab (anti-CEA antibody), anatumomab (anti-tag-72 antibody), anlukinzumab (IMA-638, anti-IL-13 antibody), apolizumab (anti-HLA-DR antibody), alsitumomab (anti-CEA antibody), aselizumab (anti-L - Selectin (CD62L) antibody), Atlizumab (also known as Tocilizumab, Actemra, RoActemra, anti-IL-6 receptor antibody), Atorolimumab (anti-rhesus monkey factor antibody), Bapineozumab (anti-β-amyloid antibody), Basiliximab (Symlect, anti-CD25 (IL-2 receptor α chain) antibody), Bavituximab (Bavitu ximab (anti-phosphatidylserine antibody), bectumomab (also known as LymphoScan, anti-CD22 antibody), belimumab (also known as BENLYSTA, LymphoStat-B, anti-BAFF antibody), benralizumab (anti-CD125 antibody), vertilimumab (anti-CCL11 (eotaxin-1) antibody), becylesomab (also known as Scintimun, anti-CEA-related antigen antibody), bevacizumab (also known as Avastin, anti-VEGF antibody), bisilomab (also known as FibriScint, anti-fibrin IIβ chain antibody), vivacuzumab (anti-CD44v6 antibody), blinatumomab (also known as BiTE, anti-CD19 antibody), brentuximab (cAC10, anti-CD30 TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), Canakinumab (also known as Ilaris, anti-IL-1 antibody), Cantuzumab (also known as C242,Anti-CanAg antibody), Capromab, Katumakisomab (also known as removeb, anti-EpCAM, anti-CD3 antibody), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD4 antibody), Certolizumab pegol (also known as CIMZIA, anti-TNF-α antibody), Cetuximab (also known as Elbitux, IMC-C225, anti-EGFR antibody), Sitatuzumab (anti-EpCAM antibody), Cixutumumab (anti-IGF-1 antibody), Clenoliximab (anti-CD4 antibody), Cliva Tuzumab (anti-MUC1 antibody), Conatumumab (anti-TRAIL-R2 antibody), CR6261 (anti-influenza A hemagglutinin antibody), Dacetuzumab (anti-CD40 antibody), Daclizumab (also known as Zenapax, anti-CD25C (IL-2 receptor α chain) antibody), Daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase) antibody), Denosumab (also known as Prolia, anti-RANKL antibody), Detumomab (anti-B-lymphoma cell antibody), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside antibody), Eculizumab (also known as Soliris, anti-C5 antibody), Edbacomab (anti-endotoxin antibody), Edrecolomab (also known as Panorex, MAb17-A1, anti-EpCAM antibody), Efarizumab (also known as Raptiva, anti-LFA-1 (CD11a) antibody), Efungumab (also known as Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody), Elsilimomab (anti-IL-6 antibody), enlimomab pegol (anti-ICAM-1 (CD54) antibody), epitumomab (anti-epiciarin antibody), epratuzumab (anti-CD22 antibody), erlizumab (anti-ITGB2 (CD18) antibody), ertumaxomab (also known as Rexomun, anti-HER2 / neu, CD3 antibody), etalacizumab (also known as Abegrin, anti-integrin αvβ3), exibivirumab (anti-hepatitis B surface antigen antibody (HBs antibody)),Fanolesomab (also known as NeutroSpec, anti-CD15 antibody), faralimomab (anti-interferon receptor antibody), farletuzumab (anti-folate receptor 1 antibody), felvizumab (antibody against RSV), fezakinumab (anti-IL-22 antibody), figitumumab (anti-IGF-1 receptor antibody), fontolizumab (anti-IFN-γ antibody) ), Foravirumab (anti-rabies virus 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 (separate) Name: CR011 (anti-GPNMB antibody), Golimumab (also known as Simponi, anti-TNF-α antibody), Gomiliximab (anti-CD23C (IgE receptor) antibody), Ibalizumab (anti-CD4 antibody), Ibritumomab (anti-CD20 antibody), Igovomab (also known as Indimacis-125, anti-CA-125 antibody), Imciromab (also known as Myoscint, anti-cardiac myosin antibody), Infliximab (also known as Remicad e, anti-TNF-α antibody), intetumumab (anti-CD51 antibody), inolimomab (anti-CD25 (IL-2 receptor α chain) antibody), inotuzumab (anti-CD22 antibody), ipilimumab (anti-CD152 antibody), iratumumab (anti-CD30 (TNFRSF8) antibody), keriximab (anti-CD4 antibody), rabetsuzumab (also known as CEA-Cide, anti-CEA antibody), lebrikizumab (anti-IL-13 antibody),Lemalesomab (anti-NCA-90 (granulocyte antigen) antibody), Lerdelimumab (anti-TGFβ-2 antibody), Lexatumumab (anti-TRAIL-R2 antibody), Libivirumab (anti-hepatitis B surface antigen antibody), Lintuzumab (anti-CD33 antibody), Lucatumumab (anti-CD40 antibody), Lumiliximab (anti-CD23 (IgE receptor) antibody), Mapatumumab (anti-TRAIL-R1 antibody), Maslimomab (anti-T cell receptor antibody), Matsuzumab Matuzumab (anti-EGFR antibody), mepolizumab (also known as Bosatria, anti-IL-5 antibody), metelimumab (anti-TGFβ-1 antibody), milatuzumab (anti-CD74 antibody), minretumomab (anti-TAG-72 antibody), mitumomab (also known as BEC-2, anti-ganglioside antibody-GD3), morolimumab (anti-rhesus monkey factor antibody), motavizumab (also known as Numax, anti-RSV antibody), muromonab-CD3 (also known as Orthoclone OKT3 (anti-CD3 antibody), Nacolomab (anti-C242 antibody), Naptumomab (anti-5T4 antibody), Natalizumab (also known as Tysabri, anti-integrin α4 antibody), Nebacumab (anti-endotoxin antibody), Necitumumab (anti-EGFR antibody), Nerelimomab (anti-TNF-α antibody), Nimotuzumab (also known as Thera cim, Theraloc, anti-EGFR antibody), nofetumomab, ocrelizumab (anti-CD20 antibody), odulimomab (also known as Afolimomab, anti-LFA-1 (CD11a) antibody), ofatumumab (also known as Arzerra, anti-CD20 antibody), olalatumab (anti-PDGF-Rα antibody), omalizumab (also known as Xolair, anti-IgEFc region antibody),Ozumab (anti-EpCAM antibody), Oregovomab (also known as OvaRex, anti-CA-125 antibody), Otelixizumab (anti-CD3 antibody), Pagibaximab (anti-LTA antibody), Palivizumab (also known as Synagis, Abbosynagis, anti-RSV antibody), Panimumab (also known as Vectibix, ABX-EGF, anti-EGFR antibody), Panobacumab (anti-Pseudomonas aeruginosa antibody), Pascolizumab mab) (anti-IL-4 antibody), pemtumomab (also known as Theragyn, anti-MUC1 antibody), pertuzumab (also known as Omnitarg, 2C4, anti-HER2 / neu antibody), pexelizumab (anti-C5 antibody), pintumomab (anti-adenocarcinoma antigen antibody), priliximab (anti-CD4 antibody), pritumumab (anti-vimentin antibody), PRO140 (anti-CCR5 antibody), racotumomab (also known as 1E10, anti (N-glycolylneuraminic acid (NeuGc,NGNA)-ganglioside (GM3) antibody), rafivirumab (anti-rabies virus glycoprotein antibody), ramucirumab (anti-VEGFR2 antibody), ranibizumab (also known as Lucentis, anti-VEGF-A antibody), laxibacumab (anti-anthrax toxin, protective antigen antibody), regavirumab (anti-CMV glycoprotein B antibody), reslizumab (anti-IL-5 antibody), rilotumumab (rilotumumab) (anti-HGF antibody), Rituximab (also known as MabThera, Rituxanmab, anti-CD20 antibody), Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-IFN-α antibody) ), Rovelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), Ruplizumab (also known as Antova, anti-CD154 (CD40L) antibody), Satumomab (anti-TAG-72 antibody),Sevilumab (anti-CMV antibody), cibrotuzumab (anti-FAP antibody), sifalimumab (anti-IFN-α antibody), siltuximab (anti-IL-6 antibody), ciprizumab (anti-CD2 antibody), (SMART) MI95 (anti-CD33 antibody), solanezumab (anti-β-amyloid antibody), so, Nepcizumab (anti-sphingosine-1-phosphate antibody), Sontuzumab (anti-epiciarin antibody), Stamulumab (anti-myostatin antibody), Sulesomab (also known as LeukoScan, (anti-NCA-90 (granulocyte antigen) antibody)), Tacatuzumab (anti-alpha-fetoprotein antibody), Tadocizumab (anti-integrin αIIbβ3 antibody), talizumab (anti-IgE antibody), tanezumab (anti-NGF antibody), taplitumomab (anti-CD19 antibody), tefibazumab (also known as Aurexis, anti-clumping factor A antibody), telimomab, tenatumomab mab) (anti-tenascin C antibody), teneliximab (anti-CD40 antibody), teplizumab (anti-CD3 antibody), TGN1412 (anti-CD28 antibody), tisilimuab (also known as Tremelimumab, anti-CTLA-4 antibody), tigatuzumab (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody), tocilizumab (also known as Atlizumab, Actemra, RoActemra (anti-IL-6 receptor antibody), Toralizumab (anti-CD154 (CD40L) antibody), Tositumomab (anti-CD20 antibody), Trastuzumab (also known as Herceptin, anti-HER2 / neu antibody), Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab cermoloykincelmoleukin (anti-EpCAM antibody), tuvirumab (anti-hepatitis B antibody), urtoxazumab (anti-E. coli antibody), ustekinumab (also known as Stellara, anti-IL-12, IL-23 antibody), vapaliximab (anti-AOC3 (VAP-1) antibody), vedolizumab (anti-integrin α4β7 antibody), bertuzumab (anti-CD20 antibody), bepalimomab (anti-AOC3 (VAP-1) antibody), bicilizumab (also known as Nuvion, anti-CD3 antibody), vitaxin (anti-angiogenic integrin avb3 antibody), voloxiximab (anti-integrin Phosphorus α5β1), Botumumab (also known as HumaSPECT, antitumor antigen CTAA16.88 antibody), Saltumumab (also known as HuMax-EGFr, (anti-EGFR antibody)), Zanolimumab (also known as HuMax-CD4, anti-CD4 antibody), Diralimumab (anti-CD147 (basic immunoglobulin) antibody), Zolimomab (anti-CD5 antibody), Etanercept (registered trademark "Enbrel"), Alefacept (registered trademark "Amevive"), Abatacept (registered trademark "Orencia"), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron regulatory protein 2) antibody], 14G2a (Nat. Cancer (Anti-ganglioside GD2 antibody for melanoma and solid tumors from Inst.), J591 (Anti-PSMA antibody for treating prostate cancer from Weill Cornell Medical School), 225.28S [Anti-HMW-MAA (high molecular weight melanoma-associated antigen) antibody for melanoma, Sorin Radiofarmaci SRL (Milan, Italy)], COL-1 (Anti-CEACAM3 antibody for colorectal and gastric cancer from Nat. Cancer Inst., CGM1), CYT-356 (Registered trademark "Oncoltad", prostate cancer), HNK20 (Ora Vax(For RSV from Inc.), ImmuRAIT (For non-Hodgkin lymphoma from IMMUNOMEDICS), Lym-1 (Anti-HLA-DR10 antibody, for tumors from Peregrine Pharm), MAK-195F [Anti-TNF (tumor necrosis factor; TNFA, TNF-α; TNFSF2) antibody for sepsis and toxic shock from Abbott / Knoll], MEDI-500 [Also known as T10B9, anti-CD3 antibody for graft-versus-host disease from MedImmune Inc., TRαβ (T cell receptor α / β)], RING SCAN [Neoprobe From Corp.: Anti-TAG72 (tumor-associated glycoprotein 72) antibody for breast cancer, colon cancer and colorectal cancer; Avicidin (anti-EPCAM (epithelial cell adhesion molecule) antibody); Anti-TACSTD1 (tumor-associated calcium signaling transducer 1) antibody; Anti-GA733-2 (gastrointestinal tumor-associated protein 2) antibody; Anti-EGP-2 (epithelial glycoprotein 2) antibody; Anti-KSA antibody; KS1 / 4 antigen; M4S; Tumor antigen 17-1A; CD326 for colon cancer, ovarian cancer, prostate cancer and non-Hodgkin lymphoma; LYMPHOCIDE (IMMUNOMEDICS, NJ); SmartID10 (Protein Design Labs); Oncolym (Techniclone Inc, CA); Allomune (BioTransplant, CA); Anti-VEGF antibody (Genentech, CA); CEAcide (Immunomedics, NJ); IMC-1C11 (ImClone Systems (NJ), and cetuximab (ImClone, NJ).

[0237] Other antibodies that function as binding ligands include, but are not limited to, antibodies against the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovary), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein (carcinoma), CA242 (colorectal), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), CD2 (Hodgkin's disease, NHL). CD3ε (Lymphoma, Multiple Myeloma), CD19 (B-cell Malignant Neoplasm), CD20 (Non-Hodgkin Lymphoma), CD22 (Leukemia, Lymphoma, Multiple Myeloma, Systemic Lupus Erythematosus), CD30 (Hodgkin Lymphoma), CD33 (Leukemia, Autoimmune Neoplasm), CD38 (Multiple Myeloma), CD40 (Lymphoma, Multiple Myeloma, Leukemia (CLL)), CD51 (Metastatic Melanoma, Sarcoma), CD52 (Leukemia), CD56 (Small Cell Lung Cancer, Ovarian Cancer, Merkel Cell Carcinoma and Humoral Neoplasms, Multiple Myeloma) ), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer and lung cancer), DLL4 (Δ-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, heme tumor, solid tumor), endoglin (CD105, solid tumor), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon cancer, NHL prostate cancer, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2; lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28G (cell surface antigen glycolipid, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (NHL),HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor-1 receptor, solid tumors, hematological malignancies), IL-2 receptor (interleukin-2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin-6 receptor, multiple myeloma, RA, Castleman disease, IL-6 dependent tumors), integrins (αVβ3, α5β1, α6β4, αIIβ3, α5β5, αVβ5 for various cancers), MAGE-1 ( MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (transmembrane 4-domain family A member 1, non-Hodgkin B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colon), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (Transmembrane 4-domain family A member 1, small cell lung cancer, NHL), nucleolin, neurocarcinoma gene product (carcinoma), P21 (carcinoma), anti-(N-glucorylneuraminic acid paratope (breast cancer, melanoma), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukemia (AML), gastric cancer, colorectal cancer, ovarian cancer), T cell transmembrane protein (cancer), Ti e(CD202b), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related apoptosis ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A,VEGF-2 (CD309) (various cancers). Other tumor-associated antigens recognized by antibodies have already been reported (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). (See 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).

[0238] There are many other antigens, including 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, CD12w, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, C D32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49 c, 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, CD74, CD75, CD75s, CD76, CD77, CD78, C D79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85g, CD85i, CD85j, CD85k, CD85m, CD86, CD 87, 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, 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、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、CDw198、CDw199、CD200、CD201、CD202(a,b)、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210a、CDw210b、CD211、CD212、CD213、CD213a1、CD213a2、CD214、CD215、CD216、CD217、CD218、CDw218a、CD218、CD21b9、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240、CD240ce, CD240d, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, C D259, 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、CD 291、CD292、CD293、CD294、CD295、CD296、CD297、CD298、CD299、CD300、CD300 a、CD300b、CD300c、CD301、CD302、CD303、CD304、CD305、CD306、CD307、CD30 7a、CD307b、CD307c、CD307d、CD307e、CD307f、CD308、CD309、CD310、CD311、C D312、CD313、CD314、CD315、CD316、CD317、CD318、CD319、CD320、CD321、CD3 22、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, CD37 4, CD375, CD376, CD377, CD378, CD379, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CRIPTO, CR, CR1, CRGF, CRIPTO, CXCR5LY64, TDGF1, 4-1BB, APO2, ASLG659, BMPR1B, 4-1BB, 5AC, 5T4), APO2, ASLG659, BMPR1B (bone morphogenesis protein receptor), CRIPTO, Annexin A1, nucleolus, endoglin (CD105), ROBO4, aminopeptidase N, delta-like ligand 3 (DLL3), delta-like ligand (4DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, idiotype, MAGE A3, P53 non-mutant, 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 mutation, gp100, p53 mutant, proteinase 3 (PR1), BCR-abl, tetratocarcinoma 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-enhancing factor, mesoserine), CRIPTO, Sema 5b (FLJ10372, KIAA1445, Mm42015, SEMA5B, 5EMAG, semaphorin 5bHlog, sdema domain, 7 platelet repeats, cytoplasmic region), PSCA, STEAP1 (6-transmembrane epithelial prostate antigen) and STEAP2 (HGNC8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate cancer), tyrosinase, survivorbin, hTERT, sarcoma metastasis breakpoint, 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, fucose ganglioside, 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, pod protein, Tie 2, Trop2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, Fos protein-related antigen 1.

[0239] The conjugates of the present invention are suitable for targeted cancer therapy. These cancers include, but are not limited to, adrenocortical carcinoma, anal cancer, bladder cancer, brain tumors (adult, brainstem glioma, pediatric, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primordial neuroectodermal and pineal tumors, visual tract and hypothalamic glioma), breast cancer, carcinoid tumors, gastrointestinal cancer, cancers of unknown primary origin, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, extrahepatic cholangiocarcinoma, and Ewing family tumors. (PNET), extracranial malignant germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumors, extragonadal tumors, trophoblast tumors, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal carcinoma, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, pilocytic), lip and oral 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, metastatic squamous neck cancer of unknown primary origin, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative syndrome, nasopharyngeal cancer, neuroblastoma, oral cancer, pharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, germ cell tumors, low-malignancy potential tumors), pancreatic cancer (exocrine, islet cell carcinoma), sinus and nasal cavity cancer, parathyroid cancer, penile cancer, This includes pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sézary syndrome, skin cancer, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), abnormal childhood cancers, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0240] The conjugate of the present invention is suitable for the prevention and treatment of autoimmune diseases. These autoimmune diseases are not limited to, but include, autoimmune gastric acid deficiency chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GMB / TBM 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 lymphocyte proliferation syndrome, autoimmune peripheral nervous system disease, and autoimmune pancreatic disease. Inflammation, multiple autoimmune endocrine disorders types I, II, and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Barlow's disease / Barlow concentric sclerosis, Behçet's disease, Berger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, Castleman disease, Chagas disease, chronic fatigue-related immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergic granulomatous angiitis, scarring pemphigoid, celiac disease, Cogan's syndrome, Cold agglutinin disease, complement component C2 deficiency, cranial arteritis, CRESTO syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, malignant atrophic papulosis, painful steatosis, herpetiform dermatitis, dermatomyositis, type 1 diabetes, diffuse scleroderma, myocardial infarction, lupus discoid, eczema, endometriosis, enthesitis-associated arthritis, eosinophilic fasciitis, acquired epidermolysis bullosa, erythema nodosum, idiopathic mixed cryoglobulinemia, Evans syndrome, progressive ossifying fibrosis, fibromyalgia, fibromyositis, fibrous alveolar septitis, gastritis, gastrointestinal pemphigoid, giant cell carcinoma Arteritis, nephronephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes zoster of pregnancy, hidradenitis suppurativa, Hughes' syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymphadenopathy,Lambert-Eaton myasthenic syndrome, leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, linear IgA disease (LAD), Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-like hepatitis, lupus erythematosus, Blau syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fischer syndrome, mixed connective tissue disease, scleroderma, Mucher-Jakob disease, Mackle-Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, optic nerve Transmyelitis (Devic's disease), neuromuscular dystrophy, ocular scarring pemphigoid, opsoclonus-myoclonus syndrome, Odo thyroiditis, relapsing rheumatoid arthritis, Panda syndrome (child autoimmune neuropsychiatric disorder associated with streptococcal infection), tumor cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, personality-Georgia syndrome, squamous cellulitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, rheumatoid arthritis Polymyalgia plexiformis, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red blood cell aplastic anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Reiter's syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, spinal joint This includes diseases such as sticky blood syndrome, Still's disease, Stiffman syndrome, subacute bacterial endocarditis, Suzak syndrome, acute febrile neutrophilic dermatosis, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthritis, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, and Westcott-Aldrich syndrome.

[0241] In other specific embodiments, antigen-binding molecules for conjugates 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 protein antibodies; antinuclear 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-SM antibodies; anti-mitochondrial antibodies; thyroid antibodies; microsomal antibodies, T-cell antibodies; thyroglobulin antibodies, anti-scleroderma-70 antibodies (AntiSCL-70); and anti-jaw antibodies. (Anti-Jo), anti-U1RNP antibody (Anti-U1RNP); anti-La / SSB antibody; anti-SSA antibody; anti-SSB antibody; anti-parietal cell antibody; anti-histone antibody; anti-RNP antibody; C-ANCA; P-ANCA; anti-centromere antibody; anti-fibrin antibody, anti-GBM antibody, anti-ganglioside antibody; anti-desmosome glycoprotein 3 core antibody (anti-Desmogein 3); anti-p62 antibody; anti-sp100 antibody; anti-mitochondrial (M2) antibody; rheumatoid factor antibody; anti-MCV antibody; anti-topoisomerase antibody; anti-neutrophil cytoplasm (cANCA) antibody.

[0242] In certain preferred embodiments, the binding molecules used in the conjugates of the present invention can bind to receptors or receptor complexes expressed by activated lymphocytes associated with autoimmune diseases. These receptors or receptor complexes may include, for example, members of the immunoglobulin gene superfamily (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD90, CD125, CD147, CD152 / CTLA-4, PD-1, or ICOS), or the TNF receptor superfamily (e.g., CD27, CD40, CD95) Examples include / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (type C, type S, or type I), or complement regulatory proteins.

[0243] In another specific embodiment, a useful cell-binding ligand having immunospecificity for a viral or microbial antigen is a humanized or human monoclonal antibody. The term “viral antigen” as used herein includes, but is not limited to, any viral peptides, polypeptide proteins (e.g., HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLVtax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and gE), and hepatitis B surface antigen) that can induce an immune response. The term “microbial antigen” as used herein includes, but is not limited to, any microbial peptides, polypeptides, proteins, sugars, polysaccharides, or lipid molecules (e.g., bacteria, fungi, pathogenic protozoa, yeast polypeptides (e.g., LPS and capsular polysaccharide 5 / 8)) that can induce an immune response. Examples of antibodies useful in treating viral or microbial infections include, but are not limited to, palivizumab (a humanized anti-respiratory syncytial virus monoclonal antibody used to treat RVS infection), PRO542 (a CD4 fusion antibody used to treat HIV infection), Ostavir (a human antibody used to treat hepatitis B virus), PROTVIR (a humanized IgG1 antibody used to treat cytomegalovirus), and anti-LPS antibodies.

[0244] The conjugates of the present invention can be used to treat infectious diseases. These infectious diseases include, but are not limited to, Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amoebiasis, Anaplasma, anthrax, bacterial tuberculosis, Argentine hemorrhagic fever, roundworm infection, aspergillosis, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, balantidiosis, Bailey's roundworm infection, BK virus infection, black sand worm, Blastosis hominis infection, Blastomyces, Bolivian hemorrhagic fever, and Borrelia infection. Infections, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, infectious calicivirus (norovirus, sapovirus), Campylobacter infection, Candida infection (candidiasis, thrush), cat scratch disease, cellulitis, Chagas disease (trypanosomiasis), chancroid, chickenpox, chinensis, chinensis infection, choleretic fever, chromocytic fungal infections, liver fluke disease, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever, common cold (acute viral nasopharyngitis, acute rhinitis), Leutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcus, cryptosporidiosis, cutaneous larval migration, cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, dinuclear amebiasis, diphtheria, diphyllobothrium cephalomyceta, dung worm infection, Ebola hemorrhagic fever, hydatid cystosis, ehrlichiosis, pinworm (pinworm infection), enterococcal infection, enterovirus infection, typhus, erythema infectiosum (fifth disease), acute childhood rash, hypertrophic trematode, unilateral trematode, fatal familial insomnia, filariasis, food poisoning caused by Clostridium perfringens, nonparasitic amebic infection, Fusobacterium infection, gas gangrene (Clostridium myonecrosis), diotrichumosis, Gerstmann-Streussler-Scheinker syndrome, giardiasis, glandular gland, gnathostomiasis, gonorrhea, inguinal granuloma (Donovan disease), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand, foot, and mouth disease (HFMD), hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex virus, histoplasmosis, hookworm infection, human balkan virus infection,Human ehrlichiosis Evans, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, tapeworm infection, influenza, isosporiasis, Kawasaki disease, mononucleosis, Kim's bacillus infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lyme Borrelia), lymphophilia (elephantiac disease), lymphocytic choriomeningitis, malaria, maar Burg hemorrhagic fever, measles, meridianus (Whitmore's disease), meningitis, meningococcal disease, metagonimus, microsporidia, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycomas, myiasis, neonatal conjunctivitis (neonatal ophthalmitis), Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (blindness-causing filariasis), paracoccidioidomycosis (South American blastomyces), lung paragonimiasis, pasteurellosis, head lice (head lice), body lice (body lice), pubic lice (pubic lice, Crark). bice), pelvic inflammatory disease, whooping cough, epidemic, pneumococcal infection, Pneumocystis pneumonia, pneumonia, polio, Prevotella infection, PAME, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, rhinovirus infection, rickettsial infection, rickettsia, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, diarrhea (dysentery), herpes zoster, smallpox, sporotrichosis This includes staphylococcal food poisoning, staphylococcal infection, nematode infection, syphilis, tapeworm infection, tetanus (trismus), barber's sitch, tinea manuum, tinea pedis, onychomycosis, tinea versicolor, toxocariasis (ocular larval migration), toxocariasis (visceral larval migration), toxoplasmosis, trichinella, trichomoniasis, cryptobiosis (whipworm infection), pulmonary tuberculosis, tularemia, mycoplasma urea infection, Venezuelan encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobia minor, pseudotuberculosis infection, Yersiniasis, yellow fever, and zygomycosis.

[0245] The cell-binding molecules described herein are effective against pathogenic strains, including, but are not limited to, Acinetobacter baumannii, Actiomyces israeri, Actinomyces odontolyticus, Propionibacterium propionicus, Trypanosoma brusey, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma species, Bacillus anthrax, Arcanobacterium haemolyticum, and Junin virus. Roundworms, Aspergillus, Astroviridae, Babesia, Bacillus cereus, Multiple bacteria, Bacteroides, Colonic pouch ciliates, Ascaris baileyi, BK virus, Piedraia hortae, Blastocystis hominis, Dermatitis bud bacteriosis, Macpo virus, Borrelia, Clostridium botulinum, Sabia, Brucella, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter, usually Candida albican Candida species and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus, Trypanosoma cruzi, Chancroid, Varicella-zoster virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Fonsecae pedrosoi, Hepatochomiasis, Clostridium difficile, Coccidioides imitis, Coccidioides posadasi, Colorado tick fever virus, Rhinovirus, Coronavirus, Creutzfeldt-Jakob disease prion, Crimean-Congo hemorrhagic fever virus Rus, Cryptococcus neoformans, Cryptosporidium, feline hookworm, coparasite, Cyclospora, Taenia solium, cytomegalovirus, dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4)-flavivirus, binuclear amoeba, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebola virus, Echinococcus, Ehrlichia, pinworm, Enterococcus, Enterovirus, typhus rickettsia, parvovirus B19, human herpesvirus type 6,Human herpesvirus type 7, hypertrophic flukes, liver flukes and giant liver flukes, FFI prions, filaria rhodontoids, Clostridium perfringens, Fusobacterium, Clostridium perfringens, other Clostridium species, Geotrichum canzidium, GSS prions, Giardial amblia, Burkholderia bacilli, Gnathostoma nematodes, Gnathostoma rhinospermum, Neisseria gonorrhoeae, Granulomatous bacteria, Streptococcus pyogenes, Streptococcus agalactie, Haemophilus influenzae, Enteroviruses, most Coxsackie A viruses, Enterovirus type 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O158:H7, Bunyaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1, Herpes simplex virus type 2, Histoplasma capsulatum, Duodenal hookworm, American hookworm, Haemophilus influenzae, Boca human virus, Ehrlichia ewingii, Anaplasma phagocytophyllum, Human metapneumovirus, Ehrlichia schafensis, Human papillomavirus, Human parainfluenza virus, Dwarf tapeworm, Shrimp Tapeworms, Epstein-Barr virus, Orthomyxoviridae, Isosporabelli, Kingellakingae, Klebsiella pneumoniae, Klebsiella otzena, Klebsiella rhinoscleromotis, Couloprin, Lassa fever virus, Legionella pneumophila, Leishmania, Mycobacterium lepromatosis, Leptospira species, Listeria monocystis, Borrelia and other Borrelia species, Wuchereria bancroftii and Diphyllobothrium malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, measles virus, Burkholderia pseudomallei, Neisseria meningitidis, Tremora yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia ciffii, Mycoplasma pneumoniae, various bacteria (Actinomycetoma) and fungi (Mycoplasma), Diptera larvae of parasitic flies,Chlamydia trachomatis, Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, spirometra, Blastomyces brasilis, Paragonimus and other Paragonimus species, Pasteurella species, head lice, body lice, Phthirus pubis, Bordetella pertussis, Pest bacillus, Streptococcus pneumoniae, Pneumocystis cysticercosis, poliovirus, Prevotella species, Naegleria amoeba, JC virus, Chlamydia psittaci, Coxiella barnettii, rabies virus, bead chain Escherichia coli and rat bite fever spirochete, respiratory respiratory syncytial virus (RSV) Viruses, Rhinosporidium sebergii, Rhinovirus, Rickettsia species, Rickettsia mites, Rift Valley fever virus, Rocky Mountain spotted fever rickettsia, Rotavirus, Rubella virus, Salmonella species, Atypical pneumonia coronavirus, Scabies mites, Schistosoma species, Shigella, Varicella-zoster virus, Smallpox major or minor, Sporotrix schenkyi, Staphylococcus species, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, Syphilis spirochete, Tapeworm species, Tetanus, Tinea, Trichophyton tonsurans, Tinea, Epidermophyton floccosumu, Dermatophytes rubra and Dermatophytes rubra Dermatophytes, Holthea werneckii, ringworm, Malassezia, roundworms in dogs and cats, Toxoplasma, Trichinella, Trichomonas vaginalis, whipworms, Mycobacterium tuberculosis, Bacillus flavipes, Ureaplasma ureaticum, Venezuelan encephalitis virus, Vibrio cholerae, Guanalitovirus, West Nile virus, Mycobacterium leukospores, Mycobacter pseudotuberculosis, Yersinia enteritis, yellow fever virus, Mucormycetes (Mucormycetes) and Mesh mold (Entomophthora), Pseudomonas aeruginosa, Campylobacter embryo (Vibrio), Aeromonas bacteria, Edwardsierra, Tarda, Plague bacillus, Shigella, Shigella, Red Dysentery sonne, Salmonella typhi, Treponema pertenue, Treponema calateneum, Fensenburg dolferi, Borrelia burgdorferi, Leptospirosis hemorrhagic jaundice, Pneumocystis carinii, Bacillus gracilis (bovine abortifacient), Bacillus porcini (porcine abortifacient), Malta fever bacillus, Mycoplasma species, Trickettsia scutellaria, Rickettsia scutellaria, Chlamydia species, pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma maculatum); protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas ensata, Trypanosoma gambiens, Trypanosoma rhodesiae,This includes *Donovan's leishmania*, *Leishmania tropicala*, *Leishmania brizarius*, *Pneumocystis carinii* pneumonia, *Platypleura malariae*, *Platypleura malariae*, and malignant malaria; or helminths (*Schistosoma japonicum*, *Schistosoma mansoni*, *Schistosoma haematocephala*, and hookworms).

[0246] Other antibodies used as binding ligands for the treatment of viral diseases of the present invention include, but are not limited to, antibodies against pathogenic viral antigens, and such pathogenic viruses include, but are not limited to, Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, Influenza virus, Parainfluenza virus, Mumps, Measles, Respiratory syncytial virus, Rubella, Arbovirus, Rhabdoviruses, arenaviridae, non-A / non-B hepatitis viruses, rhinoviruses, coronaviruses, rotaviruses, oncoviruses [e.g., HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma), EB virus (nasopharyngeal cancer, Burkitt lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult T-cell leukemia / lymphoma)]; immune diseases caused by viruses: [e.g., human immunodeficiency virus (A)] IDS)], CNS viruses: [e.g., JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arbovirus encephalitis, orthomyxovirus (presumably) (encephalitis lethargica), RV (rabies), bullous stomatitis, herpesvirus meningitis, Ramsay Hunt syndrome type II; poliovirus (poliomyelitis, post-polio syndrome), HTLV-I (tropical spastic paralysis)]; cytomegalovirus (CMV retinitis, HSV (herpetic keratitis)); cardiovascular viruses [e.g., CBV (pericarditis, myocarditis)]; respiratory Systemic / Acute Nasopharyngitis / Viral Pneumonia: [EBV (EBV infection / infectious mononucleosis), cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome), Orthomyxovirus family: Influenza virus A / B / C (influenza / avian influenza), Paramyxovirus: Human parainfluenza virus (parainfluenza), RSV (human respiratory syncytial virus), hMPV]; Gastrointestinal viruses [MuV (mumps), cytomegalovirus (CMV esophagitis); Adenovirus (adenovirus infection);This includes pathogenic viral diseases such as rotavirus, norovirus, astrovirus, coronavirus, HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus); and genitourinary tract viruses [e.g., BK virus, MuV (mumps)].

[0247] In a further objective, the present invention also relates to pharmaceutical compositions for the treatment of cancer or autoimmune diseases, comprising the conjugate of the present invention together with a pharmaceutically acceptable carrier, diluent, or excipient. Methods for treating cancer, infections, and autoimmune diseases can be carried out in vitro, in vivo, or ex vivo. Examples of in vitro therapies include cell culture treatments to kill all cells except desirable variants that do not express a target antigen, or to kill variants that express an undesirable antigen. Examples of ex vivo therapies include treating hematopoietic stem cells (HSCs) prior to performing a transplant (HSCT) and returning them to the body of the same patient to kill diseased or malignant cells. For example, clinical ex vivo treatments to remove cancer cells or lymphocytes from bone marrow prior to autologous transplantation in the treatment of cancer and autoimmune diseases, or to remove T cells and other lymphocytes from allogeneic bone marrow or tissue prior to transplantation to prevent graft-versus-host disease, can be carried out as follows: After obtaining bone marrow cells from a patient or another individual, they are cultured at 37°C for 15 minutes to approximately 48 hours in serum-containing medium to which the conjugate of the present invention is added, so that the concentration range is 1 pM to 0.1 mM. The appropriate concentration conditions and culture time (=dose) can be easily determined by an experienced clinician. After the culture is complete, the bone marrow cells are washed with serum-containing medium and returned to the human body by known methods such as intravenous injection. If the patient is receiving other treatments (e.g., pharmacokinetic chemotherapy or total body irradiation) between the acquisition and reinfusion of bone marrow cells, the processed bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0248] For in vivo clinical use, the conjugates of the present invention are provided as a solution or as a lyophilized solid that can be redissolved in sterile water for injection. A suitable protocol for conjugate administration is as follows: The conjugates are administered intravenously as a bolus once a week for 4 to 12 weeks. The bolus dose is dissolved in 50 to 500 mL of physiological saline, to which human serum albumin may optionally be added (e.g., 0.5 to 1 mL of concentrated human serum albumin solution at 100 mg / mL). The drug dose is approximately 50 μg to 20 mg / kg body weight per week, administered by intravenous injection (each injection in the range of 10 μg to 200 mg / kg). After completion of 4 to 12 weeks of treatment, the patient may accept a second course of treatment. Detailed treatment methods, including the route of administration, excipients, diluents, dosage, and duration of treatment, can be determined by an experienced surgeon.

[0249] Examples of conditions that can be treated by selectively killing cell populations in vivo or ex vivo include any type of cancer, autoimmune diseases, transplant rejection, and infections (including those caused by viruses, bacteria, or parasites).

[0250] The amount of conjugate required to achieve the desired biological effect varies depending on many factors, including the chemical properties, efficacy, and bioavailability of the conjugate, the type of disease, the patient's race, the patient's medical condition, the route of administration, and all other factors that determine the required dosage, including the delivery and regimen used.

[0251] Generally speaking, the formulations of the present invention may be parenteral formulations dissolved in physiological buffer so as to contain the conjugate at a concentration of 0.1 to 10% w / v. Typical dose ranges are 1 μg / kg body weight to 0.1 g / kg body weight per day, preferred dose ranges are 0.01 mg / kg body weight to 20 mg / kg body weight per day, or equivalent doses for children. Preferred drug doses may appropriately depend on variables such as, for example, the type and degree of disease or disability progression, the overall health status of the individual patient, the relative biological activity of the selected drug, the dosage form of the compound, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic characteristics of the drug in the selected mode of administration, and the rate of administration (single injection or continuous infusion) and the administration schedule (frequency of administration within a set period of time).

[0252] The conjugates of the present invention can also be administered in unit doses, where "unit dose" refers to a single dose administered to one patient, and can be used in simple and convenient packaging, maintaining a physically and chemically stable unit dose as the active conjugate itself or as a pharmaceutically acceptable composition as described below. Therefore, the typical daily dose range is 0.01 to 100 mg / kg body weight. Generally, the unit dose ranges from 1 to 3000 mg per day. Preferably, the unit dose is 1 mg to 500 mg administered four times a day, or 10 mg to 500 mg administered once a day. The conjugates given herein can be prepared by adding one or more pharmaceutically acceptable excipients to a pharmaceutical composition. A unit dose of the drug may be administered orally as a tablet, simple capsule or soft capsule; intranasally as a powder, nasal spray or aerosol; or cutaneously as, for example, an ointment, cream, lotion, gel or spray or skin patch, by any method known in the art of pharmaceutical science, e.g., Remington: The Science and Practice of Pharmacy, 21 thIt can be prepared by the method described in Lippincott Williams & Wilkins: Philadelphia, PA, 2005.

[0253] Pharmaceutical formulations containing the compounds of the present invention, including pharmaceutical compositions, are preferably administered orally or parenterally. Oral formulations such as tablets, pills, powders, capsules, and lozenges may contain one or more of the following components or other compounds having similar properties: binders such as microcrystalline cellulose or tragacanth gum; diluents such as starch or lactose; disintegrants such as starch or cellulose derivatives; lubricants such as magnesium stearate; flow promoters such as colloidal silica; sweeteners such as sucrose or saccharin; and flavoring agents such as peppermint or methyl salicylate. Capsules can be hard or soft capsules and are generally made from a mixture of gelatin mixed with a plasticizer, as is the case with starch capsules. Furthermore, unit dosage forms may contain various other raw materials that alter their physical form, such as sugar coatings, shellac, or enteric coatings. Other oral dosage forms, such as syrups or elixirs, may contain sweeteners, preservatives, pigments, colorants, and flavoring agents. Furthermore, the active compound can be prepared in a rapid-release, controlled-release, or sustained-release form through various treatments and formulations, with the sustained-release form being preferably bimodal. The tablets preferably contain lactose, corn starch, magnesium silicate, croscarmellose sodium, polyvinylpyrrolidone, magnesium stearate, talc, and other combinations.

[0254] Liquid pharmaceuticals for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid compositions may contain binders, buffers, preservatives, chelating agents, sweeteners, flavorings, and colorants. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous solvents include mixtures of alcohol and water, buffering media, and physiological saline. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers can be used as excipients to control the release of active compounds. Intravenous media may include liquids and nutritional rehydration solutions, electrolyte rehydration solutions such as Ringer's dextrose base, etc. Other viable parenteral delivery systems for the active drugs of the present invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

[0255] Other dosage forms include inhalation formulations containing dry powder, aerosol, and droplets. Inhalation formulations may be, for example, aqueous solutions containing polyoxyethylene-9-lauryl ether, glycocholates, and deoxycholates, or oily solutions for administration in the form of nasal drops or gels applied into the nasal cavity. Formulations for buccal administration include, for example, lozenges or troches, which may contain flavoring agents such as sucrose or acacia, and other auxiliary materials such as glycocholates. Formulations for rectal administration are preferably provided as suppositories of a unit dose together with a solid carrier such as cocoa butter, and may contain salicylic acid. Formulations for topical application to the skin are preferably in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Petrolatum, lanolin, polyethylene glycol, alcohol, or mixtures thereof can be used as drug carriers. Suitable formulations for transdermal administration can be provided as individual patches, or as lipophilic emulsions or buffered aqueous solutions dissolved or dispersed in emulsions, buffers, polymers, or adhesives.

[0256] In certain embodiments, the conjugate of the present invention is administered concurrently with known or future therapeutic agents, such as chemotherapeutic agents, radiotherapy agents, immunotherapy agents, autoimmune disease agents, anti-infective agents, or other antibody-drug conjugates, thereby achieving a synergistic effect. In another specific embodiment, the synergistic drug or radiotherapy is administered or performed before or after the administration of the conjugate, in one embodiment, within one hour, 12 hours, one day, one week, one month, or several months before or after the administration of the conjugate of the present invention.

[0257] In other embodiments, the synergistic drugs include, but are not limited to, the following:

[0258] 1) Chemotherapy agents: a) Alkylating agents: Nitrogen mustard (chlorambucil, cyclophosphamide, ifosfamide, chlorambucil, melphalan, cyclophosphamide); nitrosourea compounds (carmustine, lomustine); alkyl sulfonates (busulfan, treosulfan); triazenes (dacarbazine); platinum-containing compounds (carboplatin, cisplatin, oxaliplatin), etc. b) Plant alkaloids: Vinca alkaloids (vincristine, vinblastine, vindesine, vinorelbine); taxoids (paclitaxel, docetaxel), etc. c) DNA topoisomerase inhibitors: Epipodophyllins (9-aminocamptothecin, camptothecin, cristinator, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan); and mitomycins (mitomycin C), etc. d) Antimetabolites: {[Antifolic acids: (e.g., DHFR inhibitors: methotrexate, trimethrexate); IMP dehydrogenase inhibitors (e.g., mycophenolic acid, thiazophrine, ribavirin, EICAR, etc.); ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine)]; [Pyrimidine analogs: Uracil analogs (e.g., 5-fluorouracil, doxifluridine, floxuridine, lacitrexed (tomudex); cytosine analogs (cytarabine, fludarabine, etc.), purine analogs: (e.g., azathioprine, mercaptopurine, thioguanine)] etc.; e) Hormone therapy agents: Receptor antagonists: [Anti-estrogens: (e.g., megestrol, raloxifene, tamoxifen); LHRH agonists: (including goserelin, leuprolide acetate); Antiandrogens: (e.g., bicalutamide, flutamide, carsterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane, and other similar androgens] (Dioxin inhibitors); Retinoids / Deltoid muscle: [e.g., Vitamin D3 analogs: (CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); Photodynamic therapy agents: (e.g., verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrelin A); Cytokines: (e.g., interferon α, interferon γ, tumor necrosis factor (TNF), TNF domain-containing human proteins)], etc. f) Kinase inhibitors: BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), moritinib, meditinib, pratinib, ponatinib (ap24534), HQP1351, bafetinib (INNO-406), bosutinib (SKI-606), sunitinib, cabotinib, volitinib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib, etc. i) Others: gemcitabine, epoxomicins (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zyprestat, PLX4032, STA-9090, Stimuvax, allobectin-7, Zygeba, Provenge, Elboy, isoprenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridine iodine ), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g., bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zolubicin), mitoxantrone, MDR inhibitors (e.g., verapamil), Ca 2+ ATP inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (including vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), bellinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A, etc.

[0259] 2) Anti-autoimmune disease drugs include, but are not limited to, cyclosporine, cyclosporine A, aminocaproic acid, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticoids (hormonal agents, e.g., betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), dehydroepiandrosterone, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolic acid, sirolimus, tacrolimus, prednisone, etc.

[0260] 3) Anti-infective drugs include, but are not limited to, the following: a) Aminoglycosides: Amikacin, Astromycin, Gentamycin (Netylmycin, Shisomycin, Isepamycin), Hygromycin B, Kanamycin (Amikacin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin), Neomycin (Furamycin, Paromomycin, Ribostamycin), Netylmycin, Spectinomycin, Streptomycin, Tobramycin, Verdamicin; b) Amphenicols: Azidamphenicol, Chloramphenicol, Florphenicol, Thiamphenicol; c) Ansamycin derivatives: geldanamycin, harbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, panipenem; e) Cephalosporins: Carbasephalm (loracalbef), cefacetril, cefaclor, cefradin, cefadroxil, cephalonium, cefaloridine, cephalothin or cephalothin, cephalexin, cephaloglysin, cephamandol, cefapillin, cefatoridine, cefazal, cefazedone, cefazolin, cefubperazone, cefcapene, cefdaroxime, cefepime, cefminox, cefoxitin, cefprodil, ceffloxazine, ceftezol, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, ce Fetamet, cefmenoxime, cefozidime, cefonisid, cefoperazone, cefolanide, cefotaxime, cefotiam, cefozopran, cephalexin, cefpimisole, cefpyramide, cefpirome, cefpodoxime, cefprodil, cefquinome, cefsulodine, ceftazidime, cefteram, ceftibuten, cefthiolen, ceftizoxime, ceftobiprol, ceftriaxone, ceffuroxime, cefzonam, cephamycin (including cefoxitin, cefotetan, and cefmetazole), oxacepham (flomoxef, latamoxef); f) Glycopeptides: Bleomycin, vancomycin (including oritabancin and teravancin), teicoplanin (darbabancin), lamopranin; g) Glycylcyclines: e.g., tigecycline; h) β-lactamase inhibitors: Penam (sulbactam, tazobactam), Clavam (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics (CDA); k) Macrolides: Azithromycin, cesromycin, clarithromycin, dilithromycin, erythromycin, flurithromycin, josamycin, ketolides (telithromycin, cesromycin), midecamycin, myokamycin, oleandmycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentin), rokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), troleandmycin, telithromycin; l) Monobactams: Aztreonam, Tigemonam; m) Oxazolidinones: Linezolids; n) Penicillins: Amoxicillin, ampicillin, pivampicillin, hetacillin, bacampicillin, methampicillin, tarampicillin, azidocillin, azurocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin, procaine benzylpenicillin, carbenicillin (kalindacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, mesilinum (pibmesilinum), mezurocillin, methicillin, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticalcillin; o) Polypeptides: bacitracin, colistin, polymyxin B; p) Quinolones: Alatrofloxacin, valofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, phloxin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, canotorobafloxacin, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) Streptogramins: Pristinamycin, quinupristin / dalfopristin; r) Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizol, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole); s) Steroidal antibacterial agents: Selected from fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, chromocycline, demeclocycline, rimecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, lolitetracycline, tetracycline, glycylcycline (e.g., tigecycline); u) Other antibiotics: annonasin, arsphenamine, bactoprenol inhibitor (bacitracin), DADAL / AR inhibitor (cycloserine), dicthiostatin, discodermolide, eleuterobin, epothilon, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laurimalid, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitor (e.g., fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), tazobactamtinidazole, uvarcin;

[0261] 4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, bicriviroc, gp41 (enfuvirtide), PRO140, 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, klevudine, dexerbucitabine, didanosine (DDI), erbucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'-fluorosubstituted 2',3'-deoxynucleoside analogs (e.g., 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluorine Ro-2',3'-dideoxyguanosine (FLG), homivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (e.g., β-L-thymidine and β-L-2'-deoxycytidine), penciclovir, lasivir, ribavirin, stampidine, stabidine set (d4T), taribavirin (viramidine), terbivudine, tenofovir, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: Amantadine, ateviridine, caplavirine, diallylpyrimidine (etravirine, rilpivirine), delaviridine, docosanol, emibilin, efavirenz, foscarnet (phosphorylformate), imiquimod, interferon α, roviride, rodenosine, methisazone, nevirapine, NOV-205, pegylated interferon α, podophyllotoxin, rifampicin, rimantadine, reciquimod (R-848), tromantadine; g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, preconalil, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other antiviral drugs: Abzyme, Arbidol, Caranolid A, Selagenin, Cyanobilin-N, Diallylpyrimidine, Epigallocatechin gallate (EGCG), Foscarnet, Griffiscin, Taribavirin (Pyramidine), Hydroxyurea, KP-1461, Miltefosin, Preconalil, Portmanto inhibitors, Ribavirin, Cericiclib.

[0262] 5) Other immunotherapeutic agents: e.g., imiquimod, interferons (e.g., α, β), granulocyte colony-stimulating factor, cytokines, interleukins (IL-1, IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, artuximab, paximab, dacliximab, oralvazin), protein conjugates (e.g., Abraxane), as well as calicheamicin derivatives, meitansine derivatives (DM1 and DM4), CC-1065 and duocalmycin, potent taxol derivatives, doxorubicin, auristatin antimitotics (e.g., trastuzumab-DM1, inotuzumab, brentuximab vedotin, glenbatumumab vedotin, lorbotuzumab meltansine, AN-152) Antibodies conjugated to drugs selected from LMB2, TP-38, VB4-845, cantuzumab meltansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, IMGN529, IMGN853, milatuzumab-doxorubicin, SGN-75 (anti-CD70), anti-CD22-MCC-DM1).

[0263] To another purpose, the present invention also relates to a method for preparing the conjugates of the present invention. The conjugates and methods of the present invention can be prepared by various methods well known to those skilled in the art. The mitotic inhibitors in the conjugates of the present invention can be synthesized, for example, by applying or modifying the following methods, or by modifications that will be understood by those skilled in the art. Appropriate modifications and substitutions will be obvious to those skilled in the art, are well known from the scientific literature, or are readily available. In particular, such methods can be found in RCLarock, Comprehensive OrganiCTransformations, 2nd edition, Wiley-VCH Publishers, 1999.

[0264] In the reactions described below, reactive functional groups required for the final product, such as hydroxyl, amino, imino, thio, or carboxyl groups, may need to be protected to prevent them from participating in unwanted chemical reactions. Conventional protecting groups are used according to standard practice. See, for example, PG Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis, Wiley-Interscience; 4th edition (2006). Some reactions can be carried out in the presence of a base, acid, or suitable solvent. There are no particular limitations on the properties of the base, acid, and solvent used in these reactions; any base, acid, or solvent conventionally used in this type of reaction can be used, as long as it does not adversely affect the reaction. The reactions can be carried out over a wide temperature range. Typically, the reactions are carried out between -80°C and 150°C (more preferably between room temperature and approximately 100°C). The reaction time can also be very long, depending on many factors, particularly the reaction temperature and the properties of the reagents. However, as long as the reactants are carried out under the preferred conditions described above, it is usually between 3 and 20 hours.

[0265] The reaction can be completed by conventional means. For example, the reaction product can be recovered by distilling off the solvent from the reaction mixture, or, if necessary, by distilling off the solvent from the reaction mixture, then pouring the residue into water, followed by extraction with a water-immiscible organic solvent and subsequent distillation. Furthermore, if necessary, the product can be purified by recrystallization, reprecipitation, or by various well-known chromatographic techniques, particularly silica gel column chromatography or preparative thin-layer chromatography. Column chromatography and preparative thin-layer chromatography are more commonly used. [Examples]

[0266] The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The cell lines described in the following examples were maintained in culture medium under conditions specified by the American Type Culture Collection (ATCC), the German Microbial Cell Culture Collection (DSMZ) in Braunschweig, Germany, or the Shanghai Institute of Cell Culture, Chinese Academy of Sciences, unless otherwise specified. Unless otherwise specified, all cell culture reagents were supplied by Invitrogen. All anhydrous reagents were commercially available and stored in nitrogen-sealed Sure-Seal bottles. All other reagents and solvents were purchased according to the highest standards and used without further purification. Separation by preparative HPLC was performed using a Varain PreStar HPLC. NMR spectra were recorded using a Bruker 500 MHz Instrument. Chemical shifts (Δ) were expressed in ppm, with tetramethylsilane as the standard at 0.00, and binding constants (J) were expressed in Hz. Mass spectrometry data were obtained using a Waters Xevo QTOF mass spectrometer equipped with a Waters Acquity UPLC separator and an Acquity TUV detector.

[0267] Example 1: Synthesis of Compound 1 [ka]

[0268] In a 10 L reactor, 2,2-diethoxyacetonitrile (1.00 kg, 7.74 mol) was dissolved in methanol (6.0 L), and (NH4)2S (48% aqueous solution, 1.41 kg, 9.29 mol, 1.2 equivalents) was added at room temperature. The temperature in the reactor rose to 33°C and then returned to room temperature. After stirring overnight, the reaction solution was concentrated. Ethyl acetate (5 L) was added to the residue, washed with saturated NaHCO3 solution (4 × 1.0 L), and the aqueous phase was back-extracted with ethyl acetate (5 × 1.0 L). The organic phases were combined, washed with saturated brine (3 L), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was slurryed with petroleum ether, filtered under vacuum, the solid was collected, and washed with petroleum ether. After concentrating the filtrate, it was slurryed with petroleum ether, and the resulting solids were collected together to obtain a total of 1.1 kg of the target product, which was a white or light yellow solid (yield 87%). 1 H NMR (500MHz, CDCl3) 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 = 7.1Hz, 6H).

[0269] Example 2; Synthesis of Compound 2 [ka]

[0270] A 5L three-necked round-bottom flask was equipped with a reflux condenser and a constant-pressure dropping funnel. Molecular sieves (3A, 500g) and thioamide (350g, 2.14mol, 1.0 equivalent) were added to ethanol (3L), and ethyl 3-bromopyruvate (80% purity, 404mL, 2.57mol) was added dropwise over 30 minutes. After the internal temperature rose slightly during the addition, the reaction mixture was heated under reflux and stirred for 30 minutes. After the reaction mixture cooled to room temperature, insoluble substances were removed by filtration through Celite, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the crude product was mixed with silica gel (1.5kg) and thoroughly mixed. The mixture was purified by silica gel column (10kg) column chromatography (10-20% ethyl acetate / petroleum ether gradient elution) to obtain the target compound, a brown oil (509g, 92% yield).

[0271] Example 3: Synthesis of Compound 3 [ka]

[0272] A solution of acetal (300 g, 1.16 mol) in acetone (3.0 L) was heated under reflux, and a 4N HCl solution (250 mL) was added dropwise over 1.0 hour. TLC indicated that the reaction was complete and the starting material was consumed. After concentrating the reaction solution under reduced pressure, the two phases were separated. The organic phase was diluted with ethyl acetate (1.5 L), washed 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 extracted with ethyl acetate. The extracts were combined and dried over anhydrous sodium sulfate. After filtering the organic solution, it was concentrated under reduced pressure. The resulting crude product was triturated with petroleum ether / ethyl acetate (5:1) solution, the precipitated solid was collected by suction 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 obtain 40 g of the target product, which was a white or bright yellow solid (yield 43%). 1H NMR (500MHz, CDCl3) δ 10.08-10.06 (m, 1H), 8.53-8.50 (m, 1H), 4.49 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H). MS ESI m / z C7H8NO3S [M+H] + Calculated value: 186.01, Measured value: 186.01.

[0273] Example 4; Synthesis of Compound 4 [ka]

[0274] Under N2 protection at room temperature, Ti(OEt)4 (345 mL, 1.82 mol) and 3-methyl-2-butanone (81 mL, 0.825 mol) were added to a tetrahydrofuran solution (1 L) of (S)-tert-butylsulfinamide (100 g, 0.825 mol). The reaction solution was heated under reflux for 16 hours, then cooled to room temperature and poured into ice water (1 L). The filter cake was filtered and washed with ethyl acetate. The organic phase in the filtrate was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was distilled under reduced pressure (15-20 Torr, 95°C) to obtain the target product 4 as a yellow oil (141 g, 90% yield). 1 H NMR (500MHz, CDCl3) 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] + Calculated value: 212.12, Measured value: 212.11.

[0275] Example 5; Synthesis of Compound 5 [ka]

[0276] Under N2 protection at -78°C, n-butyllithium solution (2.5M, 681mL, 1.70mol) was added to an anhydrous tetrahydrofuran solution (1L) of diisopropylamine (264mL, 1.87mol). The reaction mixture was warmed to 0°C for 30 minutes and then recooled to -78°C. Compound 10 (258g, 1.36mol) was added, followed by rinsing with tetrahydrofuran (50mL). After stirring for 1 hour, ClTi(O) was added to tetrahydrofuran (1.0L). i Pr)3 (834g, 3.17mol) was added dropwise. One hour after the addition was complete, a tetrahydrofuran solution (500mL) of compound 4 (210g, 1.13mol) was slowly added dropwise, which took one hour. The resulting solution was stirred at -78°C for 3 hours. After the reaction was completed, monitored by TLC, the reaction was stopped with a mixture of acetic acid and tetrahydrofuran (volume ratio 1:1, 300mL), and the reaction solution was then poured into brine (2L) and ethyl acetate (8×1L). 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 obtain compound 5 as a colorless oil (298g, yield 74%). 1 H-NMR (500MHz, CDCl3) 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 value: 397.13, Measured value: 397.11.

[0277] Example 6; Synthesis of Compound 6 [ka]

[0278] Compound 5 (509 g, 1.35 mol) was dissolved in tetrahydrofuran (200 mL), cooled to -78 °C, and Ti(OEt)4 (570 mL, 2.72 mol) was slowly added. After the addition was complete, the mixture was stirred for 1 hour. Next, NaBH4 (51.3 g, 1.36 mol) was added gradually over 90 minutes. The reaction mixture was stirred at -78 °C for 3 hours. TLC monitoring confirmed that the starting material was still present. Ethanol (50 mL) was slowly added, and stirring continued for 1.5 hours. Then the reaction solution was poured into saturated brine (2 L, containing 250 mL of acetic acid) and warmed to room temperature. After filtering through Celite, the organic phase was separated, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (ethyl acetate / petroleum ether 1:1) to obtain compound 6 as a white solid (364 g, yield 71%). 1 H NMR (500MHz, CDCl3) 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:C 16 H 28 NaN2O4S2[M+Na] + Calculated value: 399.15, Measured value: 399.14.

[0279] Example 7; Synthesis of Compound 7 [ka]

[0280] At 0°C, 4N HCl in 1,4-dioxane (590 mL) was added to an ethanol solution (590 mL) of compound 6 (600 g, 1.60 mol). The reaction mixture was gradually warmed to room temperature and then stirred for 2.5 hours. The precipitated white solid was collected by filtration and washed with ethyl acetate. The filtrate was concentrated and triturated with ethyl acetate. The two obtained white solids totaled 446 g (yield 90%).

[0281] Example 8; Synthesis of Compound 8 [ka]

[0282] Sodium azide (740 g, 11.4 mol) was dissolved in water (2.0 L), dichloromethane (2.0 L) was added, and the mixture was cooled to 0°C. Tf2O (700 mL, 4.10 mol) was added to the solution over 1.5 hours. After the addition, stirring was continued at 0°C for 3 hours. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (2 × 500 mL). The organic phases were combined and washed with saturated NaHCO3 (3 × 1.0 L). At room temperature, this dichloromethane solution was added to a mixture of (L)-isoleucine (300 g, 2.28 mol), potassium carbonate (472 g, 3.42 mol), and copper sulfate pentahydrate (5.7 g, 22.8 mmol) in water (3.0 L) and methanol (3.0 L). During the addition, the temperature in the reaction system rose slightly. The mixture was then stirred at room temperature for 16 hours. The organic solvent was evaporated under reduced pressure, the aqueous phase was acidified to pH 6-6.5 with concentrated hydrochloric acid (approx. 280 mL), then diluted with phosphate buffer (0.25 M, pH 6.2), 6.0 L, and washed with ethyl acetate (6 × 2.0 L) to remove the sulfonamide byproduct. The solution was acidified to pH 3 with concentrated hydrochloric acid (approx. 400 mL) and extracted with ethyl acetate (4 × 2.0 L). The organic phases were combined, washed with saturated brine (2.0 L), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain product 8 as a bright yellow oily substance (320 g, yield 89%). 1H NMR (500MHz, CDCl3) 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).

[0283] Example 9; Synthesis of Compound 9 [ka]

[0284] Azide-Ile-OH (8; 153 g, 0.97 mol) was dissolved in tetrahydrofuran (1.5 L), cooled to 0°C, and NMM (214 mL, 1.94 mol) and isobutyl chloroformate (95 mL, 0.73 mol) were added in sequence. The reaction mixture was stirred at 0°C for 1 hour, and then compound 7 (150 g, 0.49 mmol) was added little by little. After stirring at 0°C for 30 minutes, the mixture was gradually warmed to room temperature and stirred for 2 hours. The reaction was quenched in ice water at 0°C and extracted three times with ethyl acetate. The combined organic phase was washed with 1N HCl, saturated NaHCO3, and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (0-30% ethyl acetate / petroleum ether) to obtain a white solid (140 g, yield 70%). 1H NMR (500MHz, CDCl3) 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] + Calculated value: 412.19, Measured value: 412.19.

[0285] Example 10; Synthesis of Compound 10 [ka]

[0286] At 0°C, imidazole (94g, 1.37 mmol, 1.3 equivalents) was added to a 50 mL dichloromethane solution of compound 9 (436 g, 1.05 mol, 1.0 equivalent), followed by chlorotriethylsilane (222 mL, 1.32 mol). The reaction mixture was allowed to rise to room temperature over 1 hour, after which stirring was continued for 1 hour. The mixture was quenched with saturated saline solution, the organic phase was separated, and 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 obtain product 10 as a colorless oil (557.4 g, yield 95%). 11H NMR (500 MHz, CDCl3) 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 value: 526.28, Measured value: 526.28.

[0287] Example 11; Synthesis of Compound 11

Chemical Structure

[0288] At 0 °C, sodium hydride (60% suspension in mineral oil, 62.2 g, 1.55 mol) was added to a solution of compound 10 (408 g, 0.77 mol) and methyl iodide (145 mL, 2.32 mol) in tetrahydrofuran (4 L). The resulting reaction solution was stirred at 0 °C overnight and then poured into a vigorously stirred solution of ice - water - saturated ammonium chloride (5 L). It was extracted with ethyl acetate (3 × 500 mL), the combined organic phases were dried, filtered, concentrated, and purified by column chromatography (15 - 35% ethyl acetate / petroleum ether) to obtain product 11 as a light yellow oil (388 g, yield 93%). 1H NMR (500MHz, CDCl3) δ 8.09 (s, 1H), 4.95 (d, J = 6.6 Hz, 1H),4.41 (q, J = 7.1 Hz, 2H), 3.56 (d, J = 9.5 Hz, 1H), 2.98 (s, 3H), 2.27-2.06 (m, 4H), 1.83-1.70 (m, 2H), 1.41 (t, J = 7.2 Hz, 3H), 1.29 (ddd, J = 8.9, 6.8, 1.6 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.96 (dt, J = 8.0, 2.9 Hz, 15H), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.7 Hz,3H). MS ESI m / z C 25 H 46 N5O4SSi [M+H] + Calculated value: 540.30, Measured value: 540.30.

[0289] Example 12; Synthesis of Compound 12 [ka]

[0290] A mixture of 2-methylalanine (500 g, 4.85 mol), formaldehyde (37% aqueous solution, 1.0 L, 12.1 mol), and formic acid (1.0 L) was heated under reflux (80°C) for 3.0 hours, cooled to room temperature, and then 6N HCl (850 mL) was added to concentrate the reaction mixture. The resulting solid was collected by filtration and washed three times with ethyl acetate (1.0 L). The solid was dissolved in water (1.5 L) and neutralized to pH 7 with 4N NaOH (approximately 1.0 L). The solution was concentrated and the water was removed by co-evaporation with ethanol (2.0 L). The residue was dissolved in methanol (2.0 L), filtered to remove solid NaCl, and washed with ethyl acetate. After concentrating the filtrate, 639.2 g of a white solid containing a small amount of NaCl was obtained, which could be used directly without further purification.

[0291] Example 13; Synthesis of Compound 13 [Chemical formula]

[0292] To a solution of compound 12 (97 g, 0.74 mol) in ethyl acetate (1 L) were added pentafluorophenol (163 g, 0.88 mol) and DIC (126 mL, 0.81 mol). The reaction mixture was stirred at room temperature for 24 hours, then filtered through celite, and the filter pad was washed with 10 mL of ethyl acetate. The filtrate was used immediately without further purification or concentration.

[0293] Example 14; Synthesis of Compound 14 [Chemical formula]

[0294] To a solution of the above pentafluorophenyl ester 13 in ethyl acetate were added compound 11 (200 g, 0.37 mol) and dry Pd / C (10 wt%, 10 g). The reaction solution was stirred under hydrogen (1 atm) for 27 hours. It was filtered through celite, and the filter pad was washed with ethyl acetate. The combined organic phases were concentrated and purified by column chromatography (0 - 5% methanol / ethyl acetate) to obtain compound 14 (184 g, yield 79%). MS ESI m / z C 31 H 58 N4O5SSi [M + H] + : Calculated value: 627.39, Measured value: 627.39.

[0295] Example 15; Synthesis of Compound 15 [Chemical formula]

[0296] Compound 14 (200 g, 0.32 mmol) was dissolved in a mixed solution of acetic acid / water / tetrahydrofuran (v / v / v 3:1:1, 638 mL) and stirred at room temperature for 4 days. The reaction solution was concentrated, and toluene was added and it was concentrated again. This process was repeated twice to obtain compound 15, which was used directly in the next reaction. MS ESI m / z C 25 H 45 N4O5S [M+H] + Calculated value: 513.30, Measured value: 513.30.

[0297] Example 16; Synthesis of Compound 16 [ka]

[0298] At 0°C, a methanol solution (1.2 L) of compound 15 (160 g, 0.319 mol, 1.0 equivalent) was mixed with an aqueous lithium hydroxide solution (0.4 N, 600 mL, 2.55 mol). The reaction mixture was stirred at room temperature for 2 hours and then concentrated. The mixture was purified by column chromatography (from 100% dichloromethane to 80:20:1 dichloromethane / methanol / ammonia) to obtain compound 16 as an amorphous white solid (140 g, 91% yield in two steps). MS ESI m / z C 23 H 40 N4O5S [M+H] + Calculated value: 485.27, Measured value: 485.27.

[0299] Example 17; Synthesis of Compound 17 [ka]

[0300] Compound 16 (143 g, 0.30 mol) and DMAP (0.36 g, 2.95 mmol) were dissolved in a mixed solution of anhydrous tetrahydrofuran (1.4 L) and anhydrous DMF (75 mL). The mixture was cooled to 0°C, and triethylamine (82.2 mL, 0.59 mmol) and acetic anhydride (56 mL, 0.59 mmol) were added. The reaction mixture was gradually warmed to room temperature, stirred for 24 hours, then concentrated, and purified by column chromatography (5-50% methanol / dichloromethane) to obtain compound 17 as an amorphous white solid (147 g, 95% yield). 1 H NMR (500MHz, 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 (126MHz, 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] + Calculated value: 527.3, Measured value: 527.4.

[0301] Example 18; Synthesis of Compound 18 [ka]

[0302] Under nitrogen protection at room temperature, a 600 mL solution of compound 17 (41.0 g, 77.9 mmol, 1.0 equivalent) in anhydrous dichloromethane was mixed with EDC·HCl (44.8 g, 233 mmol, 3.0 equivalents) and pentafluorophenol (35.9 g, 194 mmol, 2.5 equivalents). 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) to obtain a white solid (43.0 g, yield 80%). 1 H NMR (500MHz, 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). 13C NMR (126MHz, 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] + Calculated value: 693.3, Measured value: 693.3.

[0303] Example 19; Synthesis of Compound 19 [ka]

[0304] At room temperature, sodium hydride (60%, 8g, 200 mmol) was added to a tetrahydrofuran solution (1 L) of HO-PEG9-OMe (42.8 g, 100 mmol). After stirring for 30 minutes, tert-butyl bromoacetate (48.8 g, 250 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then poured into ice water and extracted with dichloromethane. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (0-5% methanol / dichloromethane) to obtain compound 19 as a yellow oil (32 g, 59% yield).

[0305] Example 20; Synthesis of Compound 20 [ka]

[0306] Compound 432 (40 g, 73.8 mmol) was dissolved in dichloromethane (400 mL), then formic acid (600 mL) was added, and the mixture was stirred overnight at 25°C. All volatile substances were removed by distillation under reduced pressure to obtain a yellow oil (36 g, yield approximately 100%). ESI m / z C 21 H 43 O 12 [M+H] + Calculated value: 487.27, Measured value: 487.24.

[0307] Example 21; Synthesis of Compound 21 [ka]

[0308] Compound 20 (36 g, 73.8 mmol) was dissolved in dichloromethane (640 mL), and 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, and all volatile substances were removed by distillation under reduced pressure to obtain a yellow oil.

[0309] Example 22; Synthesis of Compound 22 [ka]

[0310] ZL-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). The mixture was cooled to 0°C, and 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, and the pH was adjusted to 3 by adding concentrated hydrochloric acid in an ice bath. The solution was extracted with dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate to obtain a yellow oil (55 g, 99% yield). ESI m / z C 35 H 60 N2O 15 [M+H]+ Calculated value: 749.40, Measured value: 749.39.

[0311] Example 23; Synthesis of Compound 23 [ka]

[0312] Benzyl bromide (1.33 kg, 7.78 mol) was slowly added to an acetonitrile solution (8.8 L) 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). The mixture was then stirred overnight at room temperature, and water (8 L) was added to dissolve the solid, which was then extracted with ethyl acetate (2 × 4 L). The combined organic phase was washed with water (4 L) and saturated brine (4 L), dried over anhydrous sodium sulfate, filtered, concentrated, and triturated with petroleum ether (20 L) to obtain a white solid 98 (2.73 kg, yield 95%). 1 H NMR (500MHz, CDCl3) 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] + Calculated value: 386.19, Measured value: 386.19.

[0313] Example 24; Synthesis of Compound 24 [ka]

[0314] NaBH4 (122 g, 3.2 mol) and LiCl (136 g, 3.2 mol) were added to a mixed solvent of 2.4 L of ethanol and 2.4 L of dichloromethane, cooled to 0°C, and a 2.4 L solution of compound 23 (616 g, 1.6 mol) in dichloromethane was added. After the addition, 2.4 L of dichloromethane was added to the reaction solution and allowed to rise naturally to room temperature, generating many bubbles, and then 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), the organic phase was combined, washed with water (2 L) and brine (2 L), dried, filtered, and concentrated to obtain 542 g of white solid (yield 95%).

[0315] Example 25; Synthesis of Compound 25 [ka]

[0316] Oxalyl chloride (1.02 kg, 8.0 mol) was dissolved in dichloromethane (4 L), cooled to -75°C, and while maintaining the temperature below -65°C, a 400 mL solution of DMSO (1.25 kg, 16 mol) in dichloromethane was added dropwise. After the addition was complete, the mixture was stirred for 30 minutes, and then a 8 L solution of compound 24 (1.90 kg, 5.33 mol) in dichloromethane was added dropwise. After the addition was complete, the temperature of the solution rose to approximately -65°C. After stirring for 30 minutes, triethylamine (1.62 kg, 16 mol) was added dropwise while maintaining the temperature below -50°C, and after the addition was complete, the mixture was stirred for 15 minutes. The reaction mixture was slowly warmed, and stirring was continued for approximately 1 hour. The reaction solution was warmed to approximately -30°C, and TLC monitoring indicated that the reaction was complete. Water (6 L) was added to the reaction solution, and the mixture was stirred to separate it into layers. The aqueous layer was washed with dichloromethane (2 L), and the organic layers were combined and washed with 10% HCl (4 L) and brine (2 L). The mixture was dried, filtered, and concentrated. The concentrated solution was triturated with petroleum ether / ethyl acetate in a 5:1 ratio, filtered under vacuum, and 1.36 kg of pale yellow solid was obtained (yield 72%).

[0317] Example 26; Synthesis of Compound 26 [ka]

[0318] A solution of tert-butyl 2-bromopropionate (255 g, 1.22 mol) and triphenylphosphine (320 g, 1.22 mol) in anhydrous acetonitrile (1 L) was stirred at room temperature for 18 hours. The acetonitrile was removed under reduced pressure, and toluene was added to precipitate a white solid. After adding the toluene, the white solid was dissolved in dichloromethane (1 L) and transferred to a separatory funnel. When 10% NaOH aqueous solution (1 L) was added, the organic phase immediately turned yellow after shaking. The organic phase was separated, and the aqueous phase was back-extracted once with dichloromethane (1 L). The dichloromethane phase was combined, washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain ylide 26 as a yellow solid (280 g, 58%).

[0319] Example 27; Synthesis of Compound 27 [ka]

[0320] To a 3 L solution of dry dichloromethane containing compound 25 (450 g, 1.27 mol), ylide 26 (546 g, 1.40 mmol) was added and the mixture was stirred overnight at room temperature. After confirming the completion of the reaction by TLC, the compound was purified by column chromatography (10-50% ethyl acetate / petroleum ether) to obtain compound 27 as a white solid (444 g, 75% yield). ESI m / z C 28 H 38 NO5 [M+H] + Calculated value: 468.27, Measured value: 468.22.

[0321] Example 28; Synthesis of Compound 28 [ka]

[0322] 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 overnight at room temperature under hydrogen (1 atm). After removing the catalyst by filtration, the filtrate was concentrated to obtain compound 28 (45.8 g, 93% yield).

[0323] Example 29; Synthesis of Compound 29 [ka]

[0324] At room temperature, tert-butyl nitrite (1.06 kg, 10.3 mol) was added to a tetrahydrofuran solution (4 L) of compound 28 (390 g, 1.03 mol). After stirring overnight, the tetrahydrofuran was removed by concentration under reduced pressure. The residue was purified by column chromatography (10-50% ethyl acetate / petroleum ether) to obtain compound 29 as a bright yellow solid (314 g, 72% yield).

[0325] Example 30; Synthesis of Compound 30 [ka]

[0326] Under nitrogen protection, 16 g of Pd / C (10 wt%) was added to a 500 mL ethyl acetate solution of compound 30 (166 g, 0.392 mol). The reaction flask was filled with hydrogen gas and the vacuum was changed three times. The reaction solution was stirred at room temperature under hydrogen (1 atm) for 16 hours. The mixture was filtered through Celite and concentrated to obtain compound 30 as a yellow foamy solid (146 g, 97% yield). 1H NMR (400MHz, CDCl3) 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 value: 394.25, Measured value: 395.25.

[0327] Example 31; Synthesis of Compound 31 [ka]

[0328] HATU (39.9 g, 105 mmol) was added to a 300 mL DMF solution of 4-(((benzyloxy)carbonyl)amino)butyric acid (26.1 g, 110 mmol). After stirring at room temperature for 30 minutes, the reaction mixture was added to compound 30 (39.4 g, 100 mmol) and triethylamine (20.2 g, 200 mmol) in 300 mL DMF. The reaction mixture was stirred at room temperature for 2 hours, diluted with water, and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over sodium sulfate. After concentration, it was purified by column chromatography (20-70% ethyl acetate / petroleum ether) to obtain a white solid (45 g, yield 73%). ESI m / z C 33 H 48 N3O8[M+H] + Calculated value: 614.34, Measured value: 614.15.

[0329] Example 32; Synthesis of Compound 32 [ka]

[0330] Compound 31 (100 g, 163 mmol) was dissolved in methanol (500 mL), and a Pd / C catalyst (10 wt%, 10 g) was added. Hydrogenation was carried out overnight at room temperature (1 atm, H2). After filtering off the catalyst, the filtrate was concentrated under reduced pressure to obtain a brown, foamy solid 32 (75.8 g, 97% yield). 1 H NMR (400MHz, CDCl3) 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] + Calculated value: 480.30, Measured value: 480.59.

[0331] Example 33; Synthesis of Compound 33 [ka]

[0332] At 0°C, triethylamine (66 mL, 474 mmol) and HATU (72 g, 190 mmol) were sequentially added to a 500 mL DMF solution of compound 22 (130 g, 174 mmol). The reaction mixture was then warmed to room temperature and stirred for 2 hours. At 0°C, compound 32 (75.8 g, 158 mmol) in a 500 mL DMF solution was added to the above solution, and the reaction mixture was stirred at room temperature for 1 hour. The reaction solution was poured into water (4 L), extracted with ethyl acetate (3 × 500 mL), the organic layers were combined, washed with saturated brine (2 L), dried over sodium sulfate, and concentrated. Crude product 33 (190 g) was used directly in the next reaction. ESI m / z C 60 H 100N5O 20 [M+H] + Calculated value: 1210.69, Measured value: 1210.69.

[0333] Example 34; Synthesis of Compound 34 [ka]

[0334] The crude product 33 (190 g) obtained in the previous step was dissolved in methanol (900 mL), and Pd / C catalyst (10 wt%, 19 g) was added. Hydrogenation was carried out overnight at room temperature (1 atm, H2). The catalyst was filtered off, the filtrate was concentrated under reduced pressure, and purified by SiO2 column (0-10% methanol / dichloromethane gradient) to obtain a brown oily substance (105 g, 2-step yield 62%). ESI m / z C 52 H 95 N5O 18 [M+H] + Calculated value: 1077.65, Measured value: 1077.65.

[0335] Example 35; Synthesis of Compound 35 [ka]

[0336] At room temperature, 4-maleimidobutyrate-N-succinimidyl (54.4 g, 194.2 mmol) and 0.1N NaH2PO4 solution (1.1 L) were added to a 5.3 L EtOH solution of compound 34 (105 g, 97.1 mmol). The reaction mixture was stirred overnight at room temperature. EtOH was removed by distillation under reduced pressure, the residue was poured into water (3 L), then extracted with ethyl acetate (4 × 500 mL), the organic phases were combined, washed with brine (2 L), dried over sodium sulfate, concentrated, and the crude product was purified by SiO2 column (0-10% methanol / dichloromethane) to obtain a yellow oil (100 g, yield 83%). 1H NMR (500MHz, DMSO) δ 9.53 (s, 0.7H), 9.52 (s, 0.3H), 9.22 (s, 0.7H), 9.21 (s, 0.3H), 7.95-7.87 (m, 2H), 7.65 (t, J = 5.9 Hz, 1H), 7.51-7.44 (m, 1H), 6.99 (s, 2H), 6.77-6.66 (m, 2H), 6.65-6.57 (m, 1H), 4.13 (dt, J = 5.4, 8.1 Hz, 1H), 3.84 (s, 2H), 3.55 (s, 2H), 3.52 (s, 2H), 3.51-3.45 (m, 30H), 3.42 (dd, J = 5.8, 3.7 Hz, 2H), 3.38 (t, J = 6.9 Hz, 2H), 3.23 (s, 3H), 3.14-3.01 (m, 4H), 2.64-2.44 (m, 1H), 2.41-2.22 (m, 4H), 2.16-2.04 (m, 2H), 1.76-1.64 (m, 4H), 1.64-1.52 (m, 2H), 1.52-1.35 (m, 2H), 1.37 (s, 3H), 1.35 (s, 6H), 1.31 (s, 9H), 0.97 (t, J = 8.5 Hz, 3H)。 13C NMR (126MHz, 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 value: 1241.7, Measured value: 1241.8.

[0337] Example 36; Synthesis of Compound 36 [ka]

[0338] Compound 35 (31.5 g, 25.4 mmol) was dissolved in dichloromethane (125 mL), and then trifluoroacetic acid (125 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated in a rotary evaporator until the solvent stopped evaporating. Then, it was concentrated in a vacuum oil pump until the weight basically stopped changing to obtain the crude product. The crude product was extracted and washed with ether (200 mL), and the product layer was separated and concentrated under reduced pressure until the solvent was gone. Then, this was concentrated in a vacuum oil pump until its weight basically stopped changing to obtain compound 36 (36.0 g, including solvent). 1H NMR (500MHz, 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)。 13C NMR (126MHz, 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 value: 1085.6, Measured value: 1085.4.

[0339] Example 37; Synthesis of Compound 37 [ka]

[0340] A 60 mL DMF solution of compound 36 (36.0 g, 25.4 mmol) was added to the reaction flask and cooled to 5°C in an ice bath. A 150 mL DMF solution of compound 18 (19.3 g, 27.9 mmol) was added, followed by the dropwise addition of DIPEA (25 mL, 139 mmol). After the addition was complete, the ice bath was removed, the mixture was warmed to room temperature, and stirred for 18 hours. The reaction mixture was concentrated using a vacuum oil pump until no more solvent evaporated. After concentration, the concentrate was diluted with dichloromethane, cooled to 5°C in an ice bath, and formic acid was slowly added dropwise to adjust the pH to 3.0-4.0. The mixture was then concentrated until no more solvent evaporated, the residue was transferred to a silica gel column, and purified by elution with n-hexane / ethyl acetate / formic acid and dichloromethane / methanol / formic acid. The fraction was concentrated to obtain the crude product. The purified crude product was dissolved in water / methanol / formic acid and further purified by preparative HPLC elution with water / acetonitrile / formic acid. The fraction was concentrated, diluted with water, and transferred evenly to a lyophilization flask. After lyophilization, a pale yellow foamy solid was obtained (24 g, 60% yield). 1H NMR (500MHz, 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] + Calculated value: 1593.9, Measured value: 1593.8.

[0341] Example 38; Synthesis of Compound 38 [ka]

[0342] Under a nitrogen atmosphere at approximately -70°C, n-butyllithium (2.5 M in n-hexane, 1.36 L, 3.4 mol, 1.1 equivalent) was added dropwise to an anhydrous tetrahydrofuran solution (8 L) of (S)-4-isopropyloxazolidine-2-one (400 g, 3.09 mol, 1.0 equivalent) in a reaction flask. After the addition was complete, the mixture was stirred at -70°C for 1 hour, and then propionyl chloride (315 g, 3.4 mol, 1.1 equivalent) was added dropwise. After the addition was complete, the mixture was stirred at -70°C for 1 hour and then slowly warmed to room temperature. The reaction solution was poured into an ice-cold saturated ammonium chloride solution (7 L) and extracted with ethyl acetate (3 × 2 L). The organic phases were combined, washed with water (2 L) and saturated brine (2 L), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (3 kg silica gel, 5:1 petroleum ether / ethyl acetate from pure petroleum ether) to obtain a colorless oil (500 g, yield 87%). MS ESI m / z C9H 16 NO3 [M+H] + Calculated value: 186.10, Measured value: 186.10. 1 H NMR (400MHz, CDCl3) 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).

[0343] Example 39; Synthesis of Compound 39 [ka]

[0344] Under a nitrogen atmosphere, compound 38 (92.6 g, 0.50 mol) was dissolved in anhydrous dichloromethane (1.5 L), the temperature was lowered to -10°C, and diisopropylethylamine (70.5 g, 0.54 mol) and n-Bu2BOTf (1.0 M dichloromethane solution, 500 mL, 0.50 mol) were added dropwise to the reaction flask. The mixture was reacted at 0°C for 1 hour, then cooled to -78°C. A dichloromethane solution of compound 25 (161 g, 0.45 mol) (1 L) was added dropwise to the reaction flask, ensuring that the solution temperature did not exceed -70°C. The reaction was completed in 2 hours, then slowly warmed to room temperature and allowed to react overnight. The following day, phosphate buffer (0.1N, pH 7.0, 2L) was added to the reaction flask, the phases were separated, the aqueous phase was extracted with dichloromethane (2 × 500 mL), the organic phase was combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in methanol (2 L), cooled to 0°C, H2O2 (30% aqueous solution, 500 mL) was added dropwise, reacted at 5°C for 1 hour, water (3 L) was added, extracted with dichloromethane (3 × 800 mL), the organic phase was combined, washed with water (500 mL), washed with saturated NaHCO3 solution (500 mL) and saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, concentrated, purified by silica gel column (from pure petroleum ether to 5:1 petroleum ether / ethyl acetate) to obtain 150 g of white solid in 60% yield. 1H NMR (400MHz, CDCl3) 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 C30H41N2O7 [M+H]+: Calculated value 541.28, Actual value: 541.30.

[0345] Example 40; Synthesis of Compound 40 [ka]

[0346] Under a nitrogen atmosphere, compound 39 (200 g, 0.37 mol) was dissolved in anhydrous tetrahydrofuran (3.5 L), dithiocarbonylimidazole (198 g, 1.11 mol) was added, and the mixture was refluxed for 8 hours. Dithiocarbonylimidazole (65 g, 0.37 mol) was added again, and the mixture was reacted overnight. The next day, the reaction mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and purified by silica gel column (from pure petroleum ether to 5:1 petroleum ether / ethyl acetate) to obtain an oily liquid (170 g, yield 83%). 1H NMR (400MHz, CDCl3) 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 value: 651.27, Measured value: 651.39.

[0347] Example 41; Synthesis of Compound 41 [ka]

[0348] Under a nitrogen atmosphere, compound 40 (210 g, 323 mmol) was dissolved in anhydrous toluene (3 L), and under a nitrogen atmosphere, tri-n-butyl stannane (182 g, 646 mmol) and azobisisobutyronitrile (0.5 g, 3.23 mmol) were added sequentially. The mixture was refluxed for 2.5 hours, cooled to room temperature, then concentrated, and purified by silica gel column (from pure petroleum ether to 5:1 petroleum ether / ethyl acetate) to obtain an oily substance (141 g, yield 83%). 1H NMR (400MHz, CDCl3) 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 value: 525.28, measured value: 525.37.

[0349] Example 42; Synthesis of compound 42

change

[0350] Compound 41 (208 g, 390 mmol) was dissolved in a mixture of tetrahydrofuran (2.1 L) and water (0.7 L), and LiOH (23.7 g, 0.99 mol) in H2O2 (30% aqueous solution, 336 mL) was added dropwise. After 3 hours of reaction, the temperature was controlled not to exceed 5°C, and a sodium sulfite (1.5 M, 2 L) solution was added dropwise to adjust the pH to 4 with 2N HCl. The mixture was extracted with ethyl acetate (3 × 800 mL), the organic phases were combined, washed once with water (500 mL) and saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column (from pure petroleum ether to 3:1 petroleum ether / ethyl acetate) to obtain an oily substance (158 g, yield 96%). 1 H NMR (400MHz, CDCl3) 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 value: 414.22, Measured value: 414.21.

[0351] Example 43; Synthesis of Compound 43 [ka]

[0352] Compound 42 (158 g, 0.38 mmol) was dissolved in methanol (1.5 L), Pd / C (10 wt%, 15 g) was added, and the mixture was subjected to catalytic hydrogenation (1 atm H2) for 16 hours. The mixture was then filtered through Celite. The filtrate was concentrated to obtain an oily substance (123 g, 100% yield). 1H NMR (400MHz, CDCl3) δ 7.00 (d, J = 7.5 Hz, 2H), 6.80 (s, 2H), 4.51 (d, J = 9.0 Hz, 1H), 3.88 (s, 1H), 2.66 (dd, J = 65.6, 22.6 Hz, 4H), 1.88 (t, J = 12.2 Hz, 1H), 1.42 (s, 9H), 1.14 (d, J = 6.6 Hz, 3H). MS ESI m / z C 17 H 26 NO5 [M+H] + Calculated value: 324.17, Measured value: 324.16.

[0353] Example 44; Synthesis of Compound 44 [ka]

[0354] Compound 43 (113 g, 0.35 mol) was dissolved in anhydrous tetrahydrofuran (1.5 L), and tert-butyl nitrite (360 g, 3.5 mol) was added dropwise. The mixture was reacted at room temperature for 3 hours. After the reaction was complete, the reaction product was concentrated and purified by silica gel column (from pure petroleum ether to 2:1 petroleum ether / ethyl acetate) to obtain a yellow solid (85 g, 61% yield). 1 H NMR (400MHz, 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 value: 369.15, Measured value: 369.14.

[0355] Example 45; Synthesis of Compound 45 [ka]

[0356] Compound 44 (80 g, 217 mmol) was dissolved in methanol (500 mL), Pd / C (10 wt%, 2.0 g) was added, and the mixture was reacted by catalytic hydrogenation (1 atm, H2) for 1 hour. The mixture was filtered through Celite. The filtrate was concentrated and dried to obtain a white solid (73 g, yield 93%). MS ESI m / z C 17 H 27 N2O5[M+H] + Calculated value: 339.18, Measured value: 339.17. 1 H NMR (400MHz, 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).

[0357] Example 46; Synthesis of Compound 46 [ka]

[0358] Octaethylene glycol monomethyl ether (115.2 g, 0.3 mol) was dissolved in dry tetrahydrofuran (3 L), sodium hydride (60 wt%, 24 g, 0.6 mol) was added at room temperature, and the mixture was stirred and reacted for 1 hour. Then tert-butyl bromoacetate (146.3 g, 0.75 mol) was added. The mixture was reacted at room temperature for 1 hour, and the reaction solution was added to 4 L of dichloromethane. 2 kg of crushed ice was added while stirring, and the aqueous phase was separated and extracted with 1 L of dichloromethane. The organic phases were combined, washed with water, concentrated, and purified by column chromatography (20% ethyl acetate / petroleum ether, then 0-5% methanol / dichloromethane) to obtain 108 g of product (yield 72%).

[0359] Example 47; Synthesis of Compound 47 [ka]

[0360] Compound 46 (210 g, 0.422 mol) was added to a mixed solvent of formic anhydride (1 L) and dichloromethane (500 mL). The mixture was stirred overnight at room temperature and then concentrated to obtain 200 g of product (100% yield).

[0361] Example 48; Synthesis of Compound 48 [ka]

[0362] Compound 48 (198 g, 0.422 mol) was dissolved in 2.6 L of dichloromethane, and then 0.5 mL of DMF and oxalyl chloride (275 mL) were added dropwise at room temperature. The reaction mixture was stirred for 3 hours and then concentrated to obtain 210 g of product.

[0363] Example 49; Synthesis of Compound 49 [ka]

[0364] 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) were mixed in 1.6 L of water and cooled to 0°C in an ice salt bath. A solution of compound 48 (210 g, crude product, 0.422 mol) in tetrahydrofuran (160 mL) was added dropwise, and the mixture was reacted at room temperature for 1 hour. 1 L of ethyl acetate was added, and after stirring, the aqueous phase was separated. The pH was adjusted to 3-4 with concentrated hydrochloric acid, extracted with dichloromethane, and the organic phase was washed with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 290 g of product (yield 97%).

[0365] Example 50; Synthesis of Compound 50 [ka]

[0366] Compound 49 (182.5 g, 0.26 mol) was dissolved in 2 L of dichloromethane, and pentafluorophenol (95.4 g, 0.52 mol) and DIC (131 g, 1.04 mol) were added at room temperature. The reaction mixture was stirred for 1 hour and concentrated to obtain 430 g of crude product.

[0367] Example 51; Synthesis of Compound 51 [ka]

[0368] 62 g, 0.39 mol of tert-butyl 4-aminobutyrate was dissolved in 1.5 L of DMF, cooled with ice water, and DIPEA (134.2 g, 1.04 mol) was added. Compound 50 (430 g, 0.26 mol, crude product) was then slowly added at 10°C to 20°C. The reaction mixture was stirred at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and extracted with dichloromethane. The organic phases were combined, washed with 0.2 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (25% to 100% ethyl acetate / petroleum ether, then 0% to 5% methanol / dichloromethane) to obtain 180 g of product in 82% yield.

[0369] Example 52; Synthesis of Compound 52 [ka]

[0370] Compound 51 (78 g, 92.3 mmol) and palladium carbon (10 wt%, 13 g) were mixed in 500 mL of methanol. The mixture was stirred overnight at room temperature under a hydrogen balloon. The reaction product was filtered, the filtrate was concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to obtain 70.2 g of product (yield 92%).

[0371] Example 53; Synthesis of Compound 53 [ka]

[0372] Compound 52 (17.3 g, 94.2 mmol) was dissolved in 500 mL of dichloromethane, and pentafluorophenol (34.7 g, 188.5 mmol) and DIC (47.5 g, 377 mmol) were added sequentially at room temperature. The reaction mixture was stirred for 1 hour and concentrated to obtain 105 g of crude product.

[0373] Example 54; Synthesis of Compound 54 [ka]

[0374] 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. At 10°C to 20°C, compound 53 (105 g, crude product, 94.2 mmol) was added to the reaction solution. The reaction mixture was allowed to react at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with 0.2 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (50% to 100% ethyl acetate / petroleum ether, followed by 10% methanol / dichloromethane) to obtain 80.5 g of product, with a yield of 98%.

[0375] Example 55; Synthesis of Compound 55 [ka]

[0376] Compound 54 (80.5 g, 91.9 mmol) was mixed with formic anhydride (400 mL) and dichloromethane (200 mL). The reaction mixture was stirred overnight at room temperature, concentrated, diluted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (0%-20% methanol / dichloromethane) to obtain 70 g of product (yield 93%).

[0377] Example 56; Synthesis of Compound 56 [ka]

[0378] 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. After reacting at room temperature for 1 hour, the reaction mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 120 g of product (100% yield).

[0379] Example 57; Synthesis of Compound 57 [ka]

[0380] Compound 56 (120 g, 127 mmol) and Compound 45 (45.0 g, 133 mmol) were mixed in 1 L of tetrahydrofuran, heated, refluxed overnight, 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, stirred for 30 minutes, the aqueous phase was separated, the ethyl acetate layer was extracted with water (600 mL x 2), the aqueous phases were combined, and then washed with ethyl acetate (700 mL) to remove impurities. Sodium chloride was added to the aqueous phase to saturate it, extracted with dichloromethane (1 L x 2), the dichloromethane layer was combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0%~20% methanol / dichloromethane) to obtain 83.6 g of product (yield 58%). 1H NMR (600MHz, DMSO) δ 9.51 (bs, 1H), 9.23 (s, 1H), 7.97-7.83 (m, 1H), 7.64 (t, J = 5.8 Hz, 1H), 7.46 (s, 1H), 6.99 (s, 1H), 6.73 (s, 1H), 6.61 (d, J = 8.7 Hz, 0H), 4.14 (dt, J = 5.4, 8.3 Hz, 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, 3.8 Hz, 2H), 3.39 (t, J = 7.0 Hz, 2H), 3.23 (s, 3H), 3.14-3.01 (m, 4H), 2.54 (dd, J = 13.5, 7.0 Hz, 1H), 2.45 (dd, J = 13.5, 7.0 Hz, 1H), 2.41-2.32 (m, 3H), 2.11 (td, J = 7.1, 1.9 Hz, 2H), 1.75-1.64 (m, 4H), 1.64-1.54 (m, 1H), 1.52-1.43 (m, 1H), 1.43-1.35 (m, 2H), 1.32 (s, 9H), 1.32-1.15 (m, 4H), 1.02 (d, J = 7.1 Hz, 3H)。 13 C NMR (151MHz, DMSO) δ 177.18, 171.68, 171.43, 171.28, 171.08, 169.02, 155.15, 145.99, 134.45, 129.41, 125.91, 125.30, 123.04, 115.71, 77.29, 71.29, 70.25, 69.95, 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.

[0381] Example 58; Synthesis of Compound 58 [ka]

[0382] Compound 57 (59.0 g, 51.7 mmol) was dissolved in dichloromethane (345 mL), and trifluoroacetic acid (172 mL) was added. After addition, the reaction was carried out at room temperature for 2 hours. At the end of the reaction, the reaction solution was concentrated in a rotary evaporator until the solvent stopped evaporating. Then, it was concentrated with a vacuum oil pump until the weight remained virtually unchanged to obtain the crude product. The crude product was extracted and washed with ether (600 mL), the product layer was separated, and concentrated under reduced pressure until the solvent was gone. Next, it was concentrated on a vacuum oil pump until its weight remained virtually unchanged to obtain compound 58 (75.5 g, including solvent). 1H NMR (600MHz, DMSO) δ 9.82 (s, 1H), 9.18 (s, 1H), 7.95-7.88 (m, 2H), 7.83 (s, 2H), 7.67 (s, 1H), 7.66 (t, J = 6.1 Hz, 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), 3.42 (dd, J = 5.6, 3.9 Hz, 2H), 3.39 (t, J = 7.0 Hz, 2H), 3.33-3.25 (m, 1H), 3.23 (s, 3H), 3.14-3.02 (m, 4H), 2.76 (dd, J = 13.9, 6.1 Hz, 1H), 2.65 (dd, J = 13.9, 6.1 Hz, 1H), 2.55 (dq, J = 14.0, 7.0 Hz, 1H), 2.38 (t, J = 7.3 Hz, 2H), 2.14-2.08 (m, 2H), 1.82 (ddd, J = 14.1, 8.7, 5.5 Hz, 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 = 7.0 Hz, 3H)。 13 C NMR (151MHz, DMSO) δ 176.54, 171.73, 171.43, 171.34, 171.11, 169.07, 146.59, 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, 36.85, 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.

[0383] Example 59; Synthesis of Compound 59 [ka]

[0384] A 160 mL DMF solution of compound 58 (75.5 g, 51.7 mmol) was added to the reaction flask and cooled to 5°C in an ice bath. A 240 mL DMF solution of compound 18 (35.8 g, 51.7 mmol) was added, followed by the dropwise addition of DIPEA (36.8 g, 285 mmol). After the addition was complete, the ice bath was removed, the mixture was warmed to room temperature, and stirred for 10 hours. After the reaction, the reaction mixture was concentrated using a vacuum oil pump until no more solvent evaporated. After concentration, the residue was diluted with dichloromethane, cooled to 5°C in an ice bath, and formic acid was slowly added dropwise to adjust the pH to 3.0-4.0. The mixture was then concentrated using a rotary evaporator until no more solvent evaporated, the residue was transferred to a silica gel column, and eluted with n-hexane / ethyl acetate / formic acid and dichloromethane / methanol / formic acid to obtain the purified crude product. The crude product was dissolved in water / methanol / formic acid, further purified by preparative HPLC, eluted with water / acetonitrile / formic acid, the fraction was collected, concentrated, diluted with water, homogeneously distributed, placed in a lyophilization bottle, and lyophilized to obtain a pale yellow foamy solid (48 g, yield 60%). 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 = 8.1 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 5.58 (d, J = 10.7 Hz, 1H), 4.65 (dd, J = 9.3, 7.1 Hz, 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, 6.7 Hz, 1H), 2.63 (dd, J = 13.5, 6.7 Hz, 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 = 6.9 Hz, 3H), 1.01 (s, 3H),0.93 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H), 0.67 (d, J = 6.1 Hz, 3H)。 13C NMR (151MHz, DMSO) δ 177.05, 175.28, 172.76, 171.74, 171.47, 171.34, 171.11, 169.78, 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, 37.43, 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.

[0385] Example 60; Synthesis of Compound 60 [ka]

[0386] Octaethylene glycol monomethyl ether (10 g, 26 mmol, 1.0 equivalent) was dissolved in 100 mL of anhydrous dichloromethane, DMAP (32 mg, 0.26 mmol, 0.01 equivalent) was added, and then triethylamine (10.5 g, 104 mmol, 4.0 equivalent) and TsCl (14.9 g, 78 mmol, 3.0 equivalent) were added dropwise in an ice bath. After stirring for 10 minutes, the reaction mixture was warmed to room temperature and stirred overnight. The reaction solution was washed with 1N HCl (100 mL), water (100 mL), and brine (100 mL), dried over anhydrous sodium sulfate, and concentrated using a rotary evaporator. The residue was dissolved in a small amount of dichloromethane, loaded onto a column, and eluted with 5%-100% petroleum ether / ethyl acetate and 1%-3% methanol / dichloromethane to obtain a yellow oil (11.6 g, yield 83%). ESI m / z C24H43O11S [M+H] + Calculated value: 539.2, Measured value: 539.2.

[0387] Example 61; Synthesis of Compound 61 [ka]

[0388] Compound 60 (11.6 g, 21.5 mmol, 1.0 equivalent) was dissolved in 20 mL of anhydrous DMF, dibenzylamine (5.5 g, 27.8 mmol, 1.5 equivalents) was added, and the mixture was stirred overnight at 100°C. The mixture was then diluted with 300 mL of dichloromethane, washed with water (300 mL x 3) and brine (300 mL), dried over anhydrous sodium sulfate, and concentrated using a rotary evaporator. The residue was dissolved in a small amount of dichloromethane, loaded onto a column, and eluted with 5%-100% petroleum ether / ethyl acetate to obtain a pale yellow oil (8.2 g, yield 66%). ESI m / z C31H50NO8 [M+H] + Calculated value: 564.3, Measured value: 564.3.

[0389] Example 62; Synthesis of Compound 62 [ka]

[0390] To a 100 mL solution of compound 61 (8.6 g, 15.2 mmol, 1.0 equivalent) in anhydrous methanol, a dry palladium-carbon catalyst (0.9 g, 10 wt%) was added and heated under a hydrogen atmosphere under reflux, with stirring overnight. The reaction mixture was filtered, washed with methanol, and concentrated using a rotary evaporator to obtain a colorless oil (5.3 g, 90% yield). ESI m / z C17H38NO8 [M+H] + Calculated value: 384.3, Measured value: 384.3.

[0391] Example 63; Synthesis of Compound 63 [ka]

[0392] At 0°C, HATU (1.2g, 3.15mmol) and DIPEA (1mL, 6.3mmol) were added to a 20mL DMF solution of ZL-tert-butyl glutamate (0.96g, 2.86mmol) and compound 62 (1.1g, 2.86mmol). The reaction mixture was warmed to room temperature, stirred for 1 hour, then poured into ice water, extracted with dichloromethane (3×50mL), combined the organic phases, washed with water (30mL), saturated sodium bicarbonate (30mL), and brine (30mL), dried over sodium sulfate, filtered, concentrated, and purified by silica gel column (mobile phase: methanol / dichloromethane) to obtain product 63 (1.5g, yield 75%). ESI m / z C34H59N2O13 [M+H] + Calculated value: 703.4, Measured value: 703.4.

[0393] Example 64; Synthesis of Compound 64 [ka]

[0394] Compound 63 (0.66 g, 0.93 mmol) was dissolved in methanol (10 mL), Pd / C (10 wt%, 60 mg) was added, and the mixture was reacted under hydrogen balloon conditions for 2 hours. Compound 64 was obtained by filtration and concentration (450 mg, 85% yield). ESI m / z C 26 H 53 N2O 11 [M+H] + Calculated value: 569.4, Measured value: 569.4.

[0395] Example 65; Synthesis of Compound 65 [ka]

[0396] Compound 64 (0.45 g, 0.79 mmol) and N-succinimidyl 4-maleimide butyrate (0.33 g, 1.18 mmol) were dissolved in ethanol (5 mL), NaH2PO4 (0.1 N, 1 mL) was added, and the mixture was reacted overnight at room temperature. The mixture was concentrated under reduced pressure to remove most of the ethanol, 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) and brine (20 mL), dried over sodium sulfate, filtered, and concentrated. The crude product was purified using a silica gel column (mobile phase: methanol / dichloromethane) to obtain compound 65 (380 mg, yield 65%). ESI m / z C 34 H 60 N3O 14 [M+H] + Calculated value: 734.4, Measured value: 734.4.

[0397] Example 66; Synthesis of Compound 66 [ka]

[0398] Compound 65 (230 mg, 0.31 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, and the mixture was reacted at room temperature for 1 hour. After concentration, the mixture was stripped three times with dichloromethane and drained using an oil pump to obtain 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.

[0399] Example 67; Synthesis of Compound 67 [ka]

[0400] Compound 66 (208 mg, 0.3 mmol) was dissolved in dichloromethane (5 mL), pentafluorophenol (113 mg, 0.6 mmol) and EDC·HCl (117 mg, 0.6 mmol) were added, and the mixture was reacted overnight at room temperature. The mixture was diluted with dichloromethane (20 mL), washed with water (5 mL), dried over sodium sulfate, filtered, and concentrated to obtain 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.

[0401] Example 68; Synthesis of Compound 68 [ka]

[0402] Compound 45 (7.3 g, 21.7 mmol) and N-Boc-alanine hydroxysuccinimide (7.2 g, 21.7 mmol) were dissolved in ethanol (300 mL), 0.1 N NaH2PO4 (150 mL) was added, and the mixture was reacted overnight at room temperature. The reaction solution was concentrated, water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated, and purified by silica gel column (mobile phase: ethyl acetate / petroleum ether) to obtain compound 68 (6.7 g, yield 55%). ESI m / z C 29 H 40 N3O8[M+H] + Calculated value: 558.3, Measured value: 558.3.

[0403] Example 69; Synthesis of Compound 69 [ka]

[0404] Compound 68 (6.7 g, 12 mmol) was dissolved in methanol (100 mL), Pd / C (10 wt%, 0.67 g) was added, and the mixture was reacted under hydrogen balloon conditions for 2 hours. After filtration, the filtrate was concentrated to obtain compound 69 (5 g, 100% yield). ESI m / z C 21 H 34 N3O6[M+H] + Calculated value: 424.2, Measured value: 424.2.

[0405] Example 70; Synthesis of Compound 70 [ka]

[0406] 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, DIPEA (0.13 mL, 0.75 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 1 hour. The mixture was diluted with water (20 mL), extracted with dichloromethane (3 × 10 mL), the organic phases were combined, washed with water (10 mL), 1N HCl (10 mL), and brine (10 mL), and dried over sodium sulfate. After concentration, the crude product was purified by silica gel column chromatography (mobile phase: methanol / dichloromethane) to obtain compound 70 (180 mg, yield 55%). ESI m / z C51H83N6O19 [M+H]+: Calculated value: 1083.6, Measured value: 1083.6.

[0407] Example 71; Synthesis of Compound 71 [ka]

[0408] Compound 70 (8.2 g, 7.6 mmol) was dissolved in dichloromethane (56.8 mL), trifluoroacetic acid (18.9 mL) was added, and the reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated, evaporated twice with dichloromethane, and drained with an oil pump. Diethyl ether (100 mL) was added to the residue, the mixture was stirred vigorously for 1 hour, allowed to stand, the supernatant was discarded, and this process was repeated twice. The lower liquid was concentrated using a rotary evaporator and drained with an oil pump to obtain compound 71 (9.2 g, yield >100%). ESI m / z C 46 H 75 N6O 17 [M+H] + Calculated value: 983.51, Measured value: 983.37.

[0409] Example 72; Synthesis of Compound 72 [ka]

[0410] Compound 71 (8.2 g, 7.6 mmol) was dissolved in DMF (80 mL), and the reaction flask was cooled to 0–5°C in an ice bath. A DMF solution of Compound 18 (5.2 g, 7.6 mmol) (20 mL) was added, and then DIPEA (4 mL, 22.8 mmol) was slowly added dropwise to the reaction flask. The dropping rate was controlled to maintain the temperature of the reaction mixture at 5–10°C throughout the entire dropping process. After the addition was complete, the ice bath was removed, the reaction mixture was warmed to room temperature, and stirred for 1.5 hours. The reaction mixture was concentrated, dichloromethane (100 mL) was added, and formic acid was added dropwise under ice bath to adjust the pH to 3–4. The mixture was concentrated again and purified by silica gel column elution with 20–100% ethyl acetate / n-hexane and 0–20% methanol / dichloromethane (each containing 0.1% formic acid). Next, the crude product (11.44 g) obtained by silica gel column purification was purified by preparative HPLC using 20-30% acetonitrile / water (each containing 0.1% formic acid) as the eluent. The fraction was concentrated and freeze-dried to obtain compound 72 (6.8 g, 60% yield). ESI m / z C 71 H 115 N 10 O 22 S [M+H] + Calculated value: 1491.78, Measured value: 1492.01.

[0411] Example 73; Synthesis of Compound 73 [ka]

[0412] Compound 63 (22.4 g, 0.03 mol) was added to a mixed solvent of formic anhydride (500 mL) and dichloromethane (250 mL). The mixture was stirred overnight at room temperature, then concentrated to obtain 19 g of product (100% yield).

[0413] Example 74; Synthesis of Compound 74 [ka]

[0414] Compound 73 (19.0 g, 0.03 mol) was dissolved in dichloromethane (200 mL), and pentafluorophenol (11.0 g, 0.06 mol) and DIC (15.1 g, 0.12 mol) were added at room temperature. The reaction mixture was stirred for 1 hour and concentrated to obtain 45 g of crude product.

[0415] Example 75; Synthesis of Compound 75 [ka]

[0416] 6.40 g, 0.04 mol of tert-butyl 4-aminobutyrate was dissolved in 500 mL of DMF, cooled with ice water, and DIPEA (15.5 g, 0.12 mol) was added. Then, compound 74 (45 g, 0.03 mol, crude product) was slowly added at a temperature of 10°C to 20°C. The mixture was reacted at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with 0.2 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was purified by column chromatography (25%-100% ethyl acetate / petroleum ether, then 0-5% methanol / dichloromethane) to obtain 19.5 g of product in 83% yield.

[0417] Example 76; Synthesis of Compound 76 [ka]

[0418] Compound 75 (19.5 g, 24.8 mmol) and palladium carbon (10 wt%, 5 g) were mixed in 200 mL of methanol. The mixture was reacted overnight at room temperature under hydrogen balloon conditions. After filtering and concentrating the filtrate, 16.7 g of product was obtained (100% yield).

[0419] Example 77; Synthesis of Compound 77 [ka]

[0420] 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 solution was maintained at 10°C to 20°C, and compound 53 (30 g, crude product, 24.8 mmol) was slowly added. The mixture was reacted at room temperature for 1 hour, concentrated, diluted with dichloromethane, washed with water, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with 0.2 N hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The solution was purified by column chromatography (50%-100% ethyl acetate / petroleum ether, then 10% methanol / dichloromethane) to obtain 20 g of product in 98% yield.

[0421] Example 78; Synthesis of Compound 78 [ka]

[0422] Compound 77 (16.8 g, 20.5 mmol) was dissolved in dichloromethane (60 mL), and formic anhydride (120 mL) was added. The mixture was reacted overnight at room temperature, concentrated, diluted with ethyl acetate (150 mL), and extracted with water (300 mL). The organic phase was discarded, solid sodium chloride was added to the aqueous phase to saturate it, and then extracted with dichloromethane (200 ml x 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to obtain 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.

[0423] Example 79; Synthesis of Compound 79 [ka]

[0424] At room temperature, compound 78 (15.6 g, 20.5 mol) was dissolved in dichloromethane (200 mL), and N-hydroxysuccinimide (NHS, 3.7 g, 32.3 mol) and EDC·HCl (8.3 g, 43 mol) were added sequentially. The mixture was reacted at room temperature for 30 minutes, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 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.

[0425] Example 80; Synthesis of Compound 80 [ka]

[0426] Method 1: 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 under reflux, stirred overnight, 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 to saturate it, and it was extracted with dichloromethane (2 × 150 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to obtain Compound 80 (4.0 g, yield 36%). ESI m / z C 51 H 83 O 19 N6[M+H] + Calculated value: 1083.56, Measured value: 1083.47.

[0427] Method 2: Compound 79 (4.4 g, 5.12 mmol) and Compound 45 (1.73 g, 5.12 mmol) were dissolved in 100 mL of EtOH. Next, 20 mL of 0.1 N NaH2PO4 solution was added, and the mixture was stirred overnight. After concentration, water (200 mL) and ethyl acetate (100 mL) were added to the crude product, and the mixture was stirred and the aqueous phase was separated. Sodium chloride was added to the aqueous phase to saturate it, and it was extracted with dichloromethane (2 × 100 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to obtain Compound 80 (2.0 g, yield 36%).

[0428] Method 3: Compound 79 (4.4 g, 5.12 mmol) and Compound 45 (1.73 g, 5.12 mmol) were dissolved in 100 mL of acetonitrile. Next, 20 mL of 0.1 N NaH2PO4 solution was added, and the mixture was stirred overnight. After concentration, water (200 mL) and ethyl acetate (100 mL) were added to the crude product, and the mixture was stirred and the aqueous phase was separated. Sodium chloride was added to the aqueous phase to saturate it, and it was extracted with dichloromethane (2 × 100 mL). The dichloromethane layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (0-20% methanol / dichloromethane) to obtain Compound 80 (2.2 g, yield 40%).

[0429] Example 81; Synthesis of Compound 73 [ka]

[0430] Compound 49 (20 g, 28.4 mmol, 1.0 equivalent) was dissolved in 350 mL of anhydrous dichloromethane and cooled in an ice bath. NHS (3.9 g, 34.1 mmol, 1.2 equivalents) and EDC (27 g, 142.0 mmol, 5.0 equivalents) were added in sequence. The reaction mixture was stirred overnight at room temperature, then washed with water (200 mL x 2) and brine (200 mL x 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 methanol / ethyl acetate / dichloromethane in a 2:49:49 to 4:48:48 ratio to obtain the product as 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.

[0431] Example 82; Synthesis of Compound 74 [ka]

[0432] Compound 69 (6.4 g, 15.1 mmol, 1.0 equivalent) was added to a mixture of 40 mL of ethanol and 10 mL of 0.1 MNaH2PO4, to which compound 73 (12.7 g, 15.9 mmol, 1.05 equivalent) was added. The reaction mixture was stirred overnight, concentrated, dissolved in dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column (3-5% methanol / dichloromethane) to obtain a white foamy substance (11.7 g, yield 70%). ESI m / z C 54 H 88 N5O 19 [M+H] + Calculated value, ■■■: 1110.6, Measured value: 1110.6.

[0433] Example 83; Synthesis of Compound 75 [ka]

[0434] Compound 74 (4.2 g, 3.79 mmol, 1.0 equivalent) and palladium carbon (0.4 g, 10 wt%) were mixed in 5 mL of methanol. The mixture was stirred overnight at room temperature under a hydrogen balloon, the catalyst was filtered, washed with methanol, and the filtrate was concentrated to obtain 0.32 g of crude product (87% yield), which was used directly in the next step. ESI m / z C46H82N5O17 [M+H] + Calculated value: 1997.1, Measured value: 1997.1.

[0435] Example 84; Synthesis of Compound 76 [ka]

[0436] In a 500 mL flask, H2N-PEG4-CH2CH2CO2H (3.0 g, 11.3 mmol, 1.0 equivalent) and K2CO3 (4.7 g, 33.93 mmol, 3.0 equivalent) were dissolved in 50 mL of water, cooled in an ice bath, and 50 mL of a tetrahydrofuran solution of Boc2O (3.2 g, 14.7 mmol, 1.3 equivalents) 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 1N KHSO4, extracted with dichloromethane (200 mL x 1, 100 mL x 3), washed with water (500 mL x 1) and brine (500 mL x 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, eluted with 2-4% methanol / dichloromethane, and the components were combined and concentrated to obtain 3.8 g of colorless oil (yield 93%). ESI m / z C 16 H 32 NO8 [M+H] + Calculated value: 366.2, Measured value: 366.2.

[0437] Example 85; Synthesis of Compound 77 [ka]

[0438] In a 50 mL single-neck flask, BocHN-PEG4-CH2CH2CO2H (0.81 g, 2.22 mmol, 1.0 equivalent), K2CO3 (0.92 g, 6.66 mmol, 3.0 equivalents), and NaI (0.033 g, 0.222 mmol, 0.1 equivalent) were mixed in 10 mL of DMF and cooled in an ice bath. BnBr (0.57 g, 3.33 mmol, 1.5 equivalents) was added dropwise, 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 x 2), washed with water (200 mL x 1) and brine (200 mL x 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 obtain 0.69 g of colorless oil (69% yield). ESI m / z C 23 H 38 NO8 [M+H] + Calculated value: 446.3, Measured value: 446.3.

[0439] Example 86; Synthesis of Compound 78 [ka]

[0440] BocHN-PEG4-CH2CH2CO2Bn (0.69 g, 1.5 mmol, 1.0 equivalent) in 6 mL of dichloromethane and 3 mL of TFA was stirred at room temperature for 30 minutes. The solvent was removed, and the residue was co-evaporated three times with dichloromethane and placed on a high vacuum pump. The crude product was used directly in the following reaction. ESI m / z C 18 H 30 NO6[M+H] + Calculated value: 356.2, Measured value: 356.2.

[0441] Example 87; Synthesis of Compound 79 [ka]

[0442] To a 50 mL dry dichloromethane solution of BocHN-PEG4-CH2CH2CO2H (3.8 g, 10.4 mmol, 1.0 equivalent), NHS (1.4 g, 12.5 mmol, 1.2 equivalents) and EDC (10.0 g, 52.0 mmol, 5.0 equivalents) were added. The mixture was stirred overnight at room temperature, then washed with water (50 mL x 2) and brine (100 mL x 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, Measured value: 463.2.

[0443] Example 88; Synthesis of Compound 80 [ka]

[0444] In a 300 mL flask, H2N-PEG4-CH2CH2CO2H (2.8 g, 10.4 mmol, 1.0 equivalent) and K2CO3 (4.3 g, 31.2 mmol, 3.0 equivalent) were dissolved in 40 mL of water, cooled in an ice bath, and 40 mL of a tetrahydrofuran solution of compound 79 (3.8 g, 10.4 mmol, 1.0 equivalent) was slowly added dropwise. The mixture was warmed to room temperature and stirred overnight. The reaction mixture was adjusted to pH 4-5 using 1N KHSO4, extracted with dichloromethane (150 mL x 1, 100 mL x 2), washed with water (200 mL x 1) and brine (200 mL x 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 4-6% methanol / dichloromethane to obtain a colorless oil (5.18 g, yield 81%). ESI m / z C 27 H 53 N2O 13 [M+H] + Calculated value: 613.3, Measured value: 613.3.

[0445] Example 89; Synthesis of Compound 81 [ka]

[0446] H2N-PEG4-CH2CH2CO2Bn (crude product from the previous step) was dissolved in 3 mL of DMF, cooled in an ice / water bath, and DIPEA (0.78 g, 6.0 mmol, 4.0 equivalents) was added. Then, compound 80 (0.93 g, 1.5 mmol, 1.0 equivalent) and HATU (1.72 g, 4.5 mmol, 3.0 equivalents) from 7 mL of DMF were added. The reaction mixture was stirred on an ice bath for 2 hours, diluted with 100 mL of water, extracted with dichloromethane (100 mL x 3), washed with 1 N KHSO4 (200 mL x 1), saturated sodium bicarbonate (200 mL x 1), and brine (200 mL x 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 0-5% methanol / dichloromethane. The fractions were combined and concentrated to obtain 1.0 g of pale yellow oil (yield 71%). ESI m / z C 45 H 80 N3O 18 [M+H] + Calculated value: 950.5, Measured value: 950.5.

[0447] Example 90; Synthesis of Compound 82 [ka]

[0448] To a 50 mL DMF 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 phase was washed once with 100 mL of brine, then dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / dichloromethane, 1:15) to obtain the title compound as a colorless oil (5.37 g, yield 88%).

[0449] Example 91; Synthesis of Compound 83 [ka]

[0450] At room temperature, compound 82 (0.64 g, 1.05 mmol, 1.0 equivalent) was stirred for 2 hours in a mixed solution of 5 mL of dichloromethane and 2 mL of TFA, and then concentrated. The residue was co-evaporated three times with dichloromethane and placed under a high vacuum pump. The crude product was redissolved in 3 mL of DMF and cooled in an ice bath. Compound 80 (0.64 g, 1.05 mmol, 1.0 equivalent) in a 7 mL solution of DMF was added, followed by DIPEA (0.54 g, 4.20 mmol, 4.0 equivalents) and HATU (1.2 g, 3.15 mmol, 3.0 equivalents). 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 x 1, 100 mL x 1). The organic phase was washed with 1N KHSO4 (200 mL x 1), saturated sodium bicarbonate (200 mL x 1), and brine (200 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was dissolved in a small amount of dichloromethane, loaded onto a silica gel column, and then eluted with 0-10% methanol / dichloromethane. The fractions were combined and concentrated to obtain 0.94 g of pale yellow oil (yield 81%). ESI m / z C 57 H 92N4O 17 [M+H] + Calculated value: 1104.6, Measured value: 1104.6.

[0451] Example 92; Synthesis of Compound 84 [ka]

[0452] At 0°C, 18 mL of DMF solution (1.03 g, 6.12 mmol) of tert-butyl 4-aminobutyrate and compound 49 (3.91 g, 5.56 mmol) was mixed with HATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol). The reaction mixture was stirred for 1 hour, then diluted with water (300 mL) and extracted with ethyl acetate (3 × 250 mL). The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (32:1 dichloromethane / methanol) to obtain the title compound (5.10 g, 99% yield). ESI MS m / z 846.50 ([M+H] + )

[0453] Example 93; Synthesis of Compound 85 [ka]

[0454] Compound 84 (1.0 g, 1.18 mmol) and Pd / C (10 wt%, 0.10 g) were added to methanol (50 mL) in a hydrogenation bottle. The mixture was shaken for 2 hours, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 85 (0.93 g, yield >100%). ESI MS m / z 712.50 ([M+H] + )

[0455] Example 94; Synthesis of Compound 86 [ka]

[0456] Compound 85 in a 95% EtOH (50 mL) and NaH2PO4 solution (0.1 M, pH 5.0, 10 mL) was mixed with N-succinimidyl 4-maleimide butyrate (0.50 g, 1.77 mmol, 1.5 equivalents), and the mixture was stirred overnight. The mixture was then concentrated, diluted with water (50 mL), extracted with dichloromethane (80 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (25:1 dichloromethane / methanol) to obtain the title compound as a pale yellow oil (0.82 g, 80%).

[0457] Example 95; Synthesis of Compound 87 [ka]

[0458] Compound 86 (0.82 g, 0.94 mmol) was dissolved in HCOOH (50 mL) and stirred at room temperature for 1 hour. The reaction mixture was concentrated and evaporated twice with toluene. The residue was placed in a vacuum pump to obtain compound 87 (0.80 g, crude product). ESI MS m / z ([M+H] + ): 820.45.

[0459] Example 96; Synthesis of Compound 88 [ka]

[0460] To a DMA solution (5.0 mL) of compound 87 (0.80 g, crude product, 0.94 mmol), NHS (0.12 g, 1.03 mmol) and EDC·HCl (0.27 g, 1.41 mmol) were added. The reaction mixture was stirred for 2 hours, diluted with water (15 mL), and extracted with ethyl acetate (3 × 10 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (10-50% ethyl acetate / petroleum ether) to obtain a colorless oily compound (0.67 g, yield 78%). ESI MS m / z ([M+H] + ):918.55

[0461] Example 97; Synthesis of Compound 89 [ka]

[0462] A mixture of N-Boc-ethylenediamine (5.6 mL, 35.4 mmol, 1.1 equivalents) and saturated NaHCO3 (60 mL) was cooled to 0°C, and N-methoxycarbonylmaleimide (5.00 g, 32.2 mmol, 1.0 equivalent) was added in part. After stirring at 0°C for 30 minutes, the reaction mixture was warmed to room temperature and stirred for 1 hour. The precipitate was collected by filtration, washed with cold water, then dissolved in ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated to obtain a white solid (6.69 g, 87% yield). ESI MS m / z ([M+H] + ):241.12.

[0463] Example 98; Synthesis of Compound 90 [ka]

[0464] In a high-pressure tube, compound 89 (6.00 g, 25.0 mmol) and a toluene solution (120 mL) of furan (18.0 mL) were heated under reflux and stirred for 16 hours. After the colorless solution turned yellow during the reaction, the mixture was cooled to room temperature and concentrated. The resulting white solid was triturated with ether to obtain compound 90 (6.5 g, 84% yield). ESI MS m / z([M+H]) + ):309.13.

[0465] Example 99; Synthesis of Compound 91 [ka]

[0466] At room temperature, a 15 mL solution of compound 90 (9.93 g, 32.2 mmol) in dioxane was treated with concentrated HCl (15 mL) for 3 hours. The resulting solid was collected by filtration, the filter cake was washed with ethyl acetate, and the solid was dried overnight in an oven (50°C) to obtain compound 91 (6.94 g, 88% yield). ESI MS m / z([M+H]) + ): 206.05.

[0467] Example 100; Synthesis of Compound 92 [ka]

[0468] At -10°C, POCl3 (0.47 mL, 5 mmol) was added to a tetrahydrofuran solution (10 mL) of compound 91 (1.22 g, 5 mmol). After stirring for 10 minutes, 2,5,8,11,14,17,20,23,26-nonaoxydioctadecane-28-amine (2.14 g, 5 mmol) was added, followed by DIPEA (0.87 mL, 5 mmol). The reaction mixture was warmed to 0°C, stirred for 3 hours, and then concentrated. The residue was diluted with dichloromethane (10 mL), filtered through Celite, and the filtrate was used directly in the next step. ESI MS m / z([M+H] + ): 716.29.

[0469] Example 101; Synthesis of Compound 93 [ka]

[0470] 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 elution with 5-25% ethyl acetate / dichloromethane to obtain the title compound (12.5 g, yield 83%). ESI MS m / z ([M+Na]+) value: 242.42.

[0471] Example 102; Synthesis of Compound 94 [ka]

[0472] To a 350 mL solution of anhydrous pyridine containing compound 93 (12.0 g, 49.56 mmol), 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 obtain the crude product (18.8 g, yield >100%). The crude product can be used in the next step without further purification. ESI MS m / z([M+H] + Value: 376.06

[0473] Example 103; Synthesis of Compound 95 [ka]

[0474] To a toluene solution (200 mL) of maleimide (10.0 g, 103.0 mmol), furan (10.0 mL, 137.4 mmol) was added. The mixture was heated at 100 °C for 8 hours, cooled to room temperature, concentrated, crystallized from ethyl acetate / hexane, and the solid was washed with methanol to obtain 16.7 g (99%) of the title compound. 1 H NMR (CDCl3): 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.

[0475] Example 104; Synthesis of Compound 96 [ka]

[0476] Compound 94 (freshly prepared, 90% purity, 8.5 g, approximately 20 mmol) in DMA solution (350 mL) was mixed with 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). 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, loaded onto a silica gel column, and eluted with 10-30% ethyl acetate / n-hexane to obtain the title compound (7.9 g, 77% yield). ESI MS m / z([M+Na] + Value: 536.4.

[0477] Example 105; Synthesis of Compound 97 [ka]

[0478] Compound 96 (3.0 g, 5.8 mmol) and trimethylstannane (4.8 g, 26.4 mmol) were refluxed at 80°C for 8 hours in 150 mL of 1,2-dichloroethane, and then cooled to room temperature. The residue was passed through a short silica gel column and 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 obtain the title compound (1.62 g, 76% yield). ESI MS m / z([M+Na] + Value: 386.2.

[0479] Example 106; Synthesis of Compound 98 [ka]

[0480] EDC·HCl (0.81 g, 4.20 mmol) was added to a DMA solution (20 mL) of compound 97 (1.62 g, 4.20 mmol) and compound 85 (2.71 g, 3.82 mmol). The reaction mixture was stirred overnight at room temperature, then poured into water (50 mL) and extracted with ethyl acetate (3 × 40 mL). The combined organic phase was 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 obtain a colorless oil (3.20 g, yield 80%). ESI MS m / z([M+H] + Value: 1057.85.

[0481] Example 107; Synthesis of Compound 99 [ka]

[0482] A 10 mL solution of compound 98 (3.20 g, 3.03 mmol) in formic acid was stirred overnight at room temperature. The solution was then concentrated and co-concentrated three times with toluene to obtain a colorless oil (3.00 g, crude product), which was used without further purification. ESI MS m / z([M+H] + Value: 1001.50.

[0483] Example 108; Synthesis of Compound 100 [ka]

[0484] To a solution of compound 99 (3.00 g, crude product, 3.03 mmol) in DMA (15.0 mL), 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 phase was 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 obtain a colorless oily substance (2.90 g, 90% yield). ESI MS m / z([M+H]) + Value: 1098.50.

[0485] Example 109; Synthesis of Compound 101 [ka]

[0486] 2,2'-(ethane-1,2-diyrbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 equivalents) in dry tetrahydrofuran (200 mL) was mixed with a sodium flake (0.1 g). The mixture was stirred until the Na disappeared, and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 equivalent) was added dropwise. The mixture was stirred overnight and then quenched with HCl solution (20.0 mL, 1 N) at 0°C. The tetrahydrofuran was removed by rotary evaporation, brine (300 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oil (30.20 g, yield 79.0%), which was used without further purification. MS ESI m / z([M+H] + Value: 278.17.

[0487] Example 110; Synthesis of Compound 102 [ka]

[0488] At 0°C, 30.20 g, 108.5 mmol, 1.0 equivalent of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propionate was dissolved in anhydrous dichloromethane (220 mL). TsCl (41.37 g, 217.0 mmol, 2.0 equivalents) and TEA (30.0 mL, 217.0 mmol, 2.0 equivalents) were added. 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) to obtain a colorless oil (39.4 g, yield 84.0%). MS ESI m / z([M+H] + Value: 433.28.

[0489] Example 111; Synthesis of Compound 103 [ka]

[0490] To a 100 mL solution of tert-butyl 3-(2-(2-(2-(toluenesulfonyloxy)ethoxy)ethoxy)ethoxy)propionate (39.4 g, 91.1 mmol, 1.0 equivalent) in anhydrous DMF, NaN3 (20.67 g, 316.6 mmol, 3.5 equivalents) was added. The mixture was stirred overnight at room temperature, diluted with water (500 mL), and extracted with ethyl acetate (3 × 300 mL). The combined organic layer was 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 obtain a colorless yellow oil (23.8 g, yield 85.53%). MS ESI m / z([M+Na] + Value: 326.2.

[0491] Example 112; Synthesis of Compound 104 [ka]

[0492] Raney Ni (7.5 g, suspended in water) was washed with water (3 times) and isopropanol (3 times), and mixed with compound 103 (5.0 g, 16.5 mmol) in isopropanol. The mixture was stirred under a hydrogen balloon at room temperature for 16 hours, then filtered through a Celite pad, washed the pad with isopropanol, concentrated the filtrate, and purified by column chromatography (5-25% methanol / dichloromethane) to obtain a pale yellow oil (2.60 g, yield 57%). MS ESI m / z([M+H] + Value: 279.19.

[0493] Example 113; Synthesis of Compound 105 [ka]

[0494] To a 30 mL DMF solution of tetradecanedioic acid (2.06 g, 8 mmol), 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 obtain the title compound 105 (1.2 g, 45% yield). ESI MS m / z([M+H]) + Value: 349.23.

[0495] Example 114; Synthesis of Compound 106 [ka]

[0496] To a 50 mL dichloromethane solution of compound 104 (2.60 g, 9.35 mmol) and compound 105 (3.91 g, 11.2 mmol), EDC·HCl (2.15 g, 11.2 mmol) and DIPEA (3.6 mL, 20.6 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 obtain the title compound (4.94 g, yield 87%). ESI m / z([M+H]) + Value: 608.40.

[0497] Example 115; Synthesis of Compound 107 [ka]

[0498] To a 20 mL solution of compound 106 (4.94 g, 8.14 mmol) in dichloromethane, 20 mL of TFA was added. The reaction mixture was stirred at room temperature for 1 hour, then concentrated to dryness. Both the compound and the dichloromethane were evaporated twice, and the residue was placed in a pump to obtain compound 107 (4.50 g, crude product). ESI MS m / z([M+H])+ Value: 552.35.

[0499] Example 116; Synthesis of Compound 108 [ka]

[0500] To a 50 mL dichloromethane solution of compound 107 (4.50 g, crude product, 8.14 mmol) and compound 104 (1.95 g, 7.00 mmol), 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 obtain the title compound 108 (5.22 g, 92% yield). ESI m / z([M+H]) + Value: 811.52.

[0501] Example 117; Synthesis of Compound 109 [ka]

[0502] To a 20 mL solution of compound 108 (5.22 g, 6.44 mmol) in dichloromethane, TFA (5 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, then concentrated to dryness. Both the compound and the dichloromethane were evaporated twice, and the residue was placed in a pump to obtain compound 109 (4.90 g, crude product). ESI MS m / z([M+H]) + The value is 755.46.

[0503] Example 118; Synthesis of Compound 110 [ka]

[0504] To a 30 mL solution of compound 109 (4.90 g, crude product, 6.44 mmol) in dichloromethane, 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 phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (10-50% ethyl acetate / petroleum ether) to obtain colorless oil 236 (4.90 g, 90% yield). ESI MS m / z([M+H] + Value: 852.48.

[0505] Example 119; Synthesis of Compound 111 [ka]

[0506] In a hydrogenation bottle, 20 mL of a tetrahydrofuran solution of compound 110 (4.90 g, 5.75 mmol) was mixed with Pd / C (10 wt%, 0.20 g). The mixture was stirred overnight under 1 atm of hydrogen, filtered through Celite (filtration aid), and the filtered solution was concentrated to obtain compound 111 (4.50 g, yield >100%). ESI MS m / z([M+H]) + Value: 762.44.

[0507] Example 120; Synthesis of Compound 112 [ka]

[0508] At 0°C, HATU (0.50 g, 1.32 mmol) and triethylamine (0.06 mL, 1.32 mmol) were added to a 10 mL dichloromethane solution of compound 111 (1.00 g, 1.32 mmol). The reaction mixture was stirred at 0°C for 30 minutes, then Z-Lys-OH (0.40 g, 1.43 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then diluted with water (20 mL), extracted with ethyl acetate (3 × 20 mL), washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (0-10% methanol / dichloromethane) to obtain compound 112 as a colorless oil (1.28 g, yield 95%). ESI MS m / z([M+H]) + Value: 1017.60.

[0509] Example 121; Synthesis of Compound 113 [ka]

[0510] To a 10 mL solution of compound 112 (1.28 g, 1.26 mmol) in dichloromethane, 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 mixture was stirred at room temperature for 2 hours, then diluted with water (20 mL) and extracted with ethyl acetate (3 × 15 mL). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (0-10% methanol / dichloromethane) to obtain compound 113 as a colorless oil (1.28 g, yield 91%). ESI MS m / z([M+H]) + Value: 1114.62.

[0511] Example 122; Synthesis of Compound 114 [ka]

[0512] A tetrahydrofuran solution (150 mL) of tert-butyl acrylate (12.81 g, 0.10 mmol) and ethyl-1,2-diamine (24.3 g, 0.40 mol) was stirred at 45°C for 24 hours. The mixture was concentrated and purified by elution with methanol / dichloromethane (triethylamine) (5%:15%:80%) using an Al2O3 gel column to obtain the title compound (17.50 g, 92% yield). ESI MS m / z([M+H]) + ): Value 189.20.

[0513] Example 123; Synthesis of Compound 115 [ka]

[0514] 17.00 g, 90.33 mmol of tert-butyl 3-((2-aminoethyl)amino)propionate in 1,4-dioxane (50 mL) and concentrated hydrochloric acid (15 mL) was stirred at room temperature for 30 minutes, then 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 by vacuum pump to obtain the title compound (18.70 g, yield 100%, purity 96% by LC-MS). ESI MS m / z([M+H]) + Value: 133.20.

[0515] Example 124; Synthesis of Compound 116 [ka]

[0516] At 0°C, maleic anhydride (8.85 g, 90.33 mmol) was added to 150 mL of tetrahydrofuran 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 obtain (Z)-4-(2-((2-carboxyethyl)amino)ethyl)amino)-4-oxobuto-2-enoic acid. Next, toluene (150 mL) and DMA (50 mL) were added. The mixture was stirred at 90°C and refluxed through a Dean-Stark trap. After collecting 30 mL of solvent in the trap, HMDS (hexamethyldisilazane, 9.0 mL, 43.15 mmol) and ZnCl2 (16 mL, 1.0 M ether solution) were added, and the mixture was heated to 115-125°C, and toluene was collected through the Dean-Stark trap. The reaction mixture was heated at 120°C for 6 hours. During this period, 2 × 40 mL of anhydrous toluene was added to maintain the volume of the mixture at approximately 50 mL. Next, the mixture was cooled, and 1 mL of 1:10 HCl (concentrated) / methanol was added. The mixture was concentrated, purified by silica gel column chromatography, and eluted with water / acetonitrile (1:15). The fraction was concentrated and dried by vacuum pump to obtain 14.75 g of the title compound (yield 77.0%). ESI MS m / z([M+H]) + Value: 213.10.

[0517] Example 125; Synthesis of Compound 117 [ka]

[0518] 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, purified by silica gel column, eluted with ethyl acetate / dichloromethane (1:2 to 4:1), concentrated, and dried by vacuum pump to obtain 40.51 g of the title compound (yield 86%). ESI MS m / z([M+H]) + Value: 443.35.

[0519] Example 126; Synthesis of Compound 118 [ka]

[0520] Compound 117 (40.40 g, 0.091 mol) was added to a mixture of acetic acid (200 mL) and 30% H2O2 (100 mL). The reaction mixture was stirred overnight at 35°C, then concentrated and diluted with pure water (200 mL) and toluene (150 mL). The layers were separated, and the organic layer was extracted with water (2 × 25 mL). The aqueous solutions were combined, concentrated, and dried using a vacuum pump to obtain 40.50 g of the title compound (yield 99%, LC-MS purity 95%). ESI MS m / z([M+H] + Value: 449.30.

[0521] Example 127; Synthesis of Compound 119 [ka]

[0522] Compound 118 (20.0 g, 44.62 mmol) was added sequentially to a mixture of tetrahydrofuran (100 mL) and dichloromethane (100 mL) with oxalyl chloride (25.21 g, 200.19 mmol) and DMF (0.015 mL). The mixture was stirred at room temperature for 2 hours, concentrated, co-evaporated with dichloromethane / toluene (1:1, 2 × 50 mL), and then dissolved in tetrahydrofuran (50 mL). A tetrahydrofuran solution of compound 116 (7.50 g, 35.36 mmol) (100 mL) was added, the mixture was stirred overnight, concentrated under vacuum, purified by silica gel column chromatography, eluted with methanol / dichloromethane (1:6~1:5), and dried with a vacuum pump to obtain 14.76 g of the title compound (yield 65%). ESI MS m / z([M+H]) + Value: 643.35.

[0523] Example 128; Synthesis of Compound 120 [ka]

[0524] A tetrahydrofuran solution (100 mL) containing compound 119 (7.50 g, 11.67 mmol), N-hydroxysuccinimide (1.50 g, 13.04 mmol), and EDC (10.10 g, 52.60 mmol) was stirred overnight, concentrated under vacuum, purified by silica gel column chromatography, eluted with ethyl acetate / dichloromethane (1:4-2:1), and dried with a vacuum pump to obtain 6.30 g of the title compound (yield 73%). ESI MS m / z([M+H]) + Value: 740.40

[0525] Example 129; Synthesis of Compound 121 [ka]

[0526] At 0°C, 3 mL of DIPEA was added to a 15 mL DMF solution of 2-(2-(2-(2-aminoacetamide)acetamide)acetamido)acetic acid (Gly-Gly-Gly) (0.50 g, 2.03 mmol) and compound 120 (1.65 g, 2.22 mmol). The reaction mixture was stirred at 0°C for 0.5 hours, then at room temperature for 4 hours. The reaction mixture was then concentrated and purified by silica gel chromatography (mobile phase: acetonitrile / water = 95:5, containing 0.1% formic acid) to obtain the title compound 121 (1.04 g, yield 63%). MS-ESI m / z C 32 H 56 N5O 17 S [M+H] + Calculated value: 14.33, Measured value: 814.46.

[0527] Example 130; Synthesis of Compound 122 [ka]

[0528] A tetrahydrofuran solution (20 mL) containing compound 121 (0.70 g, 0.86 mmol), N-hydroxysuccinimide (0.20 g, 1.73 mmol), and EDC (1.21 g, 6.36 mmol) was stirred overnight at room temperature, concentrated under vacuum, purified by silica gel column chromatography, eluted with ethyl acetate / dichloromethane (1:4-2:1), and dried with a vacuum pump to obtain 0.540 g of the title compound (yield 69%). MS-ESI m / z C 36 H 59 N6O 19 S [M+H]+: Calculated value: 911.34, Measured value: 911.42.

[0529] Example 131; Synthesis of Compound 123 [ka]

[0530] At 0°C, DIPEA (2 mL) was added to a DMF solution (8 mL) of (2S,4R)-5-(3-amino-4-hydroxyphenyl)-4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl solution-2,3,3,8-tetramethyl-4,7,13-trioxy-12-oxa-2,5,8-triazoundecane-11-yl)thiazole-4-formamide)-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). The reaction mixture was stirred at 0°C for 0.5 hours, then at room temperature for 4 hours. Next, the reaction mixture was concentrated and purified by preparative HPLC (mobile phase: acetonitrile / water = 10%-80%, containing 0.1% formic acid) to obtain compound 123 (95.5 mg, yield 58%). MS-ESI m / z C 69 H 112 N 11 O 24 S[M+H] +Calculated value: 1542.72, Measured value: 1542.76.

[0531] Example 132; Synthesis of Compound 124 [ka]

[0532] (S)-1-benzyl-5-tert-butyl2-aminopentanedioate hydrochloride (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) were stirred in dichloromethane (200 mL) at room temperature for 6 hours. The mixture was diluted with water (100 mL), and the phases were 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 by silica gel column (dichloromethane / ethyl acetate = 20:1 to 5:1) to obtain the title compound 314. MS ESI m / z C 37 H 54 NO7[M+H] + Calculated value: 624.38, Measured value: 624.38.

[0533] Example 133; Synthesis of Compound 125 [ka]

[0534] At 4°C, compound 124 (12.50 g, 20.05 mmol) was dissolved in dioxane (30 mL) and stirred with hydrochloric acid (10 mL, 36%) for 0.5 hours. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL), concentrated at 15°C to obtain 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.

[0535] Example 134; Synthesis of Compound 126 [ka]

[0536] Compound 125 (10.70 g, 18.86 mmol), a solution of tert-butyl 1-amino-15-oxo-3,6,9,12,19,22,25,28-octaoxa-16-azatriacontane-31-oate 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) were stirred overnight at room temperature and then washed with brine (100 mL). 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 by silica gel column (dichloromethane / ethyl acetate = 10:1 to 4:1) to obtain 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.

[0537] Example 135; Synthesis of Compound 127 [ka]

[0538] 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 hours. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL), concentrated at 15°C, and purified by silica gel column (dichloromethane / methanol = 10:1~6:1) to obtain the title compound (8.67 g, yield 87%). MS-ESI m / z C 55 H 88 N3O 17 [M+H] +Calculated value: 1062.60, Measured value: 1062.68.

[0539] Example 136; Synthesis of Compound 128 [ka]

[0540] A tetrahydrofuran solution (150 mL) 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) was stirred at room temperature for 6 hours and concentrated under vacuum to obtain a crude NHS ester that could be used in the next step without purification. The N-succinimide ester prepared above was added in four portions over 1 hour to (S)-6-amino-2-((tert-butoxycarbonyl)amino)hexanoate (2.75 g, 9.73 mmol) in a mixed solution of 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 using a silica gel column (dichloromethane / methanol = 10:1 to 4:1) to obtain 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.

[0541] Example 137; Synthesis of Compound 129 [ka]

[0542] At 4°C, compound 128 (8.10 g, 6.28 mmol) was dissolved in dioxane (40 mL) and stirred with hydrochloric acid (15 mL, 36%) for 0.5 hours. The reaction mixture was diluted with toluene (20 mL) and DMF (20 mL), and concentrated at 15°C to obtain the title compound (7.71 g, 100% yield), which was used in 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.

[0543] Example 138; Synthesis of Compound 130 [ka]

[0544] At 0°C, 7.10 g, 25.35 mmol, and 3.01 g, 33.80 mmol, 4-maleimide-N-succinamide ester and alanine in 50 mL of DMF were mixed with 10 mL of DIPEA. The reaction mixture was stirred at 0°C for 0.5 hours, then at room temperature for 1 hour. The reaction mixture was then concentrated and purified using a silica gel column (mobile phase: dichloromethane / methanol = 10:1, containing 0.1% formic acid) to obtain compound 130 (5.21 g, yield 81%). MS-ESI m / z C 11 H 14 N2O5[M+H] + Calculated value: 255.09, Measured value: 255.15.

[0545] Example 139; Synthesis of Compound 131 [ka]

[0546] 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) were mixed in dichloromethane (70 mL) at room temperature for 6 hours. The mixture was concentrated under vacuum and purified using a silica gel column (mobile phase: dichloromethane / ethyl acetate = 10:1) to obtain compound 131 (5.83 g, yield 82%). MS-ESI m / z C 15 H 17 N3O7[M+H] + Calculated value: 351.11, Measured value: 351.20.

[0547] Example 140; Synthesis of Compound 132 [ka]

[0548] At 0°C, DIPEA (7 mL) was added to a DMF solution (40 mL) of compound 129 (7.61 g, 6.39 mmol) and compound 311 (2.90 g, 8.280 mmol). The reaction mixture was stirred at 0°C for 0.5 hours, then at room temperature for 1 hour. The reaction mixture was then concentrated and purified using a silica gel column (mobile phase: dichloromethane / methanol = 10:1, containing 0.1% formic acid) to obtain compound 132 (7.10 g, yield 78%). MS-ESI m / z C 11 H 14 N2O5[M+H] + Calculated value: 1426.7782, Measured value: 1426.7820.

[0549] Example 141; Synthesis of Compound 133 [ka]

[0550] A tetrahydrofuran solution (50 mL) 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) was stirred at room temperature for 6 hours and concentrated under vacuum to obtain the NHS ester, which was used in the next step without purification.

[0551] The above compound was added in four portions over 1 hour to 2-(2-(2-aminoacetamide)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 (dichloromethane / methanol = 10:1~7:1) to obtain the title compound (8.16 g, yield 79%). MS-ESI m / z C 11 H 14 N2O5[M+H] + Calculated value: 597.84, Measured value: 1597.84.

[0552] Example 142; Synthesis of Compound 134 [ka]

[0553] EDC (100 mg, 0.526 mmol) was added to a DMA solution (5 mL) 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). The reaction mixture was stirred at room temperature for 6 hours, concentrated under vacuum, redissolved in methanol / dichloromethane (0.5 mL:3 mL), passed through a short silica gel column, eluted with methanol / dichloromethane (1:3), concentrated under vacuum to obtain the crude compound, which was used in the next step. MS-ESI m / z value: 2326.25.

[0554] A solution of the above compound in methylene chloride (1 mL) 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% acetonitrile aqueous solution containing 0.1% formic acid) to obtain compound 134 (69.0 mg, yield 72%). MS-ESI m / z C 11 H 14 N2O5[M+H]+: Calculated value: 2146.1497, Measured value: 2146.1588.

[0555] Example 143; Synthesis of Compound 135 [ka]

[0556] (S)-30-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butanamide)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriacontano-31-acid (20 mg, 0.029 mmol) was dissolved in dichloromethane (5 mL), to which EDC (11 mg, 0.059 mmol) and pentafluorophenol (10.8 mg, 0.059 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, concentrated, purified by silica gel column chromatography, and eluted with ethyl acetate / dichloromethane (1:4) to obtain 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.

[0557] Example 144; Synthesis of Compound 136 [ka]

[0558] A methanol solution (100 mL) of (S)-tert-butyl 2-((S)-2-(((benzyloxy)carbonyl)amino)propanamide)propanoate (10 g, 0.028 mol) and 10% palladium 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 obtain a colorless oily product (6.1 g, yield 100%). ESI m / z C 10 H 20 N2O3[M+H] + Calculated value: 217.15, Measured value: 217.15.

[0559] Example 145; Synthesis of Compound 137 [ka]

[0560] (S)-30-(((benzyloxy)carbonyl)amino)-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azahexatriacontane-31-acid (250) (100 mg, 0.154 mmol) was dissolved in dichloromethane (5 mL), to which EDC (59 mg, 0.309 mmol) and pentafluorophenol (PFP) (57 mg, 0.309 mmol) were added. 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 by short silica gel column elution with methanol / dichloromethane (1:10) to obtain title compound 137 (80 mg, yield 61%). ESI m / z C 40 H 68 N4O 15 [M+H] + Calculated value: 845.47, Measured value: 845.47.

[0561] Example 146; Synthesis of Compound 138 [ka]

[0562] A methanol solution (5 mL) of compound 137 (80 mg, 0.094 mmol) and 10% palladium-carbon (10 mg) was stirred under hydrogen (5 psi) for 2 hours. The solid was filtered, and the filtrate was concentrated to obtain a colorless oily product (66 mg, 100% yield), which was used in 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.

[0563] Example 147; Synthesis of Compound 139 [ka]

[0564] Compound 138 (66 mg, 0.094 mmol) in ethanol (5 mL) was mixed with 2,5-dioxopyrrolidine-1-yl-4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butanoate (39 mg, 0.141 mmol) and PBS (0.1 M, pH 7.5, 1.0 mL). The reaction mixture was stirred overnight, concentrated, and purified by silica gel column (dichloromethane / methanol = 100:0 to 10:1) to obtain the title compound 139 (37 mg, yield 45%). ESI m / z C 40 H 69 N5O 16 [M+H] + Calculated value: 876.47, Measured value: 876.47.

[0565] Example 148; Synthesis of Compound 140 [ka]

[0566] A 3 mL solution of compound 139 (50 mg, 0.057 mmol) in dichloromethane was stirred with TFA (1 mL) at room temperature for 2 hours. The reaction mixture was concentrated to dryness, 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 by silica gel column (dichloromethane / ethyl acetate = 100:10 to 3:1) to obtain the title compound 140 (41 mg, yield 73%). ESI m / z C 42 H 60 F5N5O 16 [M+H] + Calculated value: 986.40, Measured value: 986.42.

[0567] Example 149; Synthesis of Compound 141 [ka]

[0568] To a 5 mL solution of 4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanoic acid (100 mg, 0.27 mmol) in dichloromethane, 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 by silica gel column chromatography (dichloromethane / ethyl acetate = 20:1 to 5:1) to obtain the title compound 141 (114 mg, yield 80%). MS-ESI m / z C 22 H 16 F5N3O7[M+H] + Calculated value: 530.0, Measured value: 530.09.

[0569] Example 150; General method for conjugating the disulfide bond of a reduced antibody with a tubulicin derivative

[0570] 10 mg / ml of HER2 antibody was added to 2.0 mL of PBS buffer at pH 6.0–8.0, along with 0.70–2.0 mL of 100 mM NaH2PO4, buffer at pH 6.5–8.5, and TCEP (14–45 μl, 20 mM in water). After incubation at room temperature to 37.5°C for 0.5–4 hours, the same equivalent volume of azide compound (azidebenzoic acid or 2-(2-(2-hydroxyethoxy)ethoxy)ethoxyazide compound) was added, and the mixture was incubated at room temperature to 37.5°C for 1–4 hours. A tubulosine derivative having a thiol-reactive group (e.g., compounds 39, 57, 72, 123, 134, etc.) (28-32 μl, 20 mM in DMA solution) was added and incubated at room temperature to 37.5°C for 2-18 hours. Then, DHAA (135 μL, 50 mM) was added and incubated continuously at room temperature overnight. The mixture was purified using a G-25 column, cationic chromatography column, or anionic chromatography column with a 10-100 mM phosphate or citrate buffer at pH 6-7.5 containing 10-100 mM phosphate or citrate and 50-200 mM NaCl to obtain conjugates (yield 75-99%). This purification step to obtain conjugates may also be performed by dialysis and filtration using a 10-100 mM phosphate or citrate buffer at pH 6-7.5 containing 50-200 mM NaCl (3-30 times the volume of the dialysate) (yield 75-99%). The drug-to-antibody ratio (DAR) measured by HPLC-MS was 3.1–4.9. Analysis by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID × 30 cm, 0.5 ml / min, 100 min) showed that the monomer content was 95–99%. The structures of the prepared conjugates 39, 57, 72, 123, or 134 are shown below:

[0571] [ka]

[0572] Example 151; Other methods for preparing conjugates

[0573] Cell-binding molecules (antibodies) can be conjugated to the compounds of this invention via amide, thioether, or disulfide bonds. Antibodies (>5 mg / mL) were diluted in PBS buffer (pH 8.0) containing 50 mM sodium borate, and dithiothreitol (final concentration 10 mM) was added to generate free thiol groups. The mixture was treated at 35°C for 30 minutes. The mixture was purified by G-25 gel filtration column chromatography (with 1 mM EDTA added to PBS buffer). Approximately 8 thiol groups were detected in each antibody by measurement using Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid)]. Antibodies may be reacted with Trout's reagent (2-iminothiophene) (Jue, R., et al. Biochem. 1978, 17 (25): 5399-5405), SATP (N-succinimide-S-acetylthiopropionate), or N-succinimide-S-acetyl(thiotetraacetic acid) (SAT(PEG)4) under pH 7-8 conditions to generate thiol groups (Duncan, R, et al, Anal. Biochem. 1983, 132, 68-73, Fuji, N. et al, Chem. Pharm. Bull. 1985, 33, 362-367). Generally, 5-9 thiol groups are generated on each antibody molecule.

[0574] At 4°C, drug molecules containing maleimide or bromoacetamide groups were added to a cold dimethylacetamide (DMA) solution of antibody containing free thiol groups (the molar ratio of drug to thiol groups was 1.2–1.5:1) (the alkylation reaction between antibody and bromoacetamide typically requires a 0.5 M sodium borate solution (pH 9)). After 1–2 hours, the reaction was stopped by adding excess cysteine, and concentrated conjugates were obtained after ultrafiltration, gel chromatography (G-25, PBS buffer), and sterile filtration. Protein concentration and the number of drugs attached to each antibody were determined by measuring absorbance at 280 nm and 252 nm. Size exclusion HPLC could be used to determine the proportion of monomeric conjugates, and by RP-HPLC, it was measured that unbound drug was less than 0.5%. In the case of monomeric conjugates formed by thioether linkages, each antibody molecule conjugates an average of 3.2–4.8 tubulicin derivatives.

[0575] The types of conjugates are dimethyl(phenyl)silyl (DMPS), SMDP, 4-succinimidyloxycarbonylmethyl-α(2-pyridyldisulfide)toluene (SMPT), N-succinimidyl-4-(2-pyridinethio)propionate (SPP), N-succinimidyl-4-(2-pyridinethio)propionate (SPDP), N-succinimidyl-4-(2-pyridinethio)butyrate (SPDB), N-succinimidyl-4-(2-pyridinethio)butyrate (SMCC), and N-hydroxylsuccinimide-(polyethylene glycol)N-maleimide (SM(PEG)). nThe mixture includes the following: Antibody (>5 mg / mL) was dissolved in buffer (pH 6.5-7.5, 5 mM PBS, 50 mM NaCl, 1 mM EDTA), reacted with the conjugate for 2 hours, and the molar ratio of conjugate to antibody was 6-10 times or more. The reaction mixture was purified by Sephadex G25 gel chromatography, and low molecular weight molecules were removed. The mixture could also be purified by a cationic chromatography column or anionic chromatography column using 10-100 mM phosphate or citrate, 50-200 mM NaCl pH 6-7.5 buffer, to obtain the conjugate (yield 75-99%). The mixture could also be purified by dialysis and filtration using 10-100 mM phosphate or citrate, 50-200 mM NaCl pH 6-7.5 buffer (3-30 times the sample volume), to obtain the conjugate (yield 9975%-%). The antibody concentration was measured by spectrophotometry, and the conjugate contained a pyridyldithiol group. 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 (equivalent to 20 times dithiothreitol), and the extinction coefficients of the released 2-thiopyridine group at 343 nm and 280 nm were 8080 and 5100 M, respectively. -1 cm -1 For antibody modification, 1.2 to 1.5 equivalents of a tubulicin derivative molecule containing a thiol group were added. After reacting at room temperature for 5 to 18 hours, the reaction mixture was passed through Sephadex G25 gel chromatography to remove unbound drugs or other low molecular weight substances. Next, the concentration of the product was measured by measuring the absorbance at 280 nm and 252 nm. The product was monomeric, with an average of 3.2 to 4.8 drug molecules bound to each antibody molecule.

[0576] 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.

[0577] For the cytotoxicity assay, the human gastric cancer cell line NCI-N87 was used. Cells were cultured in RPMI-1640 containing 10% FBS. To perform 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. Then, cells were treated with various concentrations of the test compound (20 μl) in appropriate cell culture medium (total volume 0.2 mL). The control wells contained cells and medium but lacked the 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 f...

Claims

1. An antibody-tubulisin B derivative (congener) conjugate having the structure of formula (I), or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or a polymorphic crystalline structure represented by formula (I); or an optical isomer of the structure represented by formula (I); or one or more hydrogens ( １ H) Deuterium with 1 or more atoms ( ２ H) Substituted with or with one or more atoms １２ C atoms are one or more １３ Derivatives of these, substituted with C atoms: 【Chemistry 1】 During the ceremony, 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 These are H and C, which are independent of each other. １ -C ６ Alkyl, C １ -C ６ Alkenil, C １ -C ６ Alkoxy, C １ -C ６ Alkylcarbonyl, C １ -C ６ Alkyl ester, C １ -C ６ Alkylcarboxyl, or C １ -C ６ It is an alkylamide group; or, R １ and R ２ , R １ and R ３ , R ２ and R ３ , or R ３ and R ４ together, C ２ -C ７ Heterocyclyl or C ２ -C ７ Forms a cycloalkyl structure; R ５ H, O-C １ ~C ６ Alkyl alkyl group, C(O)-H, C(O)-C １ ~C ６ (Linear or branched) alkyl groups, C(O)-NH-C １ ~C ６ (Linear or branched) alkyl groups, or C(O)-N(C １ ~C ６ (Linear or branched alkyl) ２ It is the basis; R ６ , R ７ , and R ８ These are H and C, which are independent of each other. １ -C ６ alkyl group, C １ -C ６ Alkyl ether group, C １ -C ６ Alkylcarbonyl group, C １ -C ６ Alkyl ester group, C １ -C ６ Alkylcarboxyl group, or C １ -C ６ It is an alkylamide group; mAb is an antibody, antibody fragment, monoclonal antibody, polyclonal antibody, nanobody, prodrug antibody, or antibody and antibody fragment modified with synthetic molecules or proteins; n = 1 to 30; L is a connective including hydrophilic branches having the following structure (Ia): 【Chemistry 2】 In formula (Ia), Aa is an L- or D-natural or unnatural amino acid; r is an integer between 0 and 12; if r is not 0, (Aa) ｒ A peptide unit is composed of identical or different amino acids; I understand １ = an integer between 1 and 18; m ２ = an integer between 1 and 100; m ３ = an integer from 1 to 8; m ４ = an integer from 0 to 8; m ５ = an integer between 1 and 8; Y is C(O)NH; R ９ is H, (O=)CR １ , (O=)CNHR １ , or C1-C6 alkyl.

2. A method for producing a conjugate according to claim 1, comprising one or more of the following steps: 【Transformation 3】 In formula (II), P １ , 【Chemistry 4】 , R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ , and the mAb is as described in claim 1; The structure of L' is as follows: 【Transformation 5】 In formula (II-0), m １ , m ２ , m ３ , m ４ , m ５ , Aa, r, and R ９ This is as described in formula (Ia) of claim 1.

3. A method for producing a conjugate according to claim 2, wherein the structure of L' is as shown below: 【Transformation 6】 In the formula, m １ , m ２ , m ３ , m ４ , m ５ , Aa, r, and R ９ This is as described in claim 1.

4. A method for producing a conjugate according to claim 2 or 3, wherein the method for preparing mAb-SH includes any of the following methods: a) Reduction of disulfide bonds between heavy chains and light chains or between heavy chains, or intramolecular disulfide bonds of antibodies, antibody fragments, monoclonal antibodies, polyclonal antibodies, nanobodies, probodies or antibodies, and antibody fragments modified with synthetic molecules or proteins, by reducing agents; b) Generation of thiol groups by reaction of Traut reagent or thiolactone with the amino group of an antibody molecule; 【Transformation 7】 c) In a buffer system, introduction of readily reducible disulfide bonds into the antibody by biochemical reaction, followed by reduction by TCEP, DTT, GSH, β-MEA, or β-ME; 【Transformation 8】

5. A method for producing a conjugated compound according to any one of claims 2 to 4, wherein the buffer system used in the synthesis of the conjugated compound is a buffer solution containing 0% to 35% of a water-soluble organic solvent, which is methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, acetonitrile, acetone, DMF, DMA, or DMSO, with a pH of 5.0 to 9.5 and a concentration of 1 mM to 1000 mM of phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, Tris (trimethylolaminomethane), benzoic acid, or triethanolamine, or a mixture thereof; the reaction temperature is 0°C to 45°C and the reaction time is 5 minutes to 96 hours.

6. A method for producing a conjugate according to any one of claims 2 to 5, wherein the conjugation reaction is completed and then purified by ultrafiltration or column chromatography.

7. The method for producing a conjugate according to claim 6, wherein the purification column is a molecular sieve column, a cation column, an anion column, a hydrophobic (HIC) column, a reversed-phase column, or a protein A or G affinity column.

8. The compound of formula (II) is obtained by a condensation reaction between the tubulosine B derivative of formula (III) and the compound of formula (L'), as described in the method for producing the conjugate according to any one of claims 2 to 7: 【Chemistry 9】 In the formula, X is OH, halogen, phenol, pentachlorophenol, trifluoromethanesulfonic acid, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, p-toluenesulfonic acid, methanesulfonic acid, 2-ethyl-5-phenylisoxazole-3'-sulfonic acid, 【Chemistry 10】 , an anhydrous formed from itself or other anhydrous; or a peptide condensation reaction intermediate or a Mitsunobu reaction intermediate; The condensation reaction is carried out at -20°C to -150°C for 5 minutes to 120 hours in a solvent containing 1% to 100% by volume of pyridine, triethylamine, or diisopropylethylamine, in a mixture of two or three of these solvents, in or without inert gas protection; Alternatively, the condensation reaction may be carried out in the following buffer system under the following conditions: in a buffer system containing a water-soluble organic solvent with a pH of 5.0 to 9.5 and a volume ratio of 0% to 35%, comprising phosphoric acid, acetic acid, citric acid, boric acid, carbonic acid, barbituric acid, tris(tris-hydroxymethylaminomethane), benzoic acid, or triethanolamine, or a mixture thereof, at a concentration of 1 mM to 1000 mM; at a reaction temperature of 0°C to 45°C; and for a reaction time of 5 minutes to 96 hours.

9. NH of equation (III) ２ A method for producing a conjugate according to claim 8, wherein the group participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

10. If X is OH, the condensation reaction requires a condensation reagent selected from the following, for the method of producing the conjugate according to claim 8 or 9: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-cyclohexyl-2-morpholinoethylcarbodiimide, meso-p-toluenesulfonate (CMC or CME-CDI), carbonyldiimidazole (CDI), O-benzotriazole -N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole hexafluorophosphate-1-yloxytripyrrolidinylphosphate (PyBOP), diethyl pyrocarbonate (DEPC), N,N,N',N'-tetramethylchloroformamidine Hexafluorophosphate, 2-(7-oxobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-[(dimethylamine)(morpholino)methylene]-1[1,2,3]triazolo[4,5-b]1-pyridine-3-oxohexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolium hexafluorophosphate (CIP), chlorotripyrrolidinylphosphonium hexafluorophosphate (PyCl oP), bis(tetramethylene)fluoroformamide (BTFFH), N,N,N',N'-tetramethyl-thio-(1-oxo-2-pyridyl)thiouronium hexafluorophosphate, 2-(2-pyridon-1-yl)-1,1,3,3-tetramethylureatetrafluoroborate (TPTU), sulf-(1-oxo-2-pyridyl)-N,N,N',N'-tetramethylthiourea hexafluorophosphate, O-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-Tetramethylthiourea hexafluorophosphate fluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholine-carbonium hexafluorophosphate (COMU), (benzenetriazol-1-yloxy)dipyrrolidinecarbohexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (or on a solid support), dipyrrolidinyl (N-succinimidyloxy) hexafluorophosphate carbonyl (HSPyU) ), 1-(chloro-1-pyrrolidinylmethylene)pyrrolidine hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazole tetrafluoroborate (CIB), (benzotriazole-1-yloxy)dipiperidine carbohexafluorophosphate (HBPipU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluratetrafluoroborate (TCTU), tris(dimethylamino)phosphine hexafluorophosphate (BrOP), 1-n-propyl phosphate anhydride (PPACA, T3P( (Registered trademark)), 2-isocyanoethylmorpholine (MEI), N,N,N',N'-tetramethylurea-oxy-(N-succinimidyl)hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinetetrafluoroborate (BEP), oxy-[(ethoxycarbonyl)cyanomethylamine]-N,N,N',N'-tetramethylthioureatetrafluoroborate (TOTU), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (MMTM, DMTMM), 2-succinimidyl Lu-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)urea tetrafluoroborate (TDBTU), azodicarboxydipiperidine (ADD), bis(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), or diethyl azodicarboxylate (DEAD).

11. A method for producing a conjugate according to any one of claims 8 to 10, wherein the synthesis of a tubulicin B derivative of formula (III) comprises one or more of the following steps: 【Chemistry 11】 In the formula, R ５’ H, C １ -C ６ Alkyl, C １ -C ６ Alkenyl, or C １ -C ６ It is a linear or branched aminoalkyl group.

12. A method for producing a conjugate according to claim 11, wherein the synthesis of the tubulicine B derivative of structural formula (III) comprises one or more of the following steps: Step 1: Stir the aqueous solutions of diethoxyacetonitrile and ammonium sulfide at room temperature to obtain compound 1 (2,2-diethoxythioacetamide); 【Chemistry 12】 Step 2: Compound 1 and bromopyruvate are heated in an anhydrous solvent to condense and obtain compound 2; 【Chemistry 13】 Step 3: Compound 3 is dissolved in a solvent and hydrolyzed in the presence of a Lewis acid or protonic acid to obtain compound 3; 【Chemistry 14】 Step 4: Under low temperature conditions, the sulfinamide is dehydrogenated with n-butyllithium, and then compound 4 is obtained by condensing it with compound 3 in the presence of a Lewis acid; 【Chemistry 15】 The Lewis acid mentioned above is hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, formic acid, oxalic acid, acetic acid, p-toluenesulfonic acid, p-toluenesulfonate pyridine, AlCl ３ FeCl ３ ZnCl ２ BF ３ , BCl ３ , BBr ３ TiCl ４ , ZnBr ２ LiBF ４ ; selected from; Step 5: Under low temperature conditions, compound 4 is selectively reduced with a reducing agent to obtain compound 5, and a Lewis acid is added during the reaction to control its stereochemistry; 【Chemistry 16】 Step 6: Compound 5 is dissolved in a solvent, and the tert-butylsulfinyl group is removed with acid to obtain compound 6; 【Chemistry 17】 Step 7: Compound 6 and azidic acid are condensed in a solvent in the presence of a condensation reagent to obtain compound 7, or Alternatively, azidic acid and isobutyl chloroformate may be reacted in THF in the presence of an organic base to obtain a mixture of anhydrides, and this mixture may be condensed with the hydrochloride salt of compound 6 to obtain compound 7. Alternatively, azidic acid and oxalyl chloride are reacted in a solvent in the presence of triethylamine and a catalytic amount of DMF to obtain an acid chloride, which is then condensed with the hydrochloride salt of compound 6 to obtain compound 7; [Chemistry 18] Step 8: In the presence of an organic base, the hydroxyl group of compound 7 is reacted with a hydroxyl protecting agent in a solvent to obtain compound 8; 【Chemistry 19】 Step 9: Compound 8 in the solvent is deprotected by adding a base, and then alkylated to obtain compound 9; 【Chemistry 20】 Step 10: Compound 9 is dissolved in a solvent, the azide group is reduced to an amino group, and then compound 10 is obtained by condensation with an acid or an acid derivative having similar reactivity; 【Chemistry 21】 Step 11: Hydroxyl protecting group PG of compound 10 １ Deprotect the compound to obtain compound 11; 【Chemistry 22】 Step 12; Convert the ester compound 11 to the acid compound 12; 【Chemistry 23】 Step 13: Compound 12 is reacted with an acid anhydride or carboxylic acid chloride in the presence of a base and a catalyst to obtain compound 13, although this reaction may be carried out without a base or catalyst; 【Chemistry 24】 Step 14: Compound 13 and the hydroxyl-containing compound are condensed in the presence of a condensation reagent to obtain a reactive ester compound 14; 【Chemistry 25】 Step 15: Compound 15 and compound 14 are condensed in an aqueous phase under specific pH conditions, or in an organic phase containing an organic base or an inorganic base, to obtain compound 16; 【Chemistry 26】 Step 16: Under reducing conditions, the nitro group of compound 16 is reduced to an amino group to obtain compound III. 【Chemistry 27】

13. A method for producing a conjugate according to any one of claims 8 to 12, wherein the synthesis of the compound of formula (L') comprises one or more of the following steps: 【Chemistry 28】

14. The synthesis of the compound of formula (L') is a method for producing the conjugate according to any one of claims 8 to 12, comprising one or more of the following steps: Step 1: Compound 1a is obtained by directly condensing compound 1-1 and compound 1-2 in the presence of a condensing agent, or by reacting compound 1-2 with pentafluorophenol, nitrophenol, or N-hydroxysuccinimide in the presence of a condensing agent, and then reacting it with compound 1-1; Alternatively, compound 1b is obtained by directly condensing compounds 1-3 and 1-4 in the presence of a condensing agent, or by condensing compounds 1-3 and 1-4 through other indirect condensation reaction pathways; 【Chemistry 29】 Step 2: Deprotect the carboxyl protecting group PG of compound 1 using a deprotection reagent. ２ Remove the compound to obtain compound 2; 【Transformation 30】 Step 3: Condensing carboxyl-containing compound 2 and amino-containing compound 3 in the presence of a condensing agent or via other indirect condensation reaction pathways to obtain compound 4; 【Chemistry 31】 Step 4: Under deprotection conditions, the amino protecting group PG of compound 4 １ Remove the compound to obtain compound 5; 【Chemistry 32】 Step 5: Condensing carboxylic acid 6 and amine 5 in the presence of a condensing agent or via other indirect condensation reaction pathways to obtain compound 7; 【Transformation 33】 Step 6: Under deprotection conditions, the carboxyl protecting group PG of compound 7 ３ Remove the compound to obtain compound 8; 【Transformation 34】 Step 7: React compound 8 with a hydroxyl-containing compound, or compound 8 with another carboxylic acid-activating compound, in the presence of a condensing agent to obtain a reactive ester compound L'; 【Chemistry 35】

15. A method for producing a conjugate according to any one of claims 8 to 12, wherein the synthesis of the compound of formula (L') comprises one or more of the following steps: 【Transformation 36】

16. A method for producing a conjugate according to claim 15, wherein the synthesis of the compound of formula (L') comprises one or more of the following steps: Step 1: Under deprotection conditions, the amino protecting group PG of compound 1 １ Remove the compound to obtain compound 2; 【Chemistry 37】 Step 2: Condensing amine compound 2 and carboxylic acid 3 in the presence of a condensing agent or via other indirect condensation reaction pathways to obtain compound 4; 【Transformation 38】 Step 3: Under deprotection conditions, the carboxyl protecting group PG of compound 4 ２ Remove the compound to obtain compound 5; 【Chemistry 39】 Step 4: Condensing carboxylic acid 5 and amine 6 in the presence of a condensing agent or via other indirect condensation reaction pathways to obtain compound 7; 【Chemistry 40】 Step 5: Under deprotection conditions, the carboxyl protecting group PG of compound 7 ３ Remove the compound to obtain compound 8; 【Chemistry 41】 Step 6: Compound 8 and a hydroxyl-containing compound are reacted in the presence of a condensing agent, or compound 8 and another carboxylic acid activating compound are reacted to obtain a reactive ester compound 9. 【Chemistry 42】

17. A method for producing a conjugate according to any one of claims 2 to 10, wherein the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (IV) and the compound of formula (V): 【Chemistry 43】 Here, the definition of X and the condensation reaction conditions are as described in any one of claims 8 to 10.

18. NH of equation (V) ２ A method for producing a conjugate according to claim 17, wherein the group participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

19. A method for producing a conjugate according to claim 17 or 18, wherein the synthesis of the compound of formula (IV) comprises one or more of the following steps: 【Chemistry 44】

20. A method for producing the conjugate according to claim 19, wherein the synthesis of formula (IV) comprises any one of the following steps: In the presence of a condensation reagent, carboxylic acid compound 1 and a hydroxyl-containing compound are reacted to obtain a reactive ester; Alternatively, a reactive mixed acid anhydride is obtained by reacting carboxylic acid compound 1 with ethyl chloroformate or isobutyl chloroformate in the presence of an organic base; Alternatively, acid chlorides are obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base and a catalytic amount of DMF.

21. A method for producing a conjugate according to any one of claims 17 to 20, wherein the synthesis of the compound of formula (V) comprises one or more of the following steps: 【Chemistry 45】

22. A method for producing a conjugate according to claim 21, wherein the synthesis of formula (V) includes one or more of the following steps: Step 1: Compound 1 and Compound 2 are condensed in an aqueous phase under specific pH conditions or in an organic phase containing an organic or inorganic base to obtain Compound 3; 【Chemistry 46】 Step 2: PG (Protein Protection Group) of Compound 3 ４ Remove it under appropriate deprotection conditions to obtain compound V; 【Chemistry 47】

23. A method for producing a conjugate according to claim 21 or 22, wherein the synthesis of compound 2 comprises one or more of the following steps: 【Chemistry 48】 Here, compound 8 (compound XIVa) obtained in this synthesis step is the target compound 2 described in claim 22.

24. The method for producing the conjugate according to claim 23, wherein the synthesis of compound 2 comprises one or more of the following steps: Step 1: At 0 to 60°C, benzyl chloride, benzyl bromide, or another benzyl compound is added to the L-tyrosine ester derivative (1) in the solvent, and then an organic or inorganic base is added to obtain compound 2; Step 2: Compound 2 is dissolved in an organic solvent, and then reacted with a reducing agent to obtain compound 3; Step 3: Aldehyde 4 is obtained by oxidizing alcohol compound 3 under oxidative conditions; Step 4: Compound 5 is obtained by extending the carbon chain by reacting aldehyde 4 with a phosphate ester (Horner-Wadsworth-Emmons reaction) or a phosphate ylide (Wittig reaction); Step 5: In the presence of a homogeneous or heterogeneous catalyst, the double bond of compound 5 is reduced, thereby removing the benzyl group and yielding a stereochemically pure compound or two diastereomers; Step 6: Dissolve compound 6 in an organic solvent and perform nitration with a nitration reagent; Step 7:H ２ / Pd / C, Fe or Zn / HOAc, or SnCl ２ Under conditions containing HCl, the nitro group of compound 7 is reduced to an amino group.

25. A method for producing a conjugate according to claim 21 or 22, wherein the synthesis of compound 2 comprises one or more of the following steps: 【Chemistry 49】 In the formula, compound 8 (compound XIVb) is compound 2 as described in claim 21 or 22.

26. A method for producing a conjugate according to claim 25, wherein the synthesis of compound 2 comprises one or more of the following steps: Step 1: At -78°C to -45°C, an aldol reaction is carried out between Evans asymmetric N-acyloxazolidinone or thion 2 and compound 1 to obtain a stereochemically pure compound 3, where X=O or S;R １６ = H, methyl, or phenyl; R １７ = H, methyl, isopropyl, phenyl, or benzyl; Step 2: Under Burton-McCombee deoxygenation conditions, the hydroxyl group of compound 3 is deoxygenated, i.e., first the alcohol is converted to a thiocarbonyl derivative, and then But ３ By treating with SnH and performing radical cleavage, a dehydroxylated product is obtained, where the conditions for radical cleavage are: n-Bu ３ SnH / AIBN, n-Bu ３ SnH / AIBN / n-BuOH / PMHS, and (Bu ４ N) ２ S ２ O ８ / HCO ２ Contains Na; Step 3: Dissolve compound 4 in tetrahydrofuran and add the Evans chiral auxiliary group to LiOH / H ２ O ２ Under these conditions, it cleaves to obtain the corresponding acid 5; Step 4: Compound 5 is dissolved in an organic solvent and hydrogenated in the presence of a palladium-carbon catalyst, at which point the benzyl group is also removed to obtain compound 6; Step 5: Dissolve compound 6 in an organic solvent and perform nitration with a nitration reagent; Step 6:H ２ / Pd / C, Fe or Zn / HOAc, or SnCl ２ Under conditions containing HCl, the nitro group of compound 7 is reduced to an amino group to obtain stereochemically pure compound 8.

27. A method for producing a conjugate according to any one of claims 2 to 10, wherein the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (VI) and the compound of formula (VII): [Transformation 50] In the formula, the definition of X and the conditions for the condensation reaction are as described in any one of claims 8 to 10.

28. A method for producing a conjugate according to claim 27, wherein the synthesis of the compound of formula (VI) comprises one or more of the following steps: 【Chemistry 51】

29. A method for producing a conjugate according to claim 28, wherein the synthesis of formula (VI) includes one or more of the following steps: Step 1: Compound 1 is condensed with a suitable hydroxyl-containing compound in the presence of a condensation reagent to obtain a reactive acid derivative compound 2; 【Chemistry 52】 Step 2: Compound 2 and compound 3 are condensed in an aqueous phase under specific pH conditions or in an organic phase containing an organic or inorganic base to obtain compound 4; 【Chemistry 53】 Step 3: PG amino protecting group of compound 4 ４ The compound 5 is obtained by removing it under deprotection conditions; 【Chemistry 54】 Step 4: Compound 5 and the compound of formula (IV) are condensed in an aqueous phase under specific pH conditions, or in an organic phase containing an organic or inorganic base, to obtain compound 6; 【Transformation 55】 Step 5: Under appropriate deprotection conditions, the amino protecting group PG of compound 6 １ Remove the compound VI; 【Transformation 56】

30. A method for producing a conjugate according to any one of claims 27 to 29, wherein the synthesis of the compound of formula (VII) comprises one or more of the following steps: 【Chemistry 57】

31. A method for producing the conjugate according to claim 30, wherein the synthesis of the compound of formula (VII) comprises one or more of the following steps: Step 1: A reactive ester is obtained by reacting carboxylic acid 1 and a suitable hydroxyl-containing compound in the presence of a condensation reagent. Alternatively, in the presence of an organic base, carboxylic acid compound 1 is reacted with ethyl chloroformate or isobutyl chloroformate to obtain a reactive mixed anhydride. Alternatively, acid chlorides are obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base and a catalytic amount of DMF. 【Transformation 58】

32. A method for producing a conjugate according to any one of claims 2 to 10, wherein the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (VIII) and the compound of formula (IX): 【Chemistry 59】 In the formula, the definition of X and the conditions for the condensation reaction are as described in any one of claims 8 to 10.

33. Equation (VIII) NH ２ A method for producing a conjugate according to claim 32, wherein the group participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

34. A method for producing a conjugate according to claim 32 or 33, wherein the synthesis of formula (VIII) includes one or more of the following steps: 【Transformation 60】

35. A method for producing a conjugate according to claim 34, wherein the synthesis of the compound of formula (VIII) comprises one or more of the following steps: Step 1: Under deprotection conditions, the carboxyl protecting group PG of compound 1 ３ Remove the compound to obtain compound 2; 【Chemistry 61】 Step 2: Reactive ester compound 3 is obtained by reacting compound 2 and a hydroxyl-containing compound in the presence of a condensation reagent; 【Transformation 62】 Step 3: Compound 3 and compound 4 are condensed in an aqueous phase under appropriate pH conditions, or in an organic phase containing an organic or inorganic base, to obtain compound 5; 【Transformation 63】 Step 4: Under deprotection conditions, the amino protecting group PG of compound 5 ３ Remove the compound to obtain compound 6; 【Chemistry 64】 Step 5: Compound 6 and the compound of formula (IV) described in claims 18 to 21 are condensed in an aqueous phase under appropriate pH conditions, or in an organic phase containing an organic base or an inorganic base, to obtain compound 7; 【Transformation 65】 Step 6: Under deprotection conditions, the amino protecting group PG of compound 7 １ Remove the compound to obtain compound VIII. 【Chemical Formula 66】

36. A method for producing a conjugate according to any one of claims 32 to 35, wherein the synthesis of formula (IX) includes one or more of the following steps: Reactive ester IX is obtained by condensing carboxylic acid compound 1 and a suitable hydroxyl-containing compound in the presence of a condensation reagent. Alternatively, in the presence of an organic base, carboxylic acid compound 1 is reacted with ethyl chloroformate or isobutyl chloroformate to obtain a reactive mixed anhydride IX. Alternatively, acid chloride IX is obtained by reacting carboxylic acid compound 1 with oxalyl chloride in the presence of an organic base and a catalytic amount of DMF. 【Transformation 67】

37. A method for producing a conjugate according to any one of claims 2 to 10, wherein the compound of formula (II) is obtained by a condensation reaction between the compound of formula (X) and the compound of formula (XI): 【Transformation 68】 In the formula, Y １ and Y ２ They condense to form a Y group; Y １ and Y ２ These are NH2 and - respectively. ＋ NH ３ , COOH, COX, SO ２ Cl, P(O)Cl ２ , NHCOX, NHSO ２ Cl, NHP(O)Cl ２ , NHP(O)(OH)Cl, 【Transformation 69】 That is the case.

38. A method for producing a conjugate according to claim 37, wherein the synthesis of the compound of formula (X) comprises one or more of the following steps: 【Transformation 70】

39. A method for producing a conjugate according to claim 38, wherein the synthesis of formula (X) includes one or more of the following steps: Step 1: Compound 2 is obtained by condensing compound 1 containing a carboxyl group with compound VI in the presence of a condensing agent or by other indirect condensation reaction pathways, where Z １ Y １ It is a precursor of; 【Chemistry 71】 Step 2: Under deprotection conditions, the amino protecting group PG of compound 2 １ Remove the compound to obtain compound 3; 【Chemistry 72】 Step 3: Compound 5 is obtained by condensing carboxylic acid 4 and amine 3 in the presence of a condensing agent or by other indirect condensation reaction pathways; 【Transformation 73】 Step 4: Functional group Z of compound 5 １ functional group Y １ This is converted to obtain compound X. 【Chemistry 74】

40. A method for producing a conjugate according to any one of claims 37 to 39, wherein the synthesis of the compound of formula (XI) comprises one or more of the following steps: 【Chemistry 75】

41. A method for producing a conjugate according to claim 40, wherein the synthesis of formula (XI) includes one or more of the following steps: Step 1: Compound 1 is dissolved in an organic solvent, then dehydrogenated with a base, and then reacted with compound 2 (where X is a halogen or other leaving group) by stirring at an appropriate temperature to obtain compound 3; Step 2: Deprotection of the carboxyl protecting group PG of compound 3 １ Remove the compound XIa-1; Step 3: Compound 1 is dissolved in an organic solvent, then dehydrogenated with a base, and compound 5 is obtained by stirring with compound 4 at an appropriate temperature; Step 4: Under deprotection conditions, the carboxyl protecting group PG of compound 5 １ Remove the compound XIa-2; Step 5: Compound 6 is dissolved in an organic solvent, and then reacted with methylsulfonyl chloride or 4-toluenesulfonyl chloride at 0-5°C in the presence of an organic base to obtain compound 7; Step 6: React compound 7 with ammonia solution in water or an organic solvent to obtain compound XIb; Step 7: Compound 7 and sodium azide are reacted in an organic solvent to obtain compound 8; Step 8: Compound XIb is obtained by hydrogenation in the presence of a palladium-carbon catalyst, or by reduction of azide compound 8 under the conditions of triphenylphosphine and water; Step 9: Compound 9 is obtained by heating compound 7 and dibenzylamine in an organic solvent to 100°C; Step 10: Compound 9 is dissolved in a solvent and hydrogenated in a hydrogen atmosphere in the presence of a palladium-carbon catalyst to obtain compound XIb.

42. A method for producing a conjugate according to any one of claims 2 to 10, wherein the compound of formula (II) is synthesized by a condensation reaction between the compound of formula (XII) and the compound of formula (XIII): 【Transformation 76】 In the formula, the definition of X and the conditions for the condensation reaction are as described in any one of claims 8 to 10.

43. NH of equation (XII) ２ A method for producing a conjugate according to claim 42, wherein the group participates in the reaction in the form of a salt with trifluoroacetic acid, hydrochloric acid, formic acid, acetic acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, succinic acid, benzoic acid, or sulfonic acid.

44. A method for producing a conjugate according to claim 42 or 43, wherein the synthesis of the compound of formula (XII) comprises one or more of the following steps: 【Chemical 77】

45. A method for producing a conjugate according to claim 44, wherein the synthesis of the compound of formula (XII) comprises one or more of the following steps: Step 1: Compound 1 is reacted with a hydroxyl-containing compound in the presence of a condensation reagent to obtain a reactive carboxylic acid derivative compound 2; 【Transformation 78】 Step 2: Compound 2 and compound 3 are condensed in an aqueous phase under specific pH conditions, or in an organic phase containing an organic base or an inorganic base, to obtain compound 4; 【Transformation 79】 Step 3: PG amino protecting group of compound 4 ４ This can be selectively removed under appropriate deprotection conditions, and the Boc protecting group on the amino group can be cleaved under acidic conditions; 【Chemistry 80】 Step 4: Compound 5 is condensed with a compound of formula (IV) (i.e., formula (IV) as described in any one of claims 18 to 21) in an aqueous phase under specific pH conditions, or in an organic phase containing an organic base or an inorganic base, to obtain compound 6; 【Chemistry 81】 Step 5: Amino protecting group PG of compound 6 １ This can be selectively removed under appropriate deprotection conditions, and the Boc protecting group on the amino group can be cleaved under acidic conditions, thereby obtaining compound XII. 【Chemistry 82】

46. A method for producing a conjugate according to any one of claims 42 to 45, wherein the synthesis of the compound of formula (XIII) comprises one or more of the following steps: 【Chemistry 83】

47. A method for producing a conjugate according to claim 46, wherein the synthesis of formula (XIII) includes one or more of the following steps: Step 1: Condensing carboxylic acid 1 and amine 2 in the presence of a condensing agent or by other indirect condensation reaction pathways to obtain compound 3; 【Chemical 84】 Step 2: Under deprotection conditions, the tert-butyl ester protecting group is cleaved, thereby removing the carboxyl protecting group PG of compound 3. １ Remove the compound to obtain compound 4; 【Chemical 85】 Step 3: In the presence of a condensing agent, carboxylic acid compound 4 and a hydroxyl-containing compound are reacted to obtain a reactive ester compound of formula (XIII); Alternatively, in the presence of an organic base, carboxylic acid compound 4 is reacted with ethyl chloroformate or isobutyl chloroformate to obtain the reactive mixed acid anhydride of formula (XIII); Alternatively, carboxylic acid compound 4 and oxalyl chloride are reacted in the presence of an organic base and a catalytic amount of DMF to obtain the acid chloride of formula (XIII). 【Chemical 86】

48. A conjugate having the following structure: 【Chemistry 87】

49. Compounds having the following structure: 【Chemical 88】

50. A pharmaceutical composition comprising the conjugate described in claim 1 or 48 and a pharmaceutically acceptable excipient.

51. Use of the conjugate according to any one of claims 1 or 48 for the preparation of a drug for treating cancer, an infectious disease, or an autoimmune disease.

52. Use of the compound according to claim 49 for the preparation of a drug for treating cancer, an infectious disease, or an autoimmune disease.

53. Use of the pharmaceutical composition according to claim 50 for preparing a drug for treating cancer, an infectious disease, or an autoimmune disease.

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