Branched ligature and conjugate of amanita toxin

Abstract

Provided herein is the conjugation of an Amanita toxin compound to a cell-binding molecule with a branched linker for better targeted therapy to abnormal cells. It also relates to a branched conjugation method for conjugating an Amanita molecule to a cell-binding ligand, and methods for using the conjugate in targeted therapy of cancer, infectious diseases, and autoimmune diseases.

Description

[Technical Field]

[0001] This invention relates to the conjugation of amanita toxin compounds with cell-binding molecules via branched (side-chain) linkers for better pharmacokinetics in the delivery of conjugated compounds, resulting in highly precise targeted therapy for abnormal cells. It also relates to branched linking methods for conjugating amanita toxin analog molecules to cell-binding ligands, as well as methods for using conjugates in targeted prevention or treatment of cancer, infectious diseases, and immunological disorders. [Background technology]

[0002] Over the past two decades, antibody-drug conjugates (ADCs), which are synergistic combinations of mAbs bound to small molecule chemotherapeutic drugs via stable conjugates, have emerged as a highly promising new class of biopharmaceuticals with a large and rapidly growing clinical pipeline. The three components of an ADC work together to produce a potent oncolytic agent that can directly deliver cytotoxins that would normally be untolerant to cancer cells, thereby allowing the cytodestructive agent to be internalized and released (Non-Patent Literature 1 and 2).

[0003] Early ADC therapy had the problem of shorter treatment durations compared to standard chemotherapy agents. However, technological developments regarding conjugates and the use of cytotoxic agents that are too potent to be administered directly have significantly improved the efficacy of ADCs (Non-Patent Documents 3 and 4). However, off-target toxicity remains a major challenge in the development of ADC drugs (Non-Patent Document 5). For example, in clinical practice, ad-trastuzumab emtansine (T-DM1, "Kadcyla" registered trademark), which uses a stable (non-cleavable) MCC conjugate, has shown great benefit to patients with HER2-positive metastatic breast cancer (mBC), or patients who have already been treated for mBC or who have experienced a recurrence of HER2 tumors within 6 months of adjuvant therapy (Non-Patent Documents 6-8). However, T-DM1 has failed in clinical trials 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, because the patient benefit was small compared to the efficacy and side effects (Non-Patent Documents 9-12).

[0004] To address the issue of off-target toxicity, research and development in the chemistry and design of ADCs is now expanding beyond just potent payloads to encompass the scope of conjugate-payload compartments and conjugation chemistry, particularly the conjugate-payload activity of ADCs against targeted / target diseases (Non-Patent Documents 13 and 14). Today, many pharmaceutical developers and academic institutions are focusing on establishing reliable, novel, specific conjugates and site-specific ADC conjugation methods that appear to have long circulating half-lives, high efficacy, potentially low off-target toxicity, and a narrow range of in vivo pharmacokinetic (PK) properties of ADCs, as well as improving batch-to-batch consistency in ADC production (Non-Patent Documents 15-19). These specific binding methods reported to date include the incorporation of manipulated cysteine ​​(Non-Patent Documents 20 and 21, Patent Documents 1-5), selenocysteine ​​(Non-Patent Documents 22 and 23, Patent Document 6), cysteine-containing tags including perfluoroaromatic reagents (Non-Patent Document 24), thiolfucose (Non-Patent Document 25), unnatural amino acids (Non-Patent Documents 26-29, Patent Documents 7-17); dibromomalemide (Non-Patent Document 30), bissulfone reagents (Non-Patent Document 31, Patent Documents 18 and 1 9) Conjugation to reduced intermolecular disulfide bonds by recrosslinking with dibromopyridazine (Non-Patent Literature 32), galactosyltransferase and sialyltransferase (Non-Patent Literature 33, Patent Literature 20), formylglycine-producing enzyme (FGE) (Non-Patent Literature 34, Patent Literature 21-25), phosphopantetheinyltransferase (PPTases) (Non-Patent Literature 35), and sortase A (Non-Patent Literature 36); genetically introduced glutamine tags (mTG) using streptoverticillium movalense transglutaminase (Non-Patent Literature 37 and 38, Patent Literature 26) or microbial transglutaminase (MTGase) (Non-Patent Literature 39 and 40, Patent Literature 27 and 28); and enzymes / bacteria that form isopeptide bond-peptide bonds formed on the outside of protein backbone (Non-Patent Literature 41-43). [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 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

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Non-licensed literature

[0006] [Non-licensed document 1] L. Ducry and B Stump, Bioconjugate Chem., 2010, 21, 5-13 [Non-licensed document 2] GS Hamilton / Biologicals 2015, 43, 318-32 [Non-licensed document 3] Bander, NH et al, Clin. Adv. Hematol. Oncol., 2012, 10, 1-16;

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[0007] We have disclosed several conjugation methods for recrosslinking a pair of thiols by reduction of intermolecular disulfide bonds in natural antibodies, e.g., bromomaleimide and dibromomaleimide conjugates (WO2014 / 009774), 2,3-disubstituted succinic acid / 2-monosubstituted / 2,3-disubstituted fumaric acid or malein conjugates (WO2015 / 155753, WO20160596228), acetylenedicarboxy conjugates (WO2015 / 151080, WO20160596228), or hydrazine conjugates (WO2015 / 151081). ADCs produced by these conjugates and methods exhibit a superior therapeutic index window compared to conventional non-selective conjugates via cysteine ​​or lysine residues on the antibody. Here, we disclose an invention of a conjugate of amanita toxin, including long side-chain conjugates. Long side-chain conjugates can prevent the antibody-drug conjugate from being hydrolyzed by hydrolytic enzymes, such as proteinases or esterases, and can make the conjugate more stable in circulation, thereby reducing side effects.

[0008] Amanita toxins, mainly amatoxins, farotoxins, and virotoxins (Wieland, T., Faulstich, H., CRC Crit. Rev. Biochem. 1978, 5(3):185-260; Vetter, J., Toxicon 1998, 36 (1): 13-24; Weiland, T., and Faulstich, H. 1983. Peptide Toxins from Amanita. p. 585-635. In: Handbook of Natural Toxins, Volume I: Plant and Fungal Toxins. RF Keeler and AT Tu, Ed. Marcel Dekker, Inc. New York, NY, Wieland, T., Int J. Pept. Protein Res., 1983, 22, 257-76), can be potent cytotoxic agents for antibody-drug conjugates (Zhao, R., et al. (WO2017046658). The amanita toxin conjugates of the present invention, including long branched conjugates, can extend the half-life of the conjugate during targeted delivery, minimize exposure to non-target cells, tissues, or organs in the bloodstream, and consequently reduce off-target toxicity, resulting in a wider therapeutic window for the conjugate.

[0009] This invention provides branched binding of amanita toxin to an antibody. It also provides a method for conjugating an antibody with an amanita toxin analog having a side chain linker. [Means for solving the problem]

[0010] In one aspect of the present invention, a conjugate including side chain bonds is represented by formula (I): [ka]

[0011] During the ceremony: [ka] represents a single bond; n is from 1 to 30;

[0012] T is a cell-binding agent / molecule selected from the group consisting of an antibody, a single-chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody, a chimeric antibody fragment that binds to a target cell, a domain antibody, a domain antibody fragment that binds to a target cell, an adnectin that mimics an antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony-stimulating factor, a nutrient transport molecule (transferrin), and / or a small molecule, a cell-binding peptide, or a protein attached to albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (virus) capsid;

[0013] L1 and L2 are chains of atoms selected from C, N, O, S, Si, and P, preferably having from 0 to 500 atoms, and are bonded to W and V1, and V1 and V2. The atoms used to form L1 and L2 may be bonded in any chemically related manner that forms an alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, alkoxyamine, urethane, amino acid, peptide, acyloxyamine, or hydroxamic acid, or a combination thereof. Preferably, L1 and L2 are independently the same or different and are O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R p , p’ , 3’ , p , p , p , p ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, or the formula (OCH2CH2) p OR3, (OCH2CH(CH3)) p OR3, NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2O) p R3]-[(CH2CH2O) p’ R 3’ , (OCH2CH2) pCOOR3, or CH2CH2 (OCH2CH2) p COOR3 polyethylene oxy units (wherein p and p' are integers independently selected from 0 to about 1000), or combinations thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or (Aa) r (r = 1 to 12 (1 to 12 amino acid units), consisting of the same or different sequences of dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides, composed of natural or non-natural amino acids.) are independently selected from these;

[0014] W is an extension unit, typically a self-destructing spacer, peptide unit, hydrazone, disulfide, thioether, ester, or amide bond; w is 1, 2, or 3;

[0015] V1 and V2 are independently O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl, or alkynyl, C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, or alkylcarbonyl, or (Aa)r (r=1-12 (1-12 amino acid units), where (Aa)r consists of natural or non-natural amino acids, or dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides of the same or different sequences), or (CH2CH2O) p A spacer unit selected from (p=0~1000); v1 and v2 are independently 0, 1, or 2, provided that v1 and v2 are simultaneously 0; if v1 or v2 is 0, it means that one of the side chain Q1 or Q2 fragments is absent;

[0016] Q1 and Q2 can be expressed independently by equation (I-q1): [ka]

[0017] During the ceremony, [ka] G1 is a site that binds to L1 or L2; G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, or (Aa) q1 G3 is OH, SH, OR 12 , SR 12 ,OC(O)R 12 NHC(O)R 12 , C(O)R 12 CH3, NH2, NR 12 , + NH(R 12 ), + N(R 12 )(R 12’ ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 12’ , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2)q1 C(O)OH, OC(O)NH(CH2) q1 C(O)OH, NHCO(CH2) q1 P(O)(OH)2, NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2, NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1 S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH, OS(O)2NH(CH2) q1 C(O)OH, NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR 12 ,Ar,ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 , or (Aa) q1 p1, p2, and p3 are independently between 0 and 100, but not simultaneously 0; q1 and q2 are independently between 0 and 24;

[0018] Preferably, Q1 and Q2 are independent of C2~C 90 Polycarboxylic acid; C2~C 90 Polyalkylamine; C6~C 90 Oligosaccharides or polysaccharides; C6~C 90Zwitterionic betaine or zwitterionic poly(sulfobetaine)) (PSB) containing quaternary ammonium cations and sulfonate anions; (poly(lactic acid / glycolic acid)) (PLGA), poly(acrylate), chitosan, copolymer of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethyl phosphorylcholine)] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycol) (PLG), poly(lactide-co-glycol), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(lactide-co-glycol), poly(ethylene Poly(ethylene glycol)-modified peptides, poly(ethylene glycol) containing amino acids or peptides, poly(ethylene glycol)-modified lipids, poly(ethylene glycol)-modified alkyl carboxylic acids, poly(ethylene glycol)-modified alkylamines, poly(lactide-co-glycolide), hyaluronic acid (HA) (glycosaminoglycan), heparin / heparan sulfate (HSGAG), chondroitin sulfate / dermatan sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfates, poly(ethylene glycol)-modified alkyl phosphates, or poly(ethylene glycol)-modified alkylquaternary ammonium compounds, comprising C6-C 100 Biodegradable polymers;

[0019] D is an amanita toxin having formula (II), or an isotope of a chemical element, or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or a polymorphic crystalline structure; or its optical isomer, racemate, diastereomer, or enantiomer: [ka]

[0020] During the ceremony, [ka] This is an independent linking site that connects with W;

[0021] A single bond on an aromatic (indole) ring means that it is bonded to any one of the carbon positions on the aromatic ring;

[0022] [ka] The symbol () can optionally indicate a single bond or the absence of a bond;

[0023] R1 and R2 are independently H, OH, CH2OH, CH(OH)CH2OH, CH(CH3)CH2OH, CH(OH)CH3, C1-C8 alkyl, -OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12 (Ester), -OC(=O)OR 12 (Carbonate), -OC(=O)NHR 12 (Carbamates); selected from C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, and alkylcarbonyl;

[0024] R3 and R4 are independently H, OH, -OR 12 (Ether), -OCOR 12 (Ester), -OCOCH3 (Acetate), -OCOOR 12 (Carbonate), -OC(=O)NHR 12 (Carbamate), -OP(O)(OR 12 )(OR12')(phosphate), OP(O)(NHR 12 )(NHR 12’ (Phosphamide), O-SO3 - , or selected from O-glycosides;

[0025] R5 is H, OH, NH2, NHOH, NHNH2, -OR 12 , -NHR 12 NHNHR 12 , -NR 12 R 12’ , N(H)(R 12 )R 13 CO(Aa) r(Selected from amino acids or peptides; Aa is an amino acid or polypeptide, and r is between 0 and 100.)

[0026] R6 is H, OH, CH2OH, CH(OH)CH2OH, CH(CH2OH)2, CH(CH3)OH, CH2CH2OH, PrOH, BuOH, C1~C8 alkyl, -OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12 (Esters); selected from C3-C8 aryl, heterocyclic, or carbocyclic rings;

[0027] R7, R8, and R9 are independently H, OH, CH3, CH(CH3)2, CH(CH3)CH2CH3, CH2OH, CH(OH)CH2OH, CH2CH(OH)CH2OH, CH(CH2OH)2, CH2C(OH)(CH2OH)2, CH2C(OH)(CH3)(CH2OH), CH2C(OH)(CH(CH3)2)(CH2OH), CH2CH2OH, PrOH, BuOH, CH2COOH, CH2CH2COOH, CH(OH)COOH, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2CH2CH2NHC(=NH)NH2, C1~C8 alkyl, CH2 ar, CH2 fin, CH2SR 12 CH2SSR 12 ,CH2SSAr,CH2CH2SCH3,-OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12 (Esters); selected from C3-C8 aryl, heterocyclic, or carbocyclic rings;

[0028] R 10 and R 11 These are independently H, NH2, OH, SH, NO2, halogen, -NHOH, -N3 (azide); -CN (cyano); C1-C8 alkyl, C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic, or carbocyclic; -OR 12 (Ether), -OCOR 12 (Ester), -OCOCH3 (acetate), -OC(O)OR12 (carbonate), -OC(O)CH(R 12 )NHAa (Aa is an amino acid group), -NR 12 R 12’ (amine), -NR 12 COR 12’ (amine), -R 12 NHCOR 12’ (alkylamide), -R 12 NHR 12’ (amine), -NHR 12 NHR 12’ NHR 12’’ (amine); -R 12 NCO - NR 12’ (urea), -R 12 NCOOR 12’ (carbamate), -OCONR 12 R 12’ (carbamate); -NR 12 (C = NH)NR 12’ R 〔 12’’ (guanidinium); -R 12 NHCO(Aa) p , -R 12 NHR 12’ CO(Aa) p , -NR 12 CO(Aa) p , (amino acid or peptide, Aa is an amino acid or polypeptide, p represents 0 - 6); -N(R 12 )CONR 12’ R 12’’ (urea); -OCSNHR 12 (thiocarbamate); -R 12 SH (thiol); -R 12 SR 12’ (sulfide); -R 12 SSR 12’ (disulfide); -S(O)R 12 (sulfoxide); -S(O2)R 12 (sulfone); -SO3, HSO3, HSO2, or HSO3 - , SO3 2- ]》, or -HSO2 - salt (sulfite); -OSO3 - ; -N(R 12 )SOOR ` 12’(sulfonamide); H2S2O5 or S2O5 2- Salts of (methabisulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2, or PO3S 3- , PO2S2 3- POS3 3- PS4 3- Salts of (mono-, di-, tri-, and tetrathiophosphates); (R 12 O)2POSR 12’ (Thiophage ester); HS2O3 or S2O3 2- (thiosulfate); HS2O4 or S2O4 2- (Dithionite); (P(=S)(OR 12 )(S)(OH) or a salt formed with a cation (phosphodithioate);-N(R 12 )OR 12’ (Hydroxyamine derivative); R 12 Salts formed with C(=O)NOH or cations (hydroxamic acid); (HOCH2SO2) - or its salt (formaldehyde sulfoxylate);-N(R 12 )COR 12’ (amide); R 12 R 12’ R 12’’ NPO3H (trialkylphosphoramide or phosphoramic acid); or ArAr'Ar''NPO3H (triarylphosphonium); OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1; O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc.), NH-glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR 1’ R 2’ R 3’ And here, R 1’ , R 2’ , and R 3’ These are independently H, C1-C8 alkyl groups; Ar, Ar', and Ar'' are C3-C8 aryl or heteroaromatic groups;

[0029] R 12 , R12’ , and R 12’’ These are independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, arninyl, heteroalkyl; C3-C8 aryl, heteroaryl, heterocyclic, or carbocyclic, or none at all;

[0030] X is S, O, NH, SO, SO2, or CH2;

[0031] m' is 0 or 1; n is between 1 and 30.

[0032] In another embodiment of the present invention, a conjugate including a side chain bond is represented by formula (III): [ka]

[0033] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, n, and T have the same definitions as in formula (I).

[0034] In another aspect of the present invention, the side-chain compound is represented by formula (IV), which can readily react with cell-binding molecule T to form a conjugate of formula (I): [ka]

[0035] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, and n have the same definitions as in formula (I); Lv1 is a functional group described below:

[0036] In another aspect of the present invention, the side-chain compound is represented by formula (V), which can readily react with cell-binding molecule T to form a conjugate of formula (III): [ka]

[0037] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, and n are defined in the same way as in formula (I);

[0038] Lv1 and Lv2 are reactive groups that can react with thiols, amines, carboxylic acids, selenols, phenols, or hydroxyl groups on cell-binding molecules. Lv1 and Lv2 are selected from OH; F; Cl; Br; I; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate; anhydrides formed by themselves or with other anhydrides, such as acetic anhydride or formic anhydride; or intermediate molecules produced by condensation reagents for peptide coupling reactions or Mitsunobu reactions. The aforementioned condensation reagents are EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide meso-p-toluenesulfonate (CMC, or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate (H BTU), (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethylcyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b] Pyridine-1-ium-3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxide-2-pyridyl)thiuronium hexafluorophosphate, O -(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate (TPTU), S-(1-oxide-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbeniumhexa Fluorophosphate (COMU), O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (with or without polymer linkage), dipyrrolidino(N-succinimidyloxy)-carbenium hexafluorophosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCIU), 2-chloro-1,3-dimethylimidazolinium Tetrafluoroborate (CIB), (benzotriazole-1-yloxy)dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA, T3P®), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (MMTM,DMTMM), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uroniumtetrafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy Selected from ronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butylazodicarboxylate (DBAD), diisopropylazodicarboxylate (DIAD), and diethylazodicarboxylate (DEAD). Lv1 and Lv2 may be anhydrides formed by the acid itself or by other C1-C8 anhydrides.

[0039] The present invention further relates to a method for producing cell-binding molecule-drug conjugates of formula (I) and formula (III), and to the application of conjugates of formula (I) and formula (III). [Brief explanation of the drawing]

[0040] [Figure 1] The general synthesis of components of amatoxin analogs is shown. [Figure 2] The synthesis of components of amatoxin analogs is shown. [Figure 3] This shows the synthesis of amatoxin analogs. [Figure 4] This shows the synthesis of amatoxin analogs. [Figure 5] This shows the synthesis of amatoxin analogs including side chain links. [Figure 6] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 7] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 8] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 9] This shows the synthesis of amatoxin analogs including side chain links. [Figure 10] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 11] This shows the synthesis of amatoxin analogs including side chain links. [Figure 12] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 13] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 14] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 15] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 16] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 17] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 18] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 19] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 20] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 21] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 22] This shows the synthesis of amatoxin analogs including side chain links. [Figure 23] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 24]This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 25] This demonstrates the synthesis of amatoxin analogs, including side chain linkers, and their conjugation to antibodies. [Figure 26] This shows conjugated forms of amatoxin analogs, including side chain links. [Figure 27] This study compares the antitumor effects of conjugated compounds 78a, 146, 154, 167, 197, 198, 216, 240, S-2, and T-DM1 administered intravenously at a dose of 6 mg / kg using a human gastric cancer cell model (N87). [Figure 28] This paper presents acute toxicity tests of ADC conjugates 154, 146, 216, S-2, and T-DM1 by observing changes in mouse body weight (BW) over 12 days. [Modes for carrying out the invention]

[0041] definition

[0042] "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 low-carbon alkyl groups, such as methyl, ethyl, or propyl groups, are bonded. Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and 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.

[0043] "Halogen" refers to fluorine, chlorine, bromine, or iodine atoms, with fluorine and chlorine atoms being preferred.

[0044] 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.

[0045] A "carbocycle" refers to a saturated or unsaturated ring with 3 to 8 carbon atoms in a monocyclic system or 7 to 13 carbon atoms in a bicyclic system. Monocyclic carbocycles have 3 to 6, more typically 5 or 6, ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms and are arranged as bicyclic systems [4,5], [5,5], [5,6], or [6,6], or they have 9 to 10 ring atoms and are arranged as bicyclic systems [5,6] or [6,6]. Representative C3-C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrielinyl, -cyclooctyl, and -cyclooctadienyl.

[0046] A C3-C8 carbocycle refers to a saturated or unsaturated non-aromatic hydrocarbon carbocyclic compound having 3, 4, 5, 6, 7, or 8 carbon atoms. The C3-C8 carbocycle may be unsubstituted or substituted with one or more substituents. The substituents are not limited to these, but include -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.

[0047] "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.

[0048] "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.

[0049] "Alkylene" refers to a saturated, linear, branched, or cyclic hydrocarbon group having 1 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), and 1,4-butyl (-CH2CH2CH2CH2-).

[0050] "Alkenylene" refers to an unsaturated, linear, branched, or cyclic hydrocarbon group having 2 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkene. Typical alkenylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0051] "Alkynylene" refers to an unsaturated, linear, branched, or cyclic hydrocarbon group having 2 to 18 carbon atoms, having two monovalent centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical alkynylene groups include, but are not limited to, acetylene, propargyl, and 4-pentinyl.

[0052] "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 one or more carbon atoms, preferably 1, 2, 3, or 4 carbon atoms, are replaced by O, N, Si, Se, P, or S, preferably O, S, and N. The terms aryl or Ar also refer to a case where one or more H atoms are independently replaced by -R', -halogen, -OR', or -SR', -NR'R'', -N=NR', -N=R', -NR'R'', -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O)2OR', -PR'R'', -P(O)R'R'', -P(OR')(OR''), -P(O)(OR')(OR''), or -OP(O)(OR')(OR''). The R', R'' are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, arylalkyl, carbonyl, or pharmaceutically salt.

[0053] A "heterocycle" is a ring system 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.

[0054] The term "heteroaryl" or aromatic heterocyclic refers to an aromatic hetero, monocyclic, bicyclic, or polycyclic ring with 5 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.

[0055] 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.

[0056] "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.

[0057] "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, but are not limited to, 2-benzimidazolylmethyl and 2-furylethyl.

[0058] Examples of "hydroxy protecting groups" include, but are not limited to, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate esters, substituted acetate esters, pivaloates, benzoates, methanesulfonates, and p-toluenesulfonates.

[0059] A "leaving group" refers to a functional group that can be substituted by another functional group. Such leaving groups are well known in the art and include, but are not limited to, halides (e.g., chlorides, bromides, and iodides), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. Preferred leaving groups are selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, acid anhydrides formed with the compound itself or other acid anhydrides (e.g., acetic anhydride, formic anhydride); or intermediates produced by condensation reagents for peptide coupling reactions or Mitsunobu reactions.

[0060] 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; DEAD, diethyl azodicarboxylate; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutyl hydrogenated Aluminum; DIPEA or DEA, diisopropylethylamine; DEPC, diethylphosphoroanidiate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; ESI-MS, electro Spray mass spectrometry; Depositphotos, ethyl acetate; Fmoc, N-(9-fluorenyl methoxycarbonyl); HATU, O-(7-azabenzotriazol-1-yl)-N,N,N'-N'-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high-pressure liquid chromatography; NHS, N-hydroxysuccinimide; MeCN, acetonitrile; MeOH, methanol; MMP, 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); Ph, phenyl; phe, L-phenylalanine; PyBrop, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Val, valine; TLC, thin-layer chromatography; UV, ultraviolet light.

[0061] "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 and include, for example, hydroxyproline, lanthionine, 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (a 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 8 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.

[0062] 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.

[0063] As used herein, the term “antibody” refers to a full-length immunoglobulin molecule or a molecule comprising an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immune-specifically binds to an antigen or part thereof of a significant target, such targets including, but not limited to, cancer cells or cell populations that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may originate from any species; however, preferably, immunoglobulins may be of human, mouse, or rabbit origin. Antibodies useful in the present invention are preferably monoclonal, and include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single-chain antibodies, Fv, Fab fragments, F(ab') fragments, F(ab')2 fragments, fragments generated by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs, and any of the above epitope-binding fragments that bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens.

[0064] Just as left and right hands are identical except that they are opposite each other along a single axis (simply changing direction does not make hands appear identical), enantiomers, also known as optical isomers, are two stereoisomers that are mirror images of each other but cannot be superimposed (they are not identical). A single chiral atom or similar structural feature within a compound can result in two non-superimal structures, each being a mirror image of the other. The presence of multiple chiral elements in a particular compound increases the number of possible geometric forms, but perfect mirror-image pairs may still exist. A pure enantiomeric compound refers to a sample with only one chirality within the detection limit. In a symmetric environment, enantiomers have identical chemical and physical properties except for their ability to rotate plane-polarized light (+ / -) by equal amounts in opposite directions (although polarization can be considered an asymmetric medium). For this reason, they are sometimes called optical isomers. A mixture of equal portions of an optically active isomer and its enantiomer is called a racemic mixture, and the net rotation of plane-polarized light is zero. This is because the positive rotation of each (+) form is precisely canceled out by the negative rotation of the (-) form. Members of an enantiomer often react differently with other enantiomers. Since many biomolecules are enantiomers, there can be significant differences in the effects of two enantiomers on living organisms. For example, in drugs, often only one of the drug's enantiomers is involved in the desired physiological effect, while the other enantiomer may be less active, inactive, or even harmful. This discovery allows for the development of drugs composed of only one enantiomer ("pure enantio") to enhance pharmacological effects and sometimes eliminate some side effects.

[0065] Isotopes are variations of a particular chemical element with different numbers of neutrons. All isotopes of a given element have the same number of protons in each atom. Each atomic number identifies a particular element, but not an isotope. Atoms of a given element can have a wide range of neutron numbers. The number of nucleons (both protons and neutrons) in the nucleus is the mass number of an atom, and each isotope of a particular element has a different mass number. For example, carbon-12, carbon-13, and carbon-14 are three isotopes of carbon with mass numbers 12, 13, and 14, respectively. The atomic number of carbon is 6. This is because all carbon atoms have 6 protons, so the neutron numbers of these isotopes are 6, 7, and 8, respectively. A hydrogen atom has protium ( 1 H), deuterium ( 2 H), and tritium ( 3 There are three isotopes of protium (H), with deuterium having twice the mass of protium and tritium having three times the mass of protium. Isotope substitution can be used to determine the mechanisms of chemical reactions or to investigate kinetic isotope effects. Isotope substitution can be used to investigate not only metabolic changes of substances in the body (e.g., by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), but also how the body is affected by specific xenobiotics / chemicals after administration through mechanisms of absorption and distribution, as well as the excretion pathways and effects of drug metabolites. This study is called pharmacokinetics (PK). Isotope substitution can be used to study the biochemical and physiological effects of drugs. Effects include those that appear in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). This study is called pharmacodynamics (PD). Effects include those that appear in animals (including humans), microorganisms, or combinations of organisms (e.g., infections). Both together influence the administration, benefits, and adverse effects of a drug. Isotopes include elements that are either stable (non-radioactive) or unstable. Isotope substitution of drugs may result in therapeutic effects different from those of the original drug.

[0066] "Pharmacologically" or "pharmaceutically acceptable" means that the corresponding compound or compound composition, when administered appropriately to animals or humans, is not harmful, allergic, or otherwise adverse.

[0067] A "pharmaceutically acceptable solvate" or "solvate" refers to the association of one or more solvent molecules with the disclosed compound. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] "Administering" or "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 intends 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.

[0072] In the context of cancer, the term “treat” includes any or all of the following: inhibiting the growth of tumor cells or cancer cells, preventing the replication of tumor cells or cancer cells, reducing the overall burden of the tumor, and improving one or more symptoms associated with the disease.

[0073] In the context of autoimmune diseases, the term “treat” includes any or all of the following: preventing the replication of cells, but not limited to, that are associated with the pathogenesis of the autoimmune disease, that are capable of producing autoimmune antibodies; reducing the burden of autoimmune antibodies; and improving one or more symptoms of the autoimmune disease.

[0074] In the context of infectious diseases, the term “to treat” includes preventing the growth, proliferation, or replication of the pathogen causing the infection, and improving one or more symptoms of the infection, or all of these.

[0075] Examples of “mammals” or “animals” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry.

[0076] The novel conjugates disclosed herein utilize crosslinked connectors. Several examples of suitable connectors and their synthesis are shown in Figures 1-26.

[0077] Cytotoxic molecules via conjugation of cell binding agents' side chains

[0078] In one aspect of the present invention, a conjugate including side chain bonds is represented by formula (I): [ka]

[0079] During the ceremony, [ka] represents a single bond; n is between 1 and 30;

[0080] T is a cell-binding agent / molecule selected from the group consisting of antibodies, single-chain antibodies; antibody fragments that bind to target cells; monoclonal antibodies; single-chain monoclonal antibodies; monoclonal antibody fragments that bind to target cells; chimeric antibodies; chimeric antibody fragments that bind to target cells; domain antibodies; domain antibody fragments that bind to target cells; antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; nutrient transport molecules (transferrin); and / or small molecules attached to albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles, or (viral) capsids, cell-binding peptides, or proteins;

[0081] L1 and L2 are chains of atoms selected from C, N, O, S, Si, and P, preferably having 0 to 500 atoms, and bonded to W and V1, and V1 and V2. The atoms used to form L1 and L2 may be bonded in any chemically relevant way, such as forming alkylene, alkenylene, alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine, alkoxyamine, urethane, amino acid, peptide, acyloxyamine, or hydroxamic acid, or combinations thereof. Preferably, L1 and L2 are independently the same or different, O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R 3’ ), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, or formula (OCH2CH2) p OR3, (OCH2CH(CH3)) p OR3, NH(CH2CH2O) p R3, NH(CH2CH(CH3)O) p R3, N[(CH2CH2O) p R3]-[(CH2CH2O) p’ R 3’ ], (OCH2CH2) p COOR3, or CH2CH2 (OCH2CH2) p COOR3 polyethylene oxy units (wherein p and p' are integers independently selected from 0 to about 1000), or combinations thereof; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or (Aa) r(r = 1 to 12 (1 to 12 amino acid units), consisting of the same or different sequences of dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides, composed of natural or non-natural amino acids.) are independently selected from these;

[0082] W is C1~C 18 It is an extension unit, typically a self-destructing spacer, peptide unit, hydrazone, disulfide, thioether, ester, or amide bond; w is 1, 2, or 3;

[0083] V1 and V2 are independently O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl, or alkynyl, C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, or alkylcarbonyl, or (Aa)r (r=1-12 (1-12 amino acid units), where (Aa)r consists of natural or non-natural amino acids, or dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides of the same or different sequences), or (CH2CH2O) p A spacer unit selected from (p=0~1000); v1 and v2 are independently 0, 1, or 2, provided that v1 and v2 are simultaneously 0; if v1 or v2 is 0, it means that one of the side chain Q1 or Q2 fragments is absent;

[0084] Q1 and Q2 can be expressed independently by equation (I-q1): [ka]

[0085] During the ceremony, [ka] G1 is a site that binds to L1 or L2; G1 and G2 are independently OC(O), NHC(O), C(O), CH2, NH, OC(O)NH, NHC(O)NH, O, S, B, P(O)(OH), NHP(O)(OH), NHP(O)(OH)NH, CH2P(O)(OH)NH, OP(O)(OH)O, CH2P(O)(OH)O, NHS(O)2, NHS(O)2NH, CH2S(O)2NH, OS(O)2O, CH2S(O)2O, Ar, ArCH2, ArO, ArNH, ArS, ArNR1, or (Aa) q1 G3 is OH, SH, OR 12 , SR 12 ,OC(O)R 12 NHC(O)R 12 , C(O)R 12 CH3, NH2, NR 12 , + NH(R 12 ), + N(R 12 )(R 12’ ), C(O)OH, C(O)NH2, NHC(O)NH2, BH2, BR 12 R 12’ , P(O)(OH)2, NHP(O)(OH)2, NHP(O)(NH2)2, S(O)2(OH), (CH2) q1 C(O)OH, (CH2) q1 P(O)(OH)2, C(O)(CH2) q1 C(O)OH, OC(O)(CH2) q1 C(O)OH, NHC(O)(CH2) q1 C(O)OH, CO(CH2) q1 P(O)(OH)2, NHC(O)O(CH2) q1 C(O)OH, OC(O)NH(CH2) q1 C(O)OH, NHCO(CH2) q1 P(O)(OH)2, NHC(O)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 P(O)(OH)2, NHS(O)2(CH2) q1 C(O)OH, CO(CH2) q1S(O)2(OH), NHS(O)2NH(CH2) q1 C(O)OH, OS(O)2NH(CH2) q1 C(O)OH, NHCO(CH2) q1 S(O)2(OH), NHP(O)(OH)(NH)(CH2) q1 C(O)OH, CONH(CH2) q1 S(O)(OH), OP(O)(OH)2, (CH2) q1 P(O)(NH)2, NHS(O)2(OH), NHS(O)2NH2, CH2S(O)2NH2, OS(O)2OH, OS(O)2OR1, CH2S(O)2OR 12 ,Ar,ArR 12 , ArOH, ArNH2, ArSH, ArNHR 12 , or (Aa) q1 (Aa) q1 X1 and X2 are peptides containing identical or different sequences of natural or non-natural amino acids; X1 and X2 independently contain O, CH2, S, S(O), NHNH, NH, N(R) 12 ), + NH(R 12 ), + N(R 12 )(R 12’ ), C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH; Y2 is O, NH, NR 12 , CH2, S, NHNH, Ar; R 12 , R 12’ , R 13 , and R 13’ Y2 is independently H, C1-C8 alkyl; C2-C8 heteroalkyl, or heterocyclic; C3-C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; Y2 is O, NH, NR 12 , CH2, S, NHNH, Ar; p1, p2, and p3 are independently 0 to 100 but not simultaneously 0; q1 and q2 are independently 0 to 24; R 12 , R 12’ , R 13 , and R 13’These are independently H, C1-C8 alkyl; C2-C8 heteroalkyl or heterocyclic; C3-C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;

[0086] Preferably, Q1 and Q2 are independent of C2~C 100 Polycarboxylic acid; C2~C 90 Polyalkylamine; C6~C 90 Oligosaccharides or polysaccharides; C6~C 100 Zwitterionic betaine or zwitterionic poly(sulfobetaine)) (PSB) containing quaternary ammonium cations and sulfonate anions; (poly(lactic acid / glycolic acid)) (PLGA), poly(acrylate), chitosan, copolymer of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethyl phosphorylcholine)] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycol) (PLG), poly(lactide-co-glycol), poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(lactide-co-glycol), poly(ethylene Poly(ethylene glycol)-modified peptides, poly(ethylene glycol) containing amino acids or peptides, poly(ethylene glycol)-modified lipids, poly(ethylene glycol)-modified alkyl carboxylic acids, poly(ethylene glycol)-modified alkylamines, poly(lactide-co-glycolide), hyaluronic acid (HA) (glycosaminoglycan), heparin / heparan sulfate (HSGAG), chondroitin sulfate / dermatan sulfate (CSGAG), poly(ethylene glycol)-modified alkyl sulfates, poly(ethylene glycol)-modified alkyl phosphates, or poly(ethylene glycol)-modified alkylquaternary ammonium compounds, comprising C6-C 100 It is a biodegradable polymer;

[0087] Examples of the structures of Q1 and Q2 are shown below: [ka] TIFF0007894620000016.tif212170

[0088] In the formula, R 25 and R 25’ These are independently H;HC(O), CH3C(O), CH3C(NH), NH-(C1-C 18 )alkyl, C(O)NH-(C1-C 18 )alkyl, C(O)-(C1-C 18 ) Alkyl, C1-C 18 Alkyl, C1-C 18 Alkyl, alkyl-Y1-SO3H, C1-C 18 Alkyl-Y1-PO3H2, C1-C 18 Alkyl-Y1-CO2H, C1-C 18 Alkyl-Y1-N + R 12 R 13 R 13’ R 14 , C1-C 18 Alkyl-Y1-CONH2, C2-C 18 Alkylene, C2-C 18 Ester, C2-C 18 Ether, C2-C 18 Amine, C2-C 18 Alkylcarboxylamide, C3-C 18 Ariel, C3-C 18 Cyclic alkyl, C3-C 18 Heterocyclic ring, 1-24 amino acids; C2-C 18 lipids, C2-C 18 Fatty acids, or C2-C 18 Selected from fatty ammonium lipids; X1 and X2 are independently NH, N(R) 12’ ), O, CH2, S, C(O), S(O), S(O2), P(O)(OH), NHNH, CH=CH, Ar, or (Aa)q1 are selected, where q1 = 0 to 24 (0 to 24 amino acids, q1 = 0 means absent); X1, X2, X3, X4, Y1, Y2, and Y3 are independently NH, N(R) 12’), O, C(O), CH2, S, S(O), NHNH, C(O), OC(O), OC(O)O, OC(O)NH, NHC(O)NH, Ar or Ar or (Aa)q1 are selected, and X1, X2, X3, X4, Y1, Y2, and Y3 are independently optional; p1, p2, and p3 are independently 0 to 100 but not 0; q1, q2, and q3 are independently 0 to 24; R 12 , R 13 , R 13’ , and R 14’ (Aa) is independently selected from H and C1-C6 alkyl groups; (Aa) is a natural or non-natural amino acid; (Aa)q1 is the same or different sequence of the peptide, and q1=0 means that (Aa)q1 is absent.

[0089] D is the amanita toxin of formula (II) below, or an isotope of a chemical element, or a pharmaceutically acceptable salt, hydrate, or hydrated salt; or a polymorphic crystalline structure; or its optical isomer, racemate, diastereomer, or enantiomer: [ka]

[0090] During the ceremony [ka] This is an independent linking site that connects with W;

[0091] A single bond on an aromatic (indole) ring means that it is bonded to any one of the carbon positions on the aromatic ring;

[0092] [ka] The symbol () can optionally indicate a single bond or the absence of a bond;

[0093] R1 and R2 are independently H, OH, CH2OH, CH(OH)CH2OH, CH(CH3)CH2OH, CH(OH)CH3, C1-C8 alkyl, -OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12 (Ester), -OC(=O)OR 12 (Carbonate), -OC(=O)NHR 12 (Carbamates); selected from C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, and alkylcarbonyl;

[0094] R3 and R4 are independently H, OH, -OR 12 (Ether), -OCOR 12 (Ester), -OCOCH3 (Acetate), -OCOOR 12 (Carbonate), -OC(=O)NHR 12 (Carbamate), -OP(O)(OR 12 )(OR12')(phosphate), OP(O)(NHR 12 )(NHR 12’ (Phosphamide), O-SO3 - , or selected from O-glycosides;

[0095] R5 is H, OH, NH2, NHOH, NHNH2, -OR 12 , -NHR 12 NHNHR 12 , -NR 12 R 12’ , N(H)(R 12 )R 13 CO(Aa) p (Selected from amino acids or peptides; Aa is an amino acid or polypeptide, and p is 0-6.)

[0096] R6 is H, OH, CH2OH, CH(OH)CH2OH, CH(CH2OH)2, CH(CH3)OH, CH2CH2OH, PrOH, BuOH, C1~C8 alkyl, -OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12(Esters); selected from C3-C8 aryl, heterocyclic, or carbocyclic rings;

[0097] R7, R8, and R9 are independently H, OH, CH3, CH(CH3)2, CH(CH3)CH2CH3, CH2OH, CH(OH)CH2OH, CH2CH(OH)CH2OH, CH(CH2OH)2, CH2C(OH)(CH2OH)2, CH2C(OH)(CH3)(CH2OH), CH2C(OH)(CH(CH3)2)(CH2OH), CH2CH2OH, PrOH, BuOH, CH2COOH, CH2CH2COOH, CH(OH)COOH, CH2CONH2, CH2CH2CONH2, CH2CH2CH2CH2NH2, CH2CH2CH2NHC(=NH)NH2, C1~C8 alkyl, CH2 ar, CH2 fin, CH2SR 12 CH2SSR 12 ,CH2SSAr,CH2CH2SCH3,-OR 12 (Ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR 12 (Esters); selected from C3-C8 aryl, heterocyclic, or carbocyclic rings;

[0098] R 10 and R 11 These are independently H, NH2, OH, SH, NO2, halogen, -NHOH, -N3 (azide); -CN (cyano); C1-C8 alkyl, C2-C8 alkenyl, alkynyl, heteroalkyl; C3-C8 aryl, heterocyclic, or carbocyclic; -OR 12 (Ether), -OCOR 12 (Ester), -OCOCH3 (acetate), -OC(O)OR 12 (Carbonate), -OC(O)CH(R 12 )NHAa(Aa is an amino acid group), -NR 12 R 12’ (amine), -NR 12 COR 12’ (amine), new R 12 NHCOR 12’ (alkylamide), -R 12 NHR 12’ (amine), -NHR 12 NHR12’ NHR 12’’ (amine);-R 12 NCO-NR 12’ (Urea), -R 12 NCOOR 12’ (Carbamate), -OCONR 12 R 12’ (Carbamate); -NR 12 (C=NH)NR 12’ R 12’’ (Guanidinium);-R 12 NHCO(Aa) p ,-R 12 NHR 12’ CO(Aa) p , -NR 12 CO(Aa) p , (amino acid or peptide, Aa is an amino acid or polypeptide, p represents 0-6);-N(R 12 )CONR 12’ R 12’’ (Urea);-OCSNHR 12 (Thioccarbamate);-R 12 SH (thiol); -R 12 SR 12’ (Sulfide); -R 12 SSR 12’ (Disulfide);-S(O)R 12 (sulfoxide);-S(O2)R 12 (Sulfone); -SO3, HSO3, HSO2, or HSO3 - , SO3 2- , or -HSO2 - Salt of (sulfite); -OSO3 - ;-N(R 12 )SOOR 12’ (sulfonamide); H2S2O5 or S2O5 2- Salts of (methabisulfites); PO3SH3, PO2S2H2, POS3H2, PS4H2, or PO3S 3- , PO2S2 3- POS3 3- PS4 3- Salts of (mono-, di-, tri-, and tetrathiophosphates); (R 12 O)2POSR 12’ (Thiophage ester); HS2O3 or S2O32- (thiosulfate); HS2O4 or S2O4 2- (Dithionite); (P(=S)(OR 12 )(S)(OH) or a salt formed with a cation (phosphodithioate);-N(R 12 )OR 12’ (Hydroxyamine derivative); R 12 Salts formed with C(=O)NOH or cations (hydroxamic acid); (HOCH2SO2) - or its salt (formaldehyde sulfoxylate);-N(R 12 )COR 12’ (amide); R 12 R 12’ R 12’’ NPO3H (trialkylphosphoramide or phosphoramic acid); or ArAr'Ar''NPO3H (triarylphosphonium); OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1; O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside, fructoside, etc.), NH-glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, NR 1’ R 2’ R 3’ And here, R 1’ , R 2’ , and R 3’ These are independently H, C1-C8 alkyl groups; Ar, Ar', and Ar'' are C3-C8 aryl or heteroaromatic groups;

[0099] In the formula, R 12 , R 12’ , and R 12’’ These are independently H, C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or esters, ethers, or amides having 1 to 8 carbon atoms; or formula (OCH2CH2) p Or (OCH2CH(CH3)) pA polyethylene oxy unit (where p is an integer from 0 to approximately 1000), or a combination of the above, or none;

[0100] X is S, O, NH, SO, SO2, or CH2;

[0101] m' is either 0 or 1. n is between 1 and 30.

[0102] An example of a preferred amanita toxin structure is shown below: [ka]

[0103] More preferably, the structure of amatoxin is selected from the following structures or isotopes of one or more chemical elements, pharmaceutically acceptable salts, hydrates, or hydrated salts; polymorphic crystalline structures of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: [ka] TIFF0007894620000022.tif238170 TIFF0007894620000023.tif246170 TIFF0007894620000024.tif246170 TIFF0007894620000025.tif238170 TIFF0007894620000026.tif56170

[0104] In the formula, Z2 is oxygen or a lone pair of electrons; R 15 H, NHR 12 , OR 12, C1-C8 linear or branched alkyl or heteroalkyl; C2-C8 linear or branched alkenyl, alkynyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 linear or branched aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; carbonate (-R1C(O)OR 12 ), Carbamate (-R 12 C(O)NR 12’ R 13 ); or carboxylates, esters, ethers, or amides having 1 to 8 carbon atoms; or 1 to 8 amino acids; or formula (OCH2CH2) p Or (OCH2CH(CH3)) p A polyethylene oxy unit having (p is an integer from 0 to about 1000); Z1 is H, O, S, NH, NHNH, or R 12 It is either true or does not exist; R 21 COR 12 , NHCOR 12 COOR 12 CONHR 12 , R 12 , R 12 NH is; R 22 R 12 , SR 12 SCH(CH3)R 12 SC(CH3)2R 12 X is O, S, NH, NHNH, or CH2; R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 12’ , R 13 , and X1 are defined as above.

[0105] Furthermore, W, L1, L2, V1, and V2 may independently contain one or more of the following: 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PAB), 4-thiopentanoate (SPP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid (MCC), (4-acetyl)aminobenzoic acid (SIAB), 4-thio-butyrate (SPDB), 4-thio-2-hydroxysulfonyl-butyrate (2-sulfo-SPDB), or one or more linked components of natural or unnatural peptides having 1 to 12 natural or unnatural amino acids. The aforementioned natural amino acids are preferably selected from aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenylalanine, glycine, proline, tryptophan, and alanine.

[0106] [ka] TIFF0007894620000028.tif254170 TIFF0007894620000029.tif170170 Alternatively, L- or D-, natural or non-natural peptides containing 1 to 20 identical or different amino acids;

[0107] During the ceremony [ka] is a binding site; X2, X3, X4, X5, or X6 independently are NH;NHNH;N(R 12 );N(R 12 )N(R 12’);O;S;C1-C6 alkyl;C2-C6 heteroalkyl, alkylcycloalkyl, heterocycloalkyl;C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl;or selected from 1 to 8 amino acids;wherein R 12 and R 12’ These are independently H; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl, heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl; or esters, ethers, or amides having 1 to 8 carbon atoms; or formula (OCH2CH2) p Or (OCH2CH(CH3)) p A polyethylene oxy unit having (p is an integer from 0 to approximately 1000); or a combination thereof.

[0108] W, L1, L2, V1, and V2 may also independently contain self-destructing or non-self-destructing components, peptide units, hydrazone bonds, disulfides, esters, oximes, amides, or thioether bonds. The self-destructing units include, but are not limited to, 2-aminoimidazole-5-methanol derivatives, heterocyclic PAB analogs, β-glucuronides, and aromatic compounds whose electronic structure is similar to that of a para-aminobenzylcarbamoyl (PAB) group, such as o- or p-aminobenzyl acetals.

[0109] Preferably, the self-destructing linkage component has one of the following structures: [ka]

[0110] In the formula, ( * ) Atoms are additional spacers or releaseable linkage units, cytotoxic agents, and / or binding sites of binding molecules (CBAs); X 1 , Y 1 , Z 2 , and Z 3NH, O, or S are independent of Z 1 These are independently H, NHR1, OR1, SR1, COX1R1, where X1 and R1 are defined as above; v is 0 or 1; U 1 These are independently H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NR5R5', NO2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5R5', PO2R5R5', OPO(OR5)(OR5'), or OCH2PO(OR5(OR5')), where R5 and R5' are independently selected from H, C1-C8 alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, or amino acid; C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or glycoside; or pharmaceutically active cation salts;

[0111] W, L1, L2, V1, and V2 may also independently contain a non-self-destructing conjugate component having one of the following structures: [ka] TIFF0007894620000033.tif170170

[0112] In the formula, ( * Atoms labeled with ) are additional spacers or releaseable linkers, cytotoxic agents, and / or binding molecules; X 1 , Y 1 , U 1 , R 5 and R 5’ The definition is as above; r is between 0 and 100; m and n are independently between 0 and 30;

[0113] More preferably, W, L1, L2, V1, and V2 may independently contain releaseable conjugate components. The term "releaseable conjugate" refers to a conjugate comprising at least one bond that can be cleaved under physiological conditions such as pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability, or enzymatic instability. It is understood that such physiological conditions resulting in bond rupture do not necessarily involve biological or metabolic processes, but instead may include standard chemical reactions such as hydrolysis or substitution reactions, e.g., disulfide bond exchange reactions with intracellular thiols such as glutathione in the millimolar range, which are abundant in endosomes and / or malignant cells, where the pH is lower than the cytoplasmic pH.

[0114] Examples of releaseable components of W, L1, L2, V1, and V2 include, but are not limited to, the following:-(CR 15 R 16 ) m (Aa) r (CR 17 R 18 ) n (OCH2CH2) t -,-(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -,-(Aa) r -(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -,-(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -,-(CR 15 R 16 ) m (CR 17 =R 18 )(CR19 R 20 ) n (Aa) t (OCH2CH2) r -、-(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (CO)(Aa) t -(CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-(CR 15 R 16 ) m (OCO)(Aa)t (CR 19 R 20 ) n -(OCH2CH2) r -,-(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -,-(CR 15 R 16 ) m -phenyl-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Frill-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Oxazolyl-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -Thiazolyl-(CO)(Aa) t (CCR 17 R 18 ) n -,-(CR 15 R 16 ) t -Chienil-(CO)(CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Imidazolyl-(CO)(CR17 R 18 ) n -,-(CR 15 R 16 ) t -Morphorino-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -Piperadino-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) t -N-methylpiperazine-(CO)(Aa) t (CR 17 R 18 ) n -,-(CR 15 R 16 ) m -(Aa) t Phenyl-,-(CR 15 R 16 ) m -(Aa) t Frill-,-(CR 15 R 16 ) m -Oxazolyl (Aa) t -,-(CR 15 R 16 ) m - Thiazolyl (Aa) t -,-(CR 15 R 16 ) m -Chienil (Aa) t -,-(CR 15 R 16 ) m -Imidazolyl (Aa) t -,-(CR 15 R 16 ) m -Morphorino-(Aa) t -,-(CR 15 R 16 ) m -Piperadino-(Aa) t -,-(CR 15 R 16 )m -N-メチルピペラジノ-(Aa) t -、-K(CR 15 R 16 ) m (Aa) r (CR 17 R 18 ) n (OCH2CH2) t -、-K(CR 15 R 16 ) m (CR 17 R 18 ) n (Aa) r (OCH2CH2) t -、-K(Aa) r (CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) t -、-K(CR 15 R 16 ) m (CR 17 R 18 ) n (OCH2CH2) r (Aa) t -、-K(CR 15 R 16 ) m (CR 17 =R 18 )(CR 19 R 20 ) n (Aa) t (OCH2CH2) r -、-K(CR 15 R 16 ) m (NR 11 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR5R6) m (Aa) t (NR 21 CO)(CR 19 R 20 ) n (OCH2CH2) r-、-K(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (NR 21 CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (OCNR 17 )(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR 15 R 16 ) m (CO)(Aa) t (CR 19 R 20 ) n (OCH2CH2) r -、-K(CR15 R 16 ) m -phenyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -Frill-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -Oxazolyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) m -Thiazolyl-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Chienil-(CO)(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Imidazolyl-(CO)(CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Morphorino-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -Piperadino-(CO)(Aa) t (CR 17 R 18 ) n -, -K(CR 15 R 16 ) t -N-methylpiperazine-(CO)(Aa) t (CR17 R 18 ) n -, -K(CR 15 R 16 ) m -(Aa) t Phenyl-,-K(CR 15 R 16 ) m -(Aa) t Frill-, -K(CR 15 R 16 ) m -Oxazolyl (Aa) t -, -K(CR 15 R 16 ) m - Thiazolyl (Aa) t -, -K(CR 15 R 16 ) m -Chienil (Aa) t -, -K(CR 15 R 16 ) m -Imidazolyl (Aa) t -, -K(CR 15 R 16 ) m -Morphorino-(Aa) t -, -K(CR 15 R 16 ) m -Piperadino-(Aa) t G-, -K (CR5R6) m -N-methylpiperazino-(Aa) t -;formula Middle, m, Aa, m, n, R 13 , R 14 , and R 15 The definition is as above; t and r are independently between 0 and 100; R 16 , R 17 , R 18 , R 19 , and R 20 Independently, H; halide; C1-C8 alkyl; C2-C8 aryl, alkenyl, alkynyl, ether, ester, amine, or amide, and one or more halides, CN, NR12 R 12’ CF3, OR 12 aryl, heterocyclic ring, S(O)R 12 SO2R 12 -CO2H, -SO3H, -OR 12 , -CO2R 12 ,-CONR 12 , -PO2R 12 R 12 -PO3H, or P(O)R 12 R 12’ R 13 Selected from those arbitrarily substituted; K is NR 12 , -SS-, -C(=O)-, -C(=O)NH-, -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, Het (heterocycle or C3~C 12 A heteroaromatic ring, or a peptide containing 1 to 20 amino acids.

[0115] More preferably, components W, L1, L2, V1, and V2 are independently linear alkyl groups having 1 to 6 carbon atoms, or of the formula (OCH2CH2) p The polyethylene oxy units (p=1 to 5000) having these units, or peptides (L or D form) containing 1 to 12 amino acids, or a combination of the above.

[0116] Selectively, one or more of W, Q1, Q2, L1, L2, V1, or V2 do not need to exist independently, but Q1 and Q2 cannot be absent at the same time.

[0117] When cell-binding molecule T is linked to V1 and / or V2, or when T is directly linked to L1 and / or L2 (where V1 and V2 are absent), the conjugated bond has one or more of the following binding structures: [ka] TIFF0007894620000035.tif254170

[0118] In the formula, R20 and R 21 These are independently C1-C8 alkyl; C2-C8 heteroalkyl or heterocyclic; C3-C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic or alkylcarbonyl; or (CH2CH2O) p C2-C has the formula 100 It is polyethylene glycol, and p is as defined above; or it is absent.

[0119] In another further embodiment, Q1 and Q2 are preferably C2~C 18 Lipids, or C2-C 18 Fatty acids, or C2-C 18The side chains are selected from polyalkylene glycols containing fatty ammonium lipids. The polyalkylene glycol chains not only help the conjugates become more hydrophilic during production, but also prevent the conjugated links from being hydrolyzed by hydrolytic enzymes, such as proteinases or esterases. The lipids can help the conjugates in mammalian blood bind to albumin, and slowly dissociate the conjugates from this complex during blood circulation. Thus, the side chain links of this patent application make the conjugates more stable in circulation. Here, polyalkylene glycols include, but are not limited to, poly(ethylene glycol) (PEG), poly(propylene glycol), and copolymers of ethylene oxide and propylene oxide. Particularly preferred is PEG, and even more particularly preferred is monofunctionally activated hydroxyPEG (e.g., single-termined activated hydroxyl PEG, including hydroxyPEG-monocarboxylic acid, hydroxyPEG-monoaldehyde, hydroxyPEG-monoamine, hydroxyPEG-monohydrazide, hydroxyPEG-monocarbazate, hydroxyl reactive esters, PEG-monoiodoacetamide, hydroxyl PEG-monomaleimide, hydroxyl PEG-monoorthopyridyl disulfide, hydroxyPEG-monoxime, hydroxyPEG-monophenylcarbonate, hydroxyl PEG-monophenylglyoxal, hydroxyl PEG-monothiazolidine-2-thion, hydroxyl PEG-monothioester, hydroxyl PEG-monothiol, hydroxyl PEG-monotriazine, and hydroxyl PEG-monovylsulfone). The polyalkylene glycol has a molecular weight of about 10 Da to about 200 kDa, preferably about 88 Da to about 40 kDa; each of the two branched chains has a molecular weight of about 88 Da to about 40 kDa; more preferably, each of the two branched chains has a molecular weight of about 88 Da to about 20 kDa. In a particular embodiment, the polyalkylene glycol is poly(ethylene) glycol and has a molecular weight of about 10 kDa; about 20 kDa; or about 40 kDa. In a particular embodiment, the PEG is PEG10 kDa (linear or branched), PEG20 kDa (linear or branched), or PEG40 kDa (linear or branched).The synthesis of linear or branched "non-antigenic" PEG polymers and their derivatives or conjugates is covered by numerous U.S. patents, e.g., U.S. Patents 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,1 This information is disclosed in 31;5,900,402;5,902,588;5,919,455;5,951,974;5,965,119;5,965,566;5,969,040;5,981,709;6,011,042;6,042,822;6,113,906;6,127,355;6,132,713;6,177,087; and 6,180,095.

[0120] Examples of formula (I) are the following structures, or isotopes of one or more chemical elements, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystalline structures of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: [ka] TIFF0007894620000037.tif246170 TIFF0007894620000038.tif229170 TIFF0007894620000039.tif246170 TIFF0007894620000040.tif221170 TIFF0007894620000041.tif238170 TIFF0007894620000042.tif229170 TIFF0007894620000043.tif254170 TIFF0007894620000044.tif60170

[0121] In the formula, X8 is O, S, NH, NHNH, NHR 12 , SR 12 SSR 12SSCH(CH3)R 12 SSC(CH3)2R 12 , or R 12 X1, X2, X3, X4, X5, p1, p2, q1, q2, m, n, R 25 , and mAb are the same as above; Aa is a natural or non-natural amino acid; r is 0 to 100; if r > 2, (Aa)r is a peptide containing the same or different amino acid sequences; r = 0 means that (Aa)r does not exist.

[0122] In one aspect of the present invention, a conjugated product including side chain bonds is represented by formula (III). [ka]

[0123] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, n, and T have the same definitions as in formula (I).

[0124] Examples of formula (III) are the following structures, or isotopes of one or more chemical elements, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystalline structures of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: [ka] TIFF0007894620000047.tif254170 TIFF0007894620000048.tif221170 TIFF0007894620000049.tif221170 TIFF0007894620000050.tif229170 TIFF0007894620000051.tif68170

[0125] In the formula, X8 is O, S, NH, NHNH, NHR 12 , SR 12 SSR 12, SSCH(CH3)R 12 , SSC(CH3)2R 12 , or R 12 ; X1, X2, X3, X4, X5, R 12 , R 12’ , R 13 , R 13’ , R 25 , R[[ID=~17]] 25’ , p1, p2, q1, q2, m, m1, n, and mAb are the same as above; Aa is a natural or non-natural amino acid; r is 0 to 12; when r > 2, (Aa)r is a peptide containing the same or different amino acid sequences; r = 0 means that (Aa)r does not exist.

[0126] In another aspect of the present invention, the side chain linking compound is represented by formula (IV), which can easily react with the cell binding molecule T or the modified cell binding molecule T to form a conjugate of formula (I):

Chemical formula

[0127] Wherein, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, and n have the same definitions as in formula (I).

[0128] Lv1 is a reactive group that can react with thiols, amines, carboxylic acids, selenols, phenols, or hydroxyl groups on cell-binding molecules. The reactive groups are not limited to these, but include halides (fluorides, chlorides, bromides, and iodides), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, chlorophenoxyl, and dichlorophenoxyl. Phenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazole-yl)oxyl, 2-ethyl-5-phenylisoxazolium-3'-sulfonyl, phenyloxadiazole-sulfonyl(-sulfone-ODA), 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole-yl(ODA), oxadiazole-yl, unsaturated carbon (carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen, or double or triple bonds between carbon and oxygen), or intermediate molecules produced by condensation reagents for the Mitsunobu reaction. The aforementioned condensation reagents are EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide meso-p-toluenesulfonate (CMC, or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), N,N,N',N'-Tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate (HBTU), (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethylcyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamide Muhexafluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridinium-1-ium-3-oxidehexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) ), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl-S-(1-oxide-2-pyridyl)thiuronium hexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate (TPTU), S-( 1-Oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(benzotriazol-1-yl)-N,N,N',N'-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (with or without polymeric linkage), dipyrrolidino(N-succinimidyloxy)-carbenium hexafluorophosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCIU), 2-chloro-1,3-dimethylimidazolinium tetrafluoroborate (CIB), (benzotriazole-1-yloxy)di Piperidinocarbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA, T3P®), 2-morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium Xafluorophosphate (HSTU), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl)azodicarboxylate (DCAD), di-tert-butylazodicarboxylate (DBAD), diisopropylazodicarboxylate (DIAD), and diethylazodicarboxylate (DEAD) are selected. Furthermore, Lv1 and Lv2 may be anhydrides formed by the acid itself or by other C1-C8 anhydrides.

[0129] Preferred Lv1 compounds include halides (e.g., fluorides, chlorides, bromides, and iodides), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophenoxyl, N-succinimidyloxyl (NHS), phenoxyl; dinitrophenoxyl; pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl, 1H-imidazole-1-yl, and chlorophenoxyl. Syl, dichlorophenoxyl, trichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazole-yl)oxyl, 2-ethyl-5-phenyl-3'-sulfonyl, phenyloxadiazole-sulfonyl(-sulfone-ODA), 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole-yl(ODA), oxadiazole-yl, unsaturated carbon (carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen, or carbon-oxygen double or triple bonds), or any of the following structures:

[0130] [ka] TIFF0007894620000054.tif195170

[0131] In the formula, X 1’ is F, Cl, Br, I, or Lv3;X 2’Lv3 is O, NH, N(R1), or CH2; R3 is independently H, aromatic, heteroaromatic, or an aromatic group in which one or more H atoms are independently substituted by -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O)2R1, or -COOR1; Lv3 is F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; mo Nofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate; anhydrides formed by themselves or with other anhydrides, such as acetic anhydride or formic anhydride; or leaving groups selected from intermediate molecules produced by condensation reagents for peptide coupling reactions or Mitsunobu reactions.

[0132] Examples of formula (IV) are the following structures, or isotopes of one or more chemical elements, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystalline structures of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: [ka] TIFF0007894620000056.tif254170TIFF0007894620000057.tif254170TIFF0007894620000058.tif254170TIFF0007894620000059.tif23817 0TIFF0007894620000060.tif246170TIFF0007894620000061.tif254170TIFF0007894620000062.tif221170TIFF0007894620000063.tif60170

[0133] In the formula, X8 is O, S, NH, NHNH, NHR 12 , SR12 , SSR 12 , SSCH(CH3)R 12 , SSC(CH3)2R 12 , or R 12 ; X1, X2, X3, X4, X5, R 12 , R 12’ , R 13 , R 13’ , R T 25 , R 25’ , p, p1, p2, p3, q1, q2, Lv3, m, m1, n, and mAb are the same as above; Aa is a natural or non-natural amino acid; r is 0 to 12; when r > 2, (Aa)r is a peptide containing the same or different amino acid sequences; r = 0 means that (Aa)r does not exist.

[0134] In another aspect of the present invention, the side chain linking compound is represented by formula (V), which can easily react with the cell binding molecule T to form a conjugate of formula (III):

Chemical formula

[0135] In the formula, D, W, w, L1, L2, Q1, Q2, V1, V2, v1, v2, and n have the same definitions as in formula (I); Lv1 and Lv2 are independently the same as Lv1 in formula (IV), and in formula (V), both Lv1 and Lv2 may be the same or different.

[0136] Examples of formula (V) are the structures shown below, or isotopes of one or more chemical elements, pharmaceutically acceptable salts, hydrates, or hydrated salts; or polymorphic crystal structures of these compounds; or optical isomers, racemates, diastereomers, or enantiomers:

Chemical formula

[0137] In the formula, X1, X2, X3, X4, X5, X8, Z2, Z3, p, p1, p2, p3, q1, q2, Lv1, Lv2, Lv3, Lv 3’ , m, n, R 12 , R 12’ , R 15 , R 25 , R 25’ (Aa)r and mAb are the same as above.

[0138] The present invention further relates to a method for producing cell-binding molecule-amatoxin analog conjugates of formula (I) and formula (III), and to the application of the conjugates of formula (I) and formula (III).

[0139] The cell-binding agent / molecule T may be any currently known or known molecule that binds, complexes, or reacts with residues of a group of cells to be therapeutically or otherwise biologically modified. Preferably, the cell-binding agent / molecule is an immunotherapy protein, antibody, single-chain antibody; antibody fragment that binds to target cells; monoclonal antibody; single-chain monoclonal antibody; or monoclonal antibody fragment that binds to target cells; chimeric antibody; chimeric antibody fragment that binds to target cells; domain antibody; domain antibody fragment that binds to target cells; antibody-mimicking adnectin; DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules (transferrin); binding peptides, proteins, antibodies having 4 or more amino acids, or binding peptides having small cell-binding molecules or ligands attached to albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles, or (viral) capsids.

[0140] Preferably, Lv1, Lv2, Lv3, and Lv 3’ The thiol reacts with the thiol pair of the cell binder / molecule. The thiol is preferably a pair of disulfide bonds reduced from the interchain disulfide bonds of the cell binder by a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and / or β-mercaptoethanol (β-ME, 2-ME). The thiol of the cell binder / molecule can be generated via Traut's reagent or thiolactone, which reacts with the amine of the cell binder / molecule to form a thiol, followed simultaneously or sequentially by Lv1, Lv2, Lv3, or Lv 3’ And that's how it reacts.

[0141] [ka]

[0142] Preparation of conjugates of amatoxin analogs and cell-binding molecules via side chain linkage

[0143] Figures 1-26 show the preparation of a conjugate between the cell-binding molecule of the present invention and an amatoxin analog, and the synthetic pathway for generating the conjugate via side-chain linkage.

[0144] The conjugates of formulas (I) and (III) can be prepared via the intermediate compounds of formulas (IV) and (V), respectively. Generally, the amatoxin analogs of formulas (IV) and (V) are synthesized to have Lv1 and Lv2 functional groups that readily react with cell-binding molecules or modified cell-binding molecules. The synthesis of the amatoxin analogs of formulas (IV) and (V), as well as some preparations of formulas (I) and (III), are structurally shown in Figures 1 to 26.

[0145] To synthesize the conjugate of formula (I), the functional group Lv1 on formula (IV) is generally reacted with one or more residues on the cell-binding molecule in an aqueous medium of pH 5-9 at 0-60°C, either with or without the addition of 0-30% of a water-miscible organic solvent (such as DMA, DMF, ethanol, methanol, acetone, acetonitrile, THF, isopropanol, dioxane, propylene glycol, or ethylenediol), followed by dialysis or chromatographic purification to form the conjugate compound of formula (I). Some of the residues of the cell-binding molecule (reacting groups for conjugation) can be obtained by protein engineering.

[0146] The conjugate of formula (III) can also be obtained by a reaction between the functional groups Lv1 and Lv2 of the conjugate of formula (V) and two or more residues of the cell-binding molecule, preferably a pair of free thiols generated by reducing the disulfide bond of the cell-binding molecule in an aqueous medium of pH 5 to 9 with or without 0 to 30% water-miscible (miscible) organic solvent at 0 to 60°C. The pair of free thiols is preferably a pair of disulfide bonds reduced from the interchain disulfide bonds of the cell binder by a reducing agent selected from dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-MEA), and / or β-mercaptoethanol (β-ME, 2-ME) in an aqueous medium of pH 4 to 9 with or without 0 to 30% water-miscible (miscible) organic solvent.

[0147] The Lv1 and Lv2 reactive groups in formulas (IV) and (V) are, independently, disulfide, thiol, maleimide, halogen-substituted maleimide, haloacetyl, azide, 1-yin, ketone, aldehyde, alkoxyamino, triflate, carbonylimidazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, or carboxylic acid esters of nitrophenol, N-hydroxysuccinimide (NHS), phenol; dinitrophenol, pentafluorophenol, tetrafluorophenol The compounds can be hydroxybenzotriazole, anhydrous, hydrazide groups, or other acid ester derivatives, and can react simultaneously or sequentially with one, two, or more groups of a cell-binding molecule / drug in an aqueous medium of pH 5-9 at 0-60°C, with or without the addition of 0-30% water-soluble (miscible) organic solvent, to produce conjugates of formulas (I) and (III) after column purification or dialysis. The Lv1 and Lv2 reactive groups on formulas (IV) and (V) react immediately with the modified cell-binding molecule in different ways. For example, in the cell binder-tubulicine analog conjugate of formula (I), the disulfide bond-containing bond is achieved by disulfide exchange between the disulfide bond in the modified cell binder and Lv1 and Lv2 having free thiol groups, or by disulfide exchange between the free thiol group of the modified cell binder and the disulfide bond on Lv1 and / or Lv2. To shift the disulfide exchange reaction, the disulfide group is typically a group such as disulfanylpyridine, disulfanyl-nitropyridine, disulfanyl-nitrobenzene, disulfanyl-nitrobenzoic acid, or disulfanyl-dinitrobenzene. The thioether-containing bonds in the conjugates of formulas (I) and (III) are achieved by the reaction of maleimide, haloacetyl, or ethylsulfonyl on a modified cell binder or on the tubulicine analogs of formulas (IV) and (V) with a free thiol group on the tubulicine analogs of formulas (IV) and (V) or on the respective modified cell binders.Bonds containing acid-unstable hydrazones in the conjugate are achieved by methods known in the art (e.g., P. Hamann et al., Cancer Res. 53, 3336-34, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-55, 1959; P. Trail et al., Cancer Res., 57; 100-5, 1997) by the reaction of a carbonyl group on a drug- or modified cell-binding molecule of formula (IV) and formula (V) with a hydrazide residue on a drug- or modified cell-binding molecule of formula (IV) and formula (V). Bonds containing triazoles in the conjugate are achieved via click chemistry (Huisgen cycloaddition) (Lutz, JF. et al., 2008, Adv. Drug Del. Rev.60, 958-70; Sletten, EM et al. This is achieved by the reaction of a 1-yine group on the drug or cell-binding molecule of formulas (IV) and (V) with the azide moiety of another counterpart, via (2011, AccChem. Research 44, 666-76). The oxime bond in the oxime-linked conjugate is achieved by the reaction of a ketone or aldehyde group on the drug or cell-binding molecule of formulas (IV) and (V) with the oxiramine group of another counterpart. The thiol-containing cell-binding molecule can react with drug molecule conjugates of formulas (IV) and (V) having maleimide, haloacetyl, or ethylsulfonyl substituents in aqueous buffer at pH 5.5-9.0. The thiol-containing cell-binding molecule can obtain a conjugate with a disulfide bond by disulfide exchange with drug conjugates of formulas (IV) and (V) having pyridyldithio residues. Cell-binding molecules having a hydroxyl group or a thiol group can react with drug conjugates of formulas (IV) and (V) having a halogen, particularly an alpha-halide of a carboxylate salt, under mild basic conditions, for example, at pH 8.0 to 9.5, to obtain modified drugs having an ether or thioether linkage.The hydroxyl or amino group of the cell-binding molecule can condense with drug conjugates of formulas (IV) and (V) containing a carboxyl group in the presence of a dehydrating agent such as EDC or DCC, yielding an ester bond. The cell-binding molecule containing an amino group can condense with the carboxyl ester groups of NHS, imidazole, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, tosylate, mesylate, or 2-ethyl-5-phenylisoxazolium-3'-sulfonate on drug conjugates of formulas (IV) and (V), yielding a conjugate via an amide bond.

[0148] The synthesized conjugates can be purified by standard biochemical methods, such as gel filtration using Sephadex G25 or Sephacryl S300 columns, adsorption chromatography, ion exchange, or dialysis. In some cases, when small molecules (e.g., folic acid, melanocyte-stimulating hormone, EGF, etc.) are conjugated with small molecule drugs as cell-binding molecules, they can be purified by chromatography, such as HPLC, medium-pressure column chromatography, or ion exchange chromatography.

[0149] To achieve a higher conjugation yield between the cell-binding molecule, preferably a pair of free thiol pairs on an antibody, and formula (I) or (III), it may be required to add a small amount of water-miscible organic solvent or phase transition agent to the reaction mixture. First, the crosslinking ligator (conjugate) of formula (IV) or formula (V) can be dissolved at a high concentration, for example, 1 to 500 mM, in a water-miscible polar organic solvent, such as different alcohols like methanol, ethanol, or propanol, acetone, acetonitrile, tetrahydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), dimethylacetamide (DMA), or dimethyl sulfoxide (DMSO). On the other hand, the cell-binding molecule, such as an antibody dissolved at a concentration of 1 to 50 mg / ml in an aqueous buffer at pH 4.0 to 9.5, preferably 6.0 to 8.5, is treated with 0.5 to 20 equivalents of TCEP or DTT for 20 minutes to 48 hours. After reduction, DTT can be removed by SEC chromatography. TCEP can also be removed by SEC chromatography if desired, or it can be retained in the reaction mixture for the next step without purification, but it is preferable to neutralize TCEP with an azide compound such as 4-azidobenzoic acid, 4-(azidomethyl)benzoic acid, or azide-polyethylene glycol (e.g., 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol). Furthermore, in order to achieve cross-linking coupling of cell-binding molecules simultaneously with TCEP reduction, the reduction of antibodies or other cell-binding agents by TCEP can be carried out in the presence of existing drug conjugate molecules of formula (IV) or formula (V).

[0150] The aqueous solution for modifying the cell binding agent is buffered to a pH of 4-9, preferably 6.0-7.5, and may contain non-nucleophilic buffer salts useful within this pH range. Typical buffers include phosphates, acetates, triethanolamine HCl, HEPES, and MOPS buffers, and may further contain additional components such as cyclodextrin, hydroxypropyl-β-cyclodextrin, polyethylene glycol, sucrose, and salts (e.g., NaCl, KCl). After adding the drug conjugate of formula (IV) or formula (V) to the solution containing the reduced cell binding molecule, the reaction mixture is incubated at an ambient temperature of 4°C-45°C, preferably 15°C. The progress of the reaction can be monitored by measuring a decrease in absorption at 252 nm, an increase in absorption at 280 nm, or changes at other appropriate wavelengths. After the reaction is complete, the modified cell binder can be isolated by common methods, such as gel filtration chromatography, ion exchange chromatography, adsorption chromatography, or column chromatography on silica gel or alumina, crystallization, pre-thin layer chromatography, ion exchange chromatography, or HPLC.

[0151] The degree of modification can be evaluated by measuring the absorbance of nitropyridinethion, dinitropyridinedithion, pyridinethion, carboxyamidopyridinedithion, and dicarboxyamidopyridinedithion groups emitted via the UV spectrum. In conjugates without chromophore groups, modification or conjugation reactions can be monitored by LC-MS, preferably ULC-QTOF mass spectrometry, or capillary electrophoresis (CEMS). The side-chain crosslinking conjugates described herein have diverse functional groups that can react with any agent having appropriate substituents, preferably cytotoxic agents. For example, modified cell-binding molecules having amino or hydroxy substituents can react with agents having N-hydroxysuccinimide (NHS) esters, and modified cell-binding molecules having thiol substituents can react with agents having maleimide or haloacetyl groups. Furthermore, modified cell-binding molecules having carbonyl substituents (ketones or aldehydes) can react with agents having hydrazides or alkyloxyamines. Those skilled in the art can easily determine which conjugate to use based on the known reactivity of the available functional groups on the conjugate.

[0152] Cell binding agent

[0153] The cell-binding molecule Cb constituting the conjugate and modified cell-binding agent of the present invention may be any currently known or known molecule that binds, complexes, or reacts with residues of a group of cells to be therapeutically or otherwise biologically modified.

[0154] Cell binding agents include, but are not limited to, large molecular weight proteins such as full-length antibodies (polyclonal or monoclonal), dimers, multimers, multispecific antibodies (e.g., bispecific antibodies); single-chain antibodies; antibody fragments such as Fab, Fab', F(ab')2, Fv [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments obtained by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs, bispecific antibodies, trispecific antibodies, cancer cell antigens, viral antigens, microbial antigens, or specific antigens, and any epitope-binding fragments of the said substances that can recognize, bind to, or express desired biological activity with proteins produced by the immune system; and interferons (e.g., I, II, I). Type II); peptides; lymphokines, e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, or interferon-gamma (IFN-γ); hormones, e.g., insulin, TRH (thyroid-stimulating hormone-releasing hormone), MSH (cytostimulating hormone), or steroid hormones such as androgens, estrogens, or melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors, e.g., epidermal growth factor (EFG), granulocyte-macrophage colony-stimulating factor (GM-CSF); transforming growth factors (TGF), e.g., TGFα, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF, and GM-CSF [Burgess, Immunology] [Today, 5, 155-158 (1984)]; vaccine growth factor (VGF); fibroblast growth factor (FGF); small molecular weight proteins, polypeptides, peptides, and peptide hormones, e.g., bombesin, gastrin, and gastrin-releasing peptides; platelet-derived growth factor; interleukins and cytokines, e.g., interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitors, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folic acid; apolipoproteins and glycoproteins, e.g., transferrin [O'Keefe et al, J. Bio. Chem.]260,932-927(1985)]; sugar-binding proteins or lipoproteins such as lectins; cellular nutrient transport molecules; and small molecule inhibitors, e.g., inhibitors of prostate-specific membrane antigen (PSMA), small molecule tyrosine kinase inhibitors (TKIs), non-peptides, or other cell-binding molecules or substances, e.g., bioactive polymers (Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61), bioactive dendrimers (Lee, et al, Nat. Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci. 2009, 106, 685-90), nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et This includes (al, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008, 19, 1309-12), liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9), and viral capsids (Flenniken, et al, Viruses Nanotechnol. 2009, 327, 71-93).

[0155] Generally, if a suitable monoclonal antibody is available, it is preferred as a cell surface binding molecule. The antibody may be derived from mouse, human, humanized, chimeric, or other species.

[0156] The production of antibodies used in this invention includes in vivo, in vitro, or a combination thereof. Methods for preparing anti-receptor peptide polyclonal antibodies are well known, for example, as shown in U.S. Patent No. 4,493,795 (Nestor et al.). A typical method for preparing monoclonal antibodies is to fuse mouse spleen cells isolated from a mouse immunized with a specific antigen 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 antigen of interest, and examples of the antigen of interest 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 specific target cell receptors or by suppressing receptor activity.

[0157] 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.

[0158] 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 g m / L of glucose, 0–20 mM of glutamine, 0–20% of fetal bovine serum, ppm amounts of several heavy metals (e.g., Cu, Mn, Fe, or Zn) or / and heavy metals added in salt form, and an antifoaming agent (e.g., polyoxyethylene-polyoxypropylene block copolymer).

[0159] 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.

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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.

[0164] Antibodies used for drug conjugation by the conjugates of the present invention to treat cancer, autoimmune diseases, and / or 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 (Alacizumab 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) 4 antibodies), Clivatuzumab (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), Detumoma Dorlizumab (anti-B lymphoma cell antibody), dorlizumab, dorlizumab, 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 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 antibody (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), F 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 anhydride antibody) (Anti-9 antibody), Glembatumumab (also known as 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 (Anticardiocardial myosin antibody), infliximab (also known as Remicade, 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), Matuzumab (anti-EGFR antibody), Mepolizumab (also known as Bosatria, anti-IL-5 antibody), Metelimumab (anti-TGFβ-1 antibody), Miratuzumab (anti-CD74 antibody), Minretumomab (anti-TAG-72 antibody), Mitumomab (also known as BEC-2, anti-ganglioside antibody-GD3), Morolimmumab (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 :Theracim, Theraloc, anti-EGFR antibody), Nofetumomab, Ocrelizumab (anti-CD20 antibody), Odurimomab (also known as Afolimomab, anti-LFA-1 (CD11a) antibody), Ofatumumab (also known as Arzerra, anti-CD20 antibody), Olaratumab (anti-PDGF-Rα antibody), Omalizumab (also known as Xolair,Anti-IgE Fc region antibody), oporuzumab (anti-EpCAM antibody), olegovomab (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), panitumumab (also known as Vectibix, ABX-EGF, anti-EGFR antibody), panobacumab (anti-Pseudomonas aeruginosa antibody), Pascolizumab (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), Lacothomomab (r acotumomab (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 (Res Lizumab (anti-IL-5 antibody), rilotumumab (anti-HGF antibody), rituximab (also known as MabThera, Rituxanmab, anti-CD20 antibody), lobatumumab (anti-IGF-1 receptor antibody), rontalizumab (anti-IFN-α antibody), lovelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), ruplizumab (also known as Antova, anti-CD154 (CD40L) antibody),Satumomab (anti-TAG-72 antibody), Sevirumab (anti-CMV antibody), Sibrotuzumab (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), Sonepcizumab (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-α-fetoprotein antibody), Tadocizumab (anti-integrin αIIbβ3 antibody), Talizumab (anti-IgE antibody), Tanezumab (anti-NGF antibody), Tapritumomab (tap litumomab (anti-CD19 antibody), tefibazumab (also known as Aurexis, anti-clumping factor A antibody), terimomab, tenatumomab (anti-tenascin C antibody), teneliximab (anti-CD40 antibody), teplizumab (anti-CD3 antibody), TGN1412 (anti-CD28 antibody), tisilimmab (also known as Tremelimumab, anti-CTLA-4 antibody), tigatuzumab (Tiga Tucotuzumab (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 celmoleukin (anti-EpCAM antibody), Tubirumab (anti-hepatitis B antibody),Urtoxazumab (anti-E. coli antibody), Ustekinumab (also known as, Stelara (anti-IL-12, IL-23 antibody), Vapaliximab (anti-AOC3 (VAP-1) antibody), Vedolizumab (anti-integrin α4β7 antibody), Vertuzumab (anti-CD20 antibody), Vepalimomab (anti-AOC3 (VAP-1) antibody), Bicilizumab (also known as Nuvion, anti-CD3 antibody), Vitaxin (anti-angiogenic integrin avb3 antibody), Voloximab (anti-integrin α5β1), Botumumab (also known as HumaSPECT, antitumor antigen CTAA16.88) Antibodies), Saltumumab (also known as HuMax-EGFr, (anti-EGFR antibody), Zanolimumab (also known as HuMax-CD4, anti-CD4 antibody), Ziralimumab (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, CGM1 from Nat. Cancer Inst.), CYT-356 (Registered trademark "Oncoltad", prostate cancer), HNK20 (Ora Vax Inc.)(for RSV), 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).

[0165] Other antibodies as cell-binding molecules / 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).

[0166] The cell-binding agent, more preferably the antibody, may be any agent capable of targeting tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells, B cells, or melanocytes. More specifically, the cell-binding agent may be any agent / molecule capable of targeting any one of the following antigens or receptors: CD2, CD2R, CD3, CD3gd, CD3e, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD12w, CD13, CD14, CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17, CD18, CD19, C D20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD 42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD47R, CD48, CD49a, CD49b, CD49c, CD49 e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD 64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD74, CD75, CD75s, C D76, CD77, CD78, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw92, CD9 3, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD107a, CD107b, CD108, CD109,CD110、CD111、CD112、CD113、CDw113、CD114、CD115、CD116、CD117、CD118、CD119、CDw119、CD120a、CD120b、CD121a、CD121b、CDw121b、CD122、CD123、CDw123、CD124、CD125、CDw125、CD126、CD127、CD128、CDw128、CD129、CD130、CD131、CDw131、CD132、CD133、CD134、CD135、CD136、CDw136、CD137、CDw137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CDw156c、CD157, CD158a、CD158b、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CDw198、CD199、CDw199、CD200、CD200a、CD200b、CD201、CD202、CD202b、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210、CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235ab、CD235b、CD236、CD236R, CD238, CD239, CD240, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD265, CD266, CD267, CD268, CD269, CD271, CD273, CD274, CD275, CD276(B7-H3), CD277, CD278, CD279, CD280, CD281, CD28 2, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306 , CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, CD339, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activator inhibitor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, α-integrin, αvβ6, aminopeptidase N, amyloid β, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin protective antigen, anti-transferrin receptor, AOC3 (VAP-1) , B7-H3, Bacillus anthrax, BAFF (B-cell activator), BCMA, B-lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, canine IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen),CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clamping Factor A, cMet, CRIPTO, FCSF1R (Colony-Stimulating Factor 1 Receptor, CD115), CSF2 (Colony-Stimulating Factor 2, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)), CSP4, CTLA4 (Cytotoxic T Lymphocyte-Associated Protein 4), CTAA16.88 Tumor Antigen, CXCR4 (CD184), CXC Chemokine Receptor Type 4, cADP Ribose Hydrolase, Cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL3 (delta-like ligand 3), DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4), DR5 (death receptor 5), Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, ED-B, EGFL7 (EGF-like domain containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, epicyalin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast-activating protein α), FCGR1, α-fetoprotein, fibrin II, β-chain, fibronectin external domain B, FOLR (folate receptor), folate receptor α, folate Dololase, RS virus Fos-related antigen 1F protein, Frizzled receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α chain, growth differentiation factor 8, GP100, GPNMB (transmembrane protein NMB), GUCY2C (guanylate cyclase 2C, guanylate cyclase C (GC-C), enteric guanylate cyclase, guanylate cyclase-C receptor, heat-stable enterotoxin receptor (hSTAR)), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (stem cell growth factor / cell dispersal factor), HHGFR, HIV-1, histone complex,HLA-DR (Human Leukocyte Antigen), HLA-DR10, HLA-DRB, HMWMAA, Human Chorionic Gonadotropin, HNGF, Human Cell Scattering Factor Receptor Kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (Cell Adhesion Molecule 1), Idiotype, IGF1R (IGF-1, Insulin-like Growth Factor 1 Receptor), IGHE, IFN-γ, Influenza Hemagglutinin, IgE, IgE Fc region, IGHE, interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, L-12, IL-13, IL-15, IL- 17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, or IL-28), IL-31RA, ILGF2 (insulin-like growth factor 2), integrin (α4, α, IIIbβ3, αvβ3, α4β7, α5β1, α6β4, α7β7, αIIβ3, α5β5, αvβ5), interferon-gamma-inducing protein, ITAGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Regmine, Lewis Y antigen, LFA-1 (Lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGEA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (Macrophage migration inhibitor or glycosylation inhibitor (GIF)), MS4A1 (Transmembrane 4-domain subfamily A member 1), MSLN (Mesothelin), MUC1 (Mucin 1, cell surface-associated (MUC1) or Polymorphic epithelial mucin (PEM), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (transmembrane 4-domain subfamily A), MYCN, myelin-related glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neuronal apoptosis regulatory proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, Page4, PAP, anti-(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDC D1 (PD-1, programmed cell death protein 1), PDGF-Rα, (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate cotransporter, PMEL17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), rhesus factor (Rhesus factor), RANKL, PhoC, Ras variant, RG55, ROBO4, RS virus, RON, ROR1, sarcoma metastasis breakpoint, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syndecane 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (prostate 1 6-transmembrane epithelial antigen), STEAP2, STn, TAG-22 (tumor-associated glycoprotein 72), survivorvin, T cell receptor, T cell transmembrane protein, TEM1 (tumor epithelial marker 1), TENB2, tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor β2), Tie (CD202b) Cells expressing any insulin growth factor receptor, or any epidermal growth factor receptor, including Tie2, TIM-1 (CDX-014), TN, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophotrophic membrane glycoprotein), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1), TRAILR2 (cell death receptor 5 (DR5)), tumor-associated calcium signaling transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or any insulin growth factor receptor.

[0167] In another specific embodiment, the cell-binding molecule may be a ligand or receptor agonist selected from: folate derivatives (binding to folate receptors, proteins overexpressed in ovarian cancer and other malignancies) (Low, PS et al 2008, Acc. Chem. Res. 41, 120-129); glutamate urea derivatives (binding to prostate-specific membrane antigens, surface markers of prostate cancer cells) (Hillier, SM et al, 2009, Cancer Res. 69, 6932-6940); somatostatin (also known as growth hormone inhibitor (GHIH), somatotropin release inhibitor (SRIF), or somatotropin release inhibitor hormone) and their derivatives, e.g., octreotide (sandostatin) and lanreotide (somatsurin) (especially for neuroendocrine tumors, GH-producing pituitary adenomas, paraganglionic tumors, dysfunctional pituitary adenomas, pheochromocytomas) (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. USA 103, 16436-16441); Somatostatin receptor subtypes (sst1, sst2, sst3, sst4, and sst5) in GH-secreting pituitary adenoma (Reubi JC, Landolt, AM 1984 J. Clin. Endocrinol Metab 59:1148-51; Reubi JC, Landolt AM 1987 J Clin Endocrinol Metab 65:65-73; Moyse E, et al, J Clin Endocrinol Metab 61:98-103), Gastrointestinal and pancreatic tumors (Reubi JC, et al, 1987 J Clin Endocrinol Metab 65:1127-34; Reubi, J. C, et al, 1990 Cancer Res 50:5969-77), pheochromocytoma (Epel-baum J, et al 1995 J Clin Endocrinol Metab 80:1837-44; Reubi JC, et al, 1992 J Clin Endocrinol Metab 74:1082-9), neuroblastoma (Prevost G, 1996 Neuroendocrinology 63:188-197; Moertel, C. L., et al.(References: 1994 Am J Clin Path 102:752-756), thyroid carcinoma (Reubi, J. C, et al 1991 Lab Invest 64:567-573), small cell lung cancer (Sagman U, et al, 1990 Cancer 66:2129-2133), scleral cysts, scleral buds, and nerve fibromas (Reubi JC, et al 1986 J Clin Endocrinol Metab 63:433-8; Reubi JC, et al 1987 Cancer Res 47:5758-64; Fruhwald, M. C, et al 1999 Pediatr Res 45:697-708), breast cancer (Reubi JC, et al 1990 Int J Cancer) 46:416-20; Srkalovic G, et al 1990 J Clin Endocrinol Metab 70:661-669), nephrotic tumor (Reubi JC, et al 1992, Int J Cancer 50:895-900), renal cell carcinoma (Reubi JC, et al 1992, Cancer Res 52:6074-6078), mesenchymal tumor (Reubi JC, et al 1996 Cancer Res 56:1922-31), prostate gland (Reubi JC, et al 1995, J. Clin. Endocrinol Metab 80:2806-14; et al 1989, Prostate 14:191-208; Halmos G, et al J. Clin. Endocrinol Metab 85:2564-71), oviduct (Halmos, G, et al, 2000 J Clin Endocrinol Metab 85:3509-12; Reubi JC, et al 1991 Am J Pathol 138:1267-72), stomach (Reubi JC, et al 1999, Int J Cancer 81: 376-86; Miller, G. V, 1992 Br J Cancer 66:391-95), hepatocytes (Kouroumalis E, et al 1998 Gut 42:442-7; Reubi JC, et al 1999 Gut 45:66-774) and nasopharyngeal head (Loh K. S, et al, 2002)Virchows Arch 441:444-8; specific aromatic sulfonamides specific to carbonic anhydrase IX (marker of hypoxia and renal cell carcinoma) (Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-777); pituitary adenylate cyclase activating peptide (PACAP) (PAC1) for pheochromocytoma and paraganglionic tumors; vasoactive intestinal peptide (VIP / PACAP) and its receptor subtypes (VPAC1, VCAP2); α-melanocyte-stimulating hormone (α-MSH) receptor; cholecystokinin (CCK) / gastrin receptor and its receptor subtypes (CCK1 (formerly CCK-A) and CCK2); bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-H is-Leu-Met-NH2) / gastrin-releasing peptide (GRP) (BB1, GRP receptor subtypes (BB2), BB3, and BB4) (Ohlsson, B., et al, 1999, Scand. J. Gastroenterology 34 (12): 1224-9; Weber, HC, 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008, Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64); neurotensin receptors and their receptor subtypes (NTR1, NTR2, NTR3); substance P receptors and their receptor subtypes (NK1 receptor in glial tumors, etc.), Hennig IM et al, 1995 Int. J. Cancer 61, 786-792); Neuropeptide Y (NPY) receptor and its receptor subtypes (Y1-Y6); RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), dimeric and multimeric cyclic RGD peptides (e.g., cRGDfV) (Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E, et al,Anti-Cancer Agents Med Chem. 7 (5): 552-558; Kerr, JS et al, Anticancer Research, 19(2A), 959-968; Thumshirn, G, et al, 2003 Chem. Eur. J. 9, 2717-2725), as well as TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (a 31-amino acid peptide that binds to cell surface-expressed nucleolin receptors) (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrative Biol, Homing peptides containing 2(7-8), 326-337; MA Burg, 1999 Cancer Res., 59(12), 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9); cell-permeable peptides (CPPs) (Nakase I, et al, 2012, J. Control Release).159(2),181-188); Luteinizing hormone-releasing hormone (LHRH) agonists and antagonists that act by targeting peptide hormones, such as testosterone production, as well as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH) agonists, such as buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt) and gonadrelin (Pyr-His-Trp-Ser-Tyr-Gly-L). (eu-Arg-Pro-Gly-NH2), Goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), Histrelin (Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt), Leuprolide (Pyr-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt), Nafarelin (Pyr-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro- Gly-NH2), Triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), Nafarelin, Deslorerin, Abarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-Pro-DAla-NH2), Cetrorelin (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-T yr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-a minoPhe(carba-moyl)-Leu-isopropylLys-Pro-D-Ala-NH2), and Ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9,Boccon-Gibod, L.; et al, 2011, Therapeutic Advances in Urology 3 (3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A. V; Nagy, A. 1999 Eur J Endocrinol 141:1-14; Koppan M, et al 1999 Prostate 38:151-158); as well as Toll-like receptors (TLRs), type C lectins, and Nodlike receptors (NLRs) (Fukata, M., et al.) that recognize everything from small molecules (imiquimod, guanidine, and adenosine analogs) to large and complex biomacromolecules such as lipopolysaccharides (LPS), nucleic acids (CpG DNA, poly(I;C)), and lipopeptides (Pam3CSK4) (Kasturi, SP, et al, 2011, Nature 470, 543-547; Lane, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dudek, AZ, et al, 2007, Clin. Cancer Res. 13, 7119-7125). al, 2009, Semin. Immunol. 21, 242-253; Maisonneuve, C., et al, 2014, Proc. Natl. Acad. Sci. USA 111, 1-6; Botos, I., et al, 2011, Structure 19, 447-459; Means, TK, et al, 2000, Life Sci. 68, 241-258)Pattern recognition receptors (PRRs) such as the calcitonin receptor (Zaidi M, et al, 1990 Crit Rev Clin Lab Sci 28, 109-174; Gorn, AH, et al. 1995 J Clin Invest 95:2680-91), a 32-amino acid neuropeptide primarily involved in regulating calcium levels through its effects on osteoclasts and the kidney; and integrin receptors and their receptor subtypes (α V β1, α V β3, α V β5, α V β6, α6β4, α7β1, α L β2, α IIb β3, etc., is expressed on the surface of various cells, particularly osteoclasts, endothelial cells, and tumor cells (Ruoslahti, E. et al, 1994 Cell 77, 477-8; Albelda, SM et al, 1990 Cancer Res., 50, 6757-64). Short-chain peptides, GRGDSPK, and cyclic RGD pentapeptides such as cyclic (RGDfV)(L1) and their derivatives [cyclo(-N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me)V-)(silengitide)] showed high binding affinity to integrin receptors (Dechantsreiter, MA et al, 1999 J. Med.Chem. 42, 3033-40; Goodman SL et al, 2002 J. Med.Chem. 45, 1045-51).

[0168] Cell-binding molecules / ligands or cell receptor agonists can be Ig-based and non-Ig-based protein scaffold molecules. Ig-based scaffolds are not limited to, but include nanobodies (derivatives of VHH (Calamidae Ig)) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-797); domain antibodies (dAb, derivatives of VH or VL domains) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); bispecific T cell engagers (BiTE, bispecific dimer) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol. Ther. 11, 22-30); biaffinity retargeting (DART, bispecific dimer) (Moore PA P, et al. 2011, Blood 117(17), 4542-4551); tetravalent tandem antibodies (TandAb, bispecific dimer) (Cochlovius, You can choose from B, et al. 2000, Cancer Res. 60(16):4336-4341). Non-Ig scaffolds are not limited to anticarin (a derivative of lipocalin) (Skerra A. 2008, FEBS J., 275(11): 2677-2683; Beste G, et al, 1999 Proc. Nat. Acad. USA. 96(5):1898-1903; Skerra, A. 2000 Biochim Biophys Acta, 1482(1-2):337-350; Skerra, A. 2007, Curr Opin Biotechnol. 18(4):295-304; Skerra, A. 2008, FEBS J. 275(11):2677-2683); adnectins (10FN3 (fibronectin)) (Koide, A, et al, 1998 J. Mol. Biol., 284(4):1141-1151; Batori V, 2002, Protein Eng. 15(12): 1015-1020; Tolcher, A. W, 2011, Clin. Cancer Res.17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4):211-219); designed ankyrin repeat proteins (DARPins) (derivatives of ankrin repeat (AR) proteins) (Boersma, YL, et al, 2011 Curr Opin Biotechnol. 22(6): 849-857), e.g., DARPinC9, DARPinEc4, and DARPinE69_LZ3_E01 (Winkler J, et al, 2009 Mol Cancer Ther. 8(9), 2674-2683; Patricia MK. M., et al, Clin Cancer Res. 2011;17(1):100-110; Boersma Y. L, et al, 2011 J. Biol. Chem. You can choose from the following: 286(48),41273-41285; Avimer (domain A / low-density lipoprotein (LDL) receptor) (Boersma Y. L, 2011 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-1561).

[0169] Examples of the structures of the small molecules of the cell-binding molecules / ligands or cell receptor agonists of this application are as follows: LB01 (folate), LB02 (PMSA ligand), LB03 (PMSA ligand), LB04 (PMSA ligand), LB05 (somatostatin), LB06 (somatostatin), LB07 (octreotide, somatostatin analog), LB08 (ramareotide, somatostatin analog), LB09 (vapreotide (Sambar), somatostatin analog), LB10 (CAIX ligand), LB11 (CAIX ligand), LB12 (Gastrin-releasing peptide receptor (GRPr), MBA), LB13 (Luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH), LB14 (Luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand), LB15 (GnRH antagonist, Abarelix), LB16 (Cobalamin, vitamin B12 analog), LB17 (Cobalamin, vitamin B12 analog), LB18 (α v LB19 (cyclic RGD pentapeptide for β3 integrin receptor), LB20 (heterodivalent peptide ligand for VEGF receptor), LB21 (neuromedin B for G protein-coupled receptor), LB22 (TLR2 for Toll-like receptor), LB23 (for androgen receptor), LB24 (α vCilengitide / cyclo(-RGDfV-) for integrin receptors: LB23 (fludrocortisone), LB25 (rifabutin analog), LB26 (rifabutin analog), LB27 (rifabutin analog), LB28 (fludrocortisone), LB29 (dexamethasone), LB30 (fluticasone propionate), LB31 (beclomethasone dipropionate), LB32 (triamcinolone acetonide), LB33 (prednisone), LB34 (prednisolone), LB35 (methylprednisolone), LB36 (betamethasone), LB37 (irinotecan analog), LB38 (crizotinide). LB39 (bortezomib analog), LB40 (carfilzomib analog), LB41 (carfilzomib analog), LB42 (leuprolide analog), LB43 (triptorelin analog), LB44 (clindamycin), LB45 (liraglutide analog), LB46 (semaglutide analog), LB47 (letapamulin analog), LB48 (indibrine analog), LB49 (vinblastine analog), LB50 (lixisenatide analog), LB51 (osimertinib analog), LB52 (nucleoside analog), LB53 (erlotinib analog), or LB54 (lapatinib analog);

[0170] [ka] TIFF0007894620000074.tif254170TIFF0007894620000075.tif254170TIFF000 7894620000076.tif238170TIFF0007894620000077.tif254170TIFF00078946200 00078.tif238170TIFF0007894620000079.tif238170TIFF0007894620000080.t if238170TIFF0007894620000081.tif246170TIFF0007894620000082.tif246170

[0171] During the ceremony, [ka] is the linking site with the side chain linkage of this patent; X4 and Y1 are independently O, NH, NHNH, NR1, S, C(O)O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N(R1), N(R1)C(O)N(R1), CH2, C(O)NHNHC(O), and C(O)NR1; X5 is H, CH3, F, or Cl; M1 and M2 are independently H, Na, K, Ca, Mg, NH4, N(R 12 R 12’ R 13 R 13’ ) is; R 12 , R 12’ , R 13 , and R 13’ This has the same definition as equation (I).

[0172] Applications of conjugates

[0173] In certain embodiments, the cell-binding ligand-drug conjugates of the present invention, mediated by side-chain linkers, are used for targeted cancer therapy. Target 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. Cervical ulcers (PNETs), extracranial malignant germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumors, extragonadal tumors, trophoblastic tumors of pregnancy, 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 Carcinoma, 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-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.

[0174] In other specific embodiments, the cell-conjugate drug conjugates of the present invention can be used, by their components and methods, for the treatment or prevention of autoimmune diseases. These autoimmune diseases include, but are not limited to, autoimmune 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 lymphoproliferative syndrome, and autoimmune peripheral nerve disease. Transcatheter diseases, autoimmune pancreatitis, 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 Bullous pemphigoid, celiac disease, Cogan 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 fibrodysplasia, fibromyalgia Fibromyalgia, fibrotic alveolar septitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, nephroglonephritis, 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, 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, neuromyelitis optica (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, Personage-Georgia syndrome, squamous cellulitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEM Sjögren's syndrome, polyarteritis nodosa, polymyalgia rheumatica, 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 This includes spondyloarthritis, 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 (a type of idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthritis, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, and Westcott-Aldrich syndrome.

[0175] In another specific embodiment, the conjugating molecules used to conjugate via the bisconjugate of the present invention 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; microgranule antibodies, T-cell antibodies; thyroglobulin antibodies, anti-scleroderma-70 antibodies (AntiSCL-70); This includes anti-Joe antibodies, anti-U1RNP antibodies, anti-La / SSB antibodies, anti-SSA antibodies, anti-SSB antibodies, anti-parietal cell antibodies, anti-histone antibodies, anti-RNP antibodies, C-ANCA, P-ANCA, anti-centromere antibodies, anti-fibrin antibodies, anti-GBM antibodies, anti-ganglioside antibodies, anti-desmosome glycoprotein 3 core antibodies (anti-Desmogein 3), anti-p62 antibodies, anti-sp100 antibodies, anti-mitochondrial (M2) antibodies, rheumatoid factor antibodies, anti-MCV antibodies, anti-topoisomerase antibodies, and anti-neutrophil cytoplasmic (cANCA) antibodies.

[0176] In certain preferred embodiments, the binding molecule used in the conjugate of the present invention can bind to both the receptor and the receptor complex expressed by activated lymphocytes associated with autoimmune diseases. The receptor or receptor complex may be, for example, a member 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.

[0177] In another specific embodiment, a useful conjugate having immunospecificity for a viral or bacterial antigen is a humanized or human monoclonal antibody. As used herein, “viral antigen” includes, but is not limited to, any viral peptide or polypeptide protein that can induce an immune response (e.g., HIVgp120, HIVnef, 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). As used herein, “bacterial antigen” includes, but is not limited to, any microbial peptide, polypeptide, protein, sugar, polysaccharide, or lipid molecule that can induce an immune response (e.g., bacteria, fungi, pathogenic protozoa, yeast polypeptides (e.g., LPS and 5 / 8)). Type I antibodies useful for treating viral or bacterial 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.

[0178] The cell-binding molecule-drug conjugate mediated by the side chain linkage of the present invention can be used to treat infectious diseases. These infectious diseases include Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amoebiasis, Anaplasma, anthrax, bacterial tuberculosis infection, 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, Borrelia infection, and botulism. Nus poisoning (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, chondroblast, chondroblast infection of pneumonia, 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, cyclospora infection, 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, non-parasitic amebia Haeba 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 Ehrlichia Evans disease, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, tapeworm disease, 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 (elephantiasis), lymphocytic choriomeningitis, malaria Rhea, Marburg hemorrhagic fever, measles, meidiomycosis (Whitmore's disease), meningitis, meningococcal disease, metagonimus, microsporidiasis, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycotoma, 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, Crarbiomycosis) ice), 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, sporotrichum, staphylococcal food poisoning, staphylococcal infection, nematode, syphilis, tapeworm infection, tetanus (trismus), tinea folliculitis (Barber's disease) This includes itch, tinea manuum, tinea pedis, tinea pedis, onychomycosis, tinea versicolor, toxocariasis (ocular larval migrans), toxocariasis (visceral larval migrans), toxoplasmosis, trichinella, trichomoniasis, cryptobiosis (whipworm infection), pulmonary tuberculosis, tularemia, mycoplasma urea infection, Venezuelan encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobia nigricans, pseudotuberculosis infection, yersiniasis, yellow fever, and zygomycosis,This is not limited to these.

[0179] The conjugate of the present invention is more preferably effective against pathogenic strains, including Acinetobacter baumannii, Actiomyces israeri, Actinomyces odontrichus gerenseria, Propionibacterium propionicus, Trypanosoma brusey, HIV (Human Immunodeficiency Virus), Entamoeba histolytica, Anaplasma species, Bacillus anthrax, and Methylshemorrhagicum (Arcanobacterium). Haemolyticum), Junin virus, roundworm, Aspergillus, Astroviridae, Babesia, Bacillus cereus, Multiple bacteria, Bacteroides, Colon pouch ciliate, Ascaris baileyi, BK virus, Piedraia hortae, Blastocystis hominis, Dermatitis spuratosa, Macpo virus, Borrelia, Clostridium botulinum, Sabia, Brucella, usually Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter, usually Candida albicans and other Candida species, Bartonella henselae 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, Cryptococcus neoformans, Cryptosporidium, Cat's claw Insects, co-parasites, Cyclospora, Taenia solium, cytomegalovirus, dengue fever virus (DEN-1, DEN-2, DEN-3 and DEN-4)-flavivirus, binuclear amoeba, Corynebacterium diphtheriae, Diphyllobothrium species, Dracunculus medinensis, Ebola virus, Echinococcus species, Ehrlichia species, pinworms, Enterococcus species, Enterovirus species, typhus rickettsia, parvovirus B19, human herpesvirus type 6, human herpesvirus type 7, hypertrophic flukes, liver flukes and giant liver flukes, FFI prions,Filariohead superfamily, Clostridium perfringens, Fusobacterium, Clostridium perfringens, other Clostridium genera, Geotrichum candidum, GSS prion, Giardia lamblia Lamblia, Burkholderia glanders, Gnathostoma nematode, Gnathostoma brachiosa, Neisseria gonorrhoeae, Granuloma granuloma, Streptococcus pyogenes, Streptococcus agalactie, Haemophilus influenzae, Enterovirus, most Coxsackie A viruses, Enterovirus 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, Hookworm, American hookworm, Haemophilus influenzae, Boca human virus, Ehrlichia ewingi ewingii), Anaplasma phagocytophyllum, human metapneumovirus, Ehrlichia schaffensis, human papillomavirus, human parainfluenza virus, tapeworm, tapeworm, Epstein-Barr virus, Orthomyxoviridae, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella otzena, Klebsiella rhinoscleromotis, Couloprin, Lassa fever virus, Legionella pneumophila, Legionella pneumophila, Leishmania, Mycobacterium lepromatosis Lepromatosis), Leptospira species, Listeria monocystis, Borrelia and other Borrelia species, Wuchereria bancroftis and Diphyllobothrium malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitidis, Tremoraria yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia cyfii, Mycoplasma pneumoniae, various bacteria (Actinomycetoma) and fungi (Mycoplasma), Diptera larvae of parasitic flies, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, filarial parasites, Blastomyces brasilis, lung flukes and other lung fluke genera, Pasteurella genera, head lice, body lice, Phthirus pubis, Bordetella pertussis, Pest bacillus, Streptococcus pneumoniae, Pneumocystis cysticercosis, poliovirus, Prevotella genera, Naegleria amoeba, JC virus, Chlamydia psittaci, Coxiella barnettii, rabies virus, bead chain Escherichia coli and rat bite fever spirochete, respiratory respiratory syncytial virus (RSV), Rhinosporidium sebergii, rhinovirus Rickettsia, Rickettsia mites, Rift Valley fever virus, Rocky Mountain spotted fever rickettsia, rotavirus, rubella virus, Salmonella, atypical pneumonia coronavirus, scabies mites, Schistosoma, Shigella, varicella-zoster virus, smallpox or smallpox, Sporotrix schenkyi, Staphylococcus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides, Syphilis spirochete, Tapeworm, Tetanus, Tinea, Trichophyton tonsurans, Tinea, Epidermophyton floccosumu, Dermatophytes rubra and Trichophyton folliculitis, Dermatophytes rubra, Horthea wernecki, Tinea, Malassezia Genus, roundworms of dogs and cats, Toxoplasma, Trichinella, Trichomonas vaginalis, whipworm, Mycobacterium tuberculosis, Bacteria toulahot Francis, 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, Edwardsiella tarda, Plague bacillus, Shigella, Shigella, Shigella sonne, Salmonella typhi, Treponema pallidum Tenue, Treponema calateneum, Fensenburgdorferi, Borrelia burgdorferi, Leptospirosis hemorrhagic jaundice, Pneumocystis carinii, Bovine abortifacient, Swine abortifacient, Malta fever bacillus, Mycoplasma, typhus rickettsia, Rickettsia scrub typhus, Chlamydia, pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma maculatum); protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas anthropoides, Trypanosoma gambiens, Rhodesia trypanosoma, Donovan's leishmania, Leishmania tropicalis)This includes, but is not limited to, Leishmania breviaria, Pneumocystis carinii pneumonia, Plasmodium vivax, Plasmodium falciparum, and malignant malaria; or helminths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma halcionis, and hookworms).

[0180] Furthermore, the conjugates of the present invention are for the treatment of viral diseases, and examples of viruses, but not limited to, include: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, Influenza virus, Parainfluenza virus, Mumps, Measles, Respiratory syncytial virus, Rubella, Arbovirus, Rhabdovirus, Arenaviridae, Non-A / Non-B Hepatitis Virus, etc. Inoviruses, 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 (AIDS)], CNS viruses: [e.g., JC V (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 (acute poliomyelitis, post-polio syndrome), HTLV-I (tropical spastic paralysis); cytomegalovirus (CMV retinitis, HSV (herpetic keratitis)); cardiovascular virus [e.g., CBV (pericarditis, myocarditis)]; respiratory system / acute nasopharyngitis / U Illegal 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 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)].

[0181] In a further objective, the present invention also relates to pharmaceutical compositions for treating cancer, infections, or autoimmune diseases, comprising together the conjugate of the present invention and 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 affected 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 30 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 approximately 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.

[0182] Chemotherapy agents / cytotoxic agents for synergistic effects

[0183] Chemotherapy agents that can be used in conjunction with the present invention for synergistic effects are small molecule drugs, including cytotoxic agents. In this specification, “small molecule drugs” is used broadly to refer to organic, inorganic, or organometallic compounds that may have molecular weights of, for example, 100 to 2500, more preferably 200 to 2000. Small molecule drugs are well characterized in the prior art, including: International Publication WO05058367A2 and U.S. Patent No. 4,956,303, as well as Chessum, N., et al, Prog Med Chem. 2015, 54: 1-63; Eder, J., et al, Nat Rev Drug Discov. 2014, 13(8): 577-87; Zhang, M.-Q., et al, Curr Opin Biotechnol. 2007, 18(6): 478-88; these are incorporated in their entirety as references. The drugs mentioned above include known drugs and those that may become known drugs.

[0184] Known drugs include, but are not limited to, the following:

[0185] (1) Chemotherapy agents:

[0186] a) Alkylating agents: Nitrogen mustard: Chlorambucil, chlornafadin, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitractol, pipobromane, nobenbitin, phenesterine, prednimustine, thiotepa, trophosphamide, uracil mustard; CC-1065 and synthetic analogs of adzeresin, karzeresin and bizeresin; duocalmycin and its synthetic analogs KW-2189 and CBI-TMI; benzodiazepine dimers or pyrrolobenzodiazepine (PBD) dimers, tomaimycin dimers, indolinobenzodiazepine dimers, imidazobe Nzothiadiazepine dimers or oxazolidinobenzodiazepine dimers; nitrosourea compounds including carmustine, lomustine, chlorozotosine, fotemustine, nimustine, and ranimustine; alkyl sulfonates including busulfan, treosulfan, improsulfan, and biposulfan; triazenes or dacarbazines; platinum-containing compounds including carboplatin, cisplatin, and oxaliplatin; aziridines, benzodopa, carboquan, metsuredopa, and uredopa; ethyleneimines, and methylamelamamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine, and trimethylolomelamine, etc.

[0187] b) Plant alkaloids: Vinca alkaloids including vincristine, vinblastine, vindesine, vinorelbine, and navelbine; taxoids including paclitaxel and docetaxel and their analogues; meitansinoids including DM1, DM2, DM3, DM4, DM5, DM6, DM7, meitansine, and anthamitosine and their analogues; cryptophycins including the group of cryptophycin 1 and cryptophycin 8; epothyrons, eruterobin, discodermolds, bryostatins, drostatins, auristatins, tubulicins, cephalostatins; pancratistatin; sarcodicuthiin; spongstatins, etc.

[0188] c) DNA topoisomerase inhibitors: 9-aminocamptothecin, camptothecin, cristinator, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinols), teniposide, topotecan, 9-nitrocamptothecin, or epipodophilins including RFS 2000; and mitomycins, and their analogues, etc.

[0189] d) Antimetabolites: {[Antifolic acid: (DHFR inhibitors: including methotrexate, trimethrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid), or other folic acid analogs); IMP dehydrogenase inhibitors (including mycophenolic acid, thiazophrine, ribavirin, EICAR); Ribonucleotide reductase inhibitors (including hydroxyurea, deferoxamine)]; [Pyrimidine analogs: uraci Purine analogs (including ancitabine, azacitidine, 6-azauridine, capecitabine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, and larcitrexed); cytosine analogs (including cytarabine, cytosine arabinoside, and fludarabine); purine analogs (including azathioprine, fludarabine, mercaptopurine, thiamiprine, and thioguanine); folic acid supplements such as folic acid, etc.

[0190] e) Hormone therapy agents: Receptor antagonists: [Anti-estrogens: (including megestrol, raloxifene, and tamoxifen); LHRH agonists: (including goserelin and leuprolide acetate); Antiandrogens: (bicalutamide, flutamide, carsterone, dromostanolone propionate, epithiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testactone, trilostane, and other similar androgen inhibitors) (Contains harmful agents.) ); Retinoids / Deltoid muscle: [Vitamin D3 analogues: (including CB1093, EB1089, KH1060, cholecalciferol, ergocalciferol); Photodynamic therapy agents: (including verteporfin, phthalocyanine, photosensitizer Pc4, demethoxyhypocrelin A); Cytokines: (including interferon α, interferon γ, tumor necrosis factor (TNF), TNF domain-containing human proteins)]} etc;

[0191] f) Kinase inhibitors: BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mblitinib, ponatinib, bafetinib, bosutinib, cabozantinib, bismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib, etc.

[0192] g) Poly(ADP-ribose) polymerase (PARP) inhibitors: olaparib, niraparib, iniparib, talazoparib, veliparib, CEP9722 (Cephalon), E7016 (Eisai), BGB-290 (Baygene), 3-aminobenzamide, etc.

[0193] h) Antibiotics: Endiyne antibiotics (calicheamicin derivatives, calicheamicin γ1, δ1, α1, or β1 (e.g., J. Med. Chem., 39(11), 2103-2117, Angew Chem Intl. Ed. Engl. 33: 183-186 (1994)); Dynemicins including dynemicin A and deoxydynemicin; selected from esperamicin, kedulcidin, C-1027, mazulopeptin, or neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore; acrasinomycins, actinomycin, anthramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, cardinophilin; chromomycins, dactinomycin, daunorubicin, detrubicin 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, eribulin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogaramycin, olibomycins, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolbicin, etc.

[0194] i) Others: Polyketides (acetogenins), especially bratacin and bratacinone; gemcitabine, epoxomicins (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zyblestat, PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, Provenge, Elboy, neurostatin inhibitors (e.g., lovastamin), 1- Methyl-4-phenylpyridine ion, cell cycle inhibitors (staurosporine, etc.), actinomycins (actinomycin D, dactinomycin, etc.), amanitins, bleomycins (bleomycin A2, bleomycin B2, peplomycin, etc.), anthracyclines (daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, etc.), mitoxantrone, MDR inhibitors (verapamil, etc.), Ca 2+ATP inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (e.g., vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat (MGCD0103), bellinostat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A, etc.); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A, anti-adrenal drugs (e.g., aminoglutethimide, mitotane, trilostance); acegraton; aldofsphamide cricoside; aminolevulinic acid; amsacrine; arabinoside, bestrabusil; bisantren; edatrexate Defofamine; Demecolsin; Diadicone; Elfornithine (DFMO), Elfomitin; Erliptinium acetate; Etocluside, Gallium nitrate, Gasitosine, Hydroxyurea; Ibandronate, Lentinan; Ronidamin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (T2 toxin, Berkarin A, Loridine A, and Anguidin, etc.); Urethanes, siRNAs, antisense drugs, and nucleic acid lysis enzymes, etc.

[0195] (2) Anti-autoimmune disease drugs:

[0196] This includes, but is not limited to, cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticoids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone propionate, fluocortolone danazol, dexamethasone, triamcinolone acetonide, and beclomethasone dipropionate), DHEA, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mofetil, mycophenolic acid, prednisone, sirolimus, and tacrolimus;

[0197] (3) Anti-infective agents (including, but not limited to, the following):

[0198] 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;

[0199] b) Amphenicols: Azidamphenicol, Chloramphenicol, Florphenicol, Thiamphenicol;

[0200] c) Ansamycin derivatives: geldanamycin, harbimycin;

[0201] d) Carbapenems: biapenem, doripenem, ertapenem, imipenem, cilastatin, meropenem, panipenem;

[0202] 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);

[0203] f) Glycopeptides: Bleomycin, vancomycin (including oritabancin and teravancin), teicoplanin (darbabancin), lamopranin;

[0204] g) Glycylcyclines: Tigecycline;

[0205] h) β-lactamase inhibitors: Penam (sulbactam, tazobactam), Clavam (clavulanic acid);

[0206] i) Lincosamides: clindamycin, lincomycin;

[0207] j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics (CDA);

[0208] 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;

[0209] l) Monobactams: Aztreonam, Tigemonam;

[0210] m) Oxazolidinones: Linezolids;

[0211] 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;

[0212] o) Polypeptides: bacitracin, colistin, polymyxin B;

[0213] 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;

[0214] q) Streptogramins: Pristinamycin, quinupristin / dalfopristin;

[0215] r) Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizol, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole);

[0216] s) Steroidal antibacterial agents: Selected from fusidic acid;

[0217] t) Tetracyclines: Doxycycline, chlortetracycline, chromocycline, demeclocycline, rimecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, lolitetracycline, tetracycline, glycylcycline (including tigecycline);

[0218] u) Other antibiotics: annonasin, arsphenamine, bactoprenol inhibitor (bacitracin), DADAL / AR inhibitor (cycloserine), dicthiostatin, discodermolide, eleuterobin, epotilon, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laurimalid, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitor (fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), tazobactamtinidazole, uvarcin;

[0219] (4) Antiviral drugs:

[0220] a) Entry / fusion inhibitors: aplaviroc, maraviroc, bicriviroc, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab);

[0221] b) Integrase inhibitors: raltegravir, elvitegravir, globoidnan A;

[0222] c) Maturation inhibitors: Bevirimat, Vivicon;

[0223] d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir;

[0224] 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 (3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'- (Including the group consisting of dideoxyguanosine (FLG)), homivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (including the group consisting of 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);

[0225] 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;

[0226] g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, preconalil, ritonavir, saquinavir, telaprevir (VX-950), tipranavir;

[0227] 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, Cericlib;

[0228] (5) Radioisotopes for radiotherapy

[0229] Examples of radioactive isotopes (radionic nuclides) include: 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 64 Cu, 68 Ga, 86 Y, 99 Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 177 Lu, 211 At, and 213Bi is an example. Radioisotope-labeled antibodies are very useful in receptor-targeted imaging experiments, or can be used directly in relative targeted therapy, for example, as in the invention of antibody-drug conjugates (Wu et al (2005) Nature Biotechnology 23(9):1137-1146). Cell-binding molecules, such as antibodies, can be labeled by binding, chelation, or forming other complex radioisotope metal bonds by the crosslinking conjugates of this application, as described above. This labeling technique is described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, NY, Pubs. (1991). Chelating agents capable of generating complex metal complexes include DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex. USAQ).

[0230] (6) Another cell-binding molecule-drug conjugate as synergistic therapy

[0231] Preferred synergistic conjugates include tubulicin analogs, meitansinoid analogs, taxanoid analogs (taxanes), CC-1065 analogs, daunorubicin and doxorubicin compounds, amatoxin analogs, benzodiazepine dimers (e.g., pyrrolobenzodiazepine (PBD), tomaimycin, anthramycin, indolinobenzodiazepines, imidazobenzothiadiazepine, or oxazolidinobenzodiazepine dimers), calicheamicin and engine antibiotics, actinomycin, azaserins, bleomycins, epirubicin, eribulin, tamoxifen, idarubicin, dorastatins, and auristatins (e.g., monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, etc.). This can be a conjugate containing cytotoxic agents such as -listatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP) and their analogs), duocalmycins, geldanamycins, methotrexates, thiotepa, vindesines, vincristines, hemiasterines, nazumamides, microginins, radiosumins, alterobactins, microsclerodermins, theonellamides, esperamicins, PNU-159682; and their analogs and derivatives.

[0232] 7) A pharmaceutically acceptable salt, acid, or derivative of any of the above drugs.

[0233] In yet another embodiment, an immunotoxin can be conjugated to a cell-binding molecule as a synergistic agent. The immunotoxins herein are typically macromolecular drugs that are cytotoxic proteins derived from bacterial or plant proteins, such as diphtheria toxin (DT), cholera toxin (CT), tricosanthine (TCS), diatine, Pseudomonas exotoxin A (ETA'), erythromycin, diphtheria toxin, AB toxin, and type III exotoxin. They may also be highly toxic bacterial pore-forming protoxins that require proteolytic treatment for activation. Examples of such protoxins are proaerolysin and its recombinant form, topsaricin. Topsaricin is a modified recombinant protein designed to be selectively activated by prostatic enzymes, causing local cell death and tissue destruction without damaging adjacent tissues or nerves.

[0234] Other synergistic immunotherapies include checkpoint inhibitors, TCR (T cell receptor) T cells, or CAR (chimeric antigen receptor) T cells, or B cell receptor (BCR), natural killer (NK) cells, or cytotoxic antibody cells, or anti-CD3, CD4, CD8, CD16 (FcγRIII), CD27, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57 bright TNFβ, Fas ligand, MHC class I molecules (HLA-A, B, C), or NKR-P1 are preferably used in conjunction with the conjugate of this patent for synergistic therapy.

[0235] Prescription and application

[0236] The patented conjugate is suitable for formulation in liquid form or for lyophilization and subsequent reconstitution into liquid formulations. The conjugate may constitute 0.01% to 99% by weight as the main component in the liquid formulation or in the formulated lyophilized powder. Generally, liquid formulations containing the conjugate active ingredient at concentrations of 0.1 g / L to 300 g / L for delivery to patients without high levels of antibody aggregation may include one or more polyols (e.g., sugars), buffers having a pH of 4.5 to 7.5, surfactants (e.g., polysorbate 20 or 80), antioxidants (e.g., ascorbic acid and / or methionine), isotonic agents (e.g., mannitol, sorbitol or NaCl), chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; preservatives (e.g., benzyl alcohol); and / or free amino acids.

[0237] Suitable buffers for use in formulations include, but are not limited to, organic acid salts such as sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalates; and Tris, tromethamine hydrochloride, sulfate, or phosphate buffers. Furthermore, amino acid cationic components can also be used as buffers. Such amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine chloride, arginine phosphate, arginine sulfate, arginine succinate, etc. In one embodiment, the arginine buffer is arginine acetate. Examples of histidine buffers include histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc. The pH of the buffer formulation is 4.5 to 7.5, preferably about 4.5 to about 6.5, and more preferably about 5.0 to about 6.2. In some embodiments, the concentration of the organic acid salt in the buffer is about 10 mM to about 500 mM.

[0238] Polyols, optionally included in pharmaceutical formulations, are substances having multiple hydroxyl groups. Polyols can be used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized formulations. Polyols can protect biopharmaceuticals from physical and chemical degradation pathways. Preferredly excluded cosolvents increase the effective surface tension of the solvent at the protein interface, thereby resulting in the most energetically favorable structural conformation having the smallest surface area. Polyols include sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. Reducing sugars contain hemiacetal groups that can reduce metal ions or react covalently with lysine or other amino groups of proteins, while non-reducing sugars lack these properties of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melegitose, and raffinose. The sugar alcohol is selected from mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, sreitol, sorbitol, and glycerol. The sugar acid includes L-gluconates and their metal salts. The polyol in the liquid formulation or the prepared lyophilized solid may be 0.0% to 20% by weight. Preferably, a non-reducing sugar, sucrose, or trehalose is selected in the formulation at a concentration of about 0.1% to 15%, with trehalose being preferred over sucrose due to its solution stability.

[0239] The surfactants optionally included in the formulation are polysorbate (polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, etc.), poloxamer (e.g., poloxamer 188, poly(ethylene oxide)-poly(propylene oxide), poloxamer 407, or polyethylene-polypropylene glycol, etc.); triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, co The following are selected: Camidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, Palmimidopropyl-, or Isostearamidopropyl-betaine (e.g., Lauroamidopropyl); Myristamidopropyl-, Palmidopropyl-, or Isostearamidopropyl-dimethylamine; Methyl cocoyl sodium, or Sodium methyl oleyl taurate; Dodecyl betaine, Dodecyldimethylamine oxide, Cocamidopropyl betaine, and Cocoamphoglycinates, the "MONAQUAT" (trademark) series (e.g., Isostearylethylimonium ethosulfate); Polyethyl glycol, Polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). Preferred surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). The concentration of the surfactant in the formulation is in the range of 0.0% by weight to about 2.0% by weight. In certain embodiments, the concentration of the surfactant is about 0.01% to about 0.2%. In one embodiment, the concentration of the surfactant is about 0.02%.

[0240] The “preservatives” optionally included in a formulation are compounds that essentially reduce bacterial activity. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The amount of preservative in a liquid formulation or prepared lyophilized powder may be 0.0% to 5.0% by weight. In one embodiment, the preservative described herein is benzyl alcohol.

[0241] Suitable free amino acids for use as isotonic or osmotic regulators in bulk raw materials or formulations are selected from, but are not limited to, arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycylcyrrhamate, or aspartic acid. Basic amino acids, i.e., those containing arginine, lysine, and / or histidine, are preferred. If the composition contains histidine, it can function as both a buffer and a free amino acid; however, if a histidine buffer is used, it typically contains non-histidine free amino acids, such as a histidine buffer and lysine. The amino acids may be D- and / or L-, but are usually L-isomers. The amino acids may exist as any stable salt, such as a hydrochloride salt of arginine-HCl. The amount of amino acids in the liquid formulation or prepared lyophilized powder can be 0.0% to 30% by weight.

[0242] The formulation may optionally contain methionine, glutathione, cysteine, cystine, or ascorbic acid as antioxidants at a concentration of up to approximately 5 mg / ml in the liquid formulation, or at a concentration of 0.0% to 5.0% by weight in the formulated lyophilized powder. The formulation may optionally contain metal chelating agents, such as EDTA or EGTA, at a concentration of up to approximately 2 mM in the liquid formulation, or at a concentration of 0.0% to 0.3% by weight in the formulated lyophilized powder.

[0243] The final formulation can be adjusted to a preferred pH with a buffering agent (e.g., acids such as HCl, H2SO4, acetic acid, H3PO4, citric acid, or bases such as NaOH, KOH, NH4OH, ethanolamine, diethanolamine, or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate, or tromethamine). The formulation needs to be controlled to be "isotonic," meaning that the formulation has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-type osmometer. The isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or NaCl. Generally, both the buffer salt and the isotonic agent can account for up to 30% by weight in the formulation.

[0244] Other excipients that may be useful in either the liquid or lyophilized formulation of the patent application include, for example, fucose, cellobiose, maltotriose, melibiose, octulose, ribose, xylitol, arginine, histidine, glycine, alanine, methionine, glutamic acid, lysine, imidazole, glycylglycine, mannosylglycerate, Triton X-100, and Pluoronic. F-127 includes cellulose, cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, dextran (10, 40 or 70 kD), polydextrose, maltodextrin, Ficol, gelatin, hydroxypropyl meta, sodium phosphate, potassium phosphate, ZnCl2, zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, fibronectin, heparin, human serum albumin, protamine, glycerin, glycerol, EDTA, metacresol, benzyl alcohol, phenol, polyhydric alcohol, or hydrogenated forms of carbohydrates having a carbonyl group reduced to a primary or secondary hydroxyl group.

[0245] Other intended excipients that can be used in the aqueous pharmaceutical composition of the patent application include, for example, flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, and casein, and salt-forming counterions such as sodium. These and additional known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art, as cited, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003)" and "Remington: The Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005)."

[0246] Pharmaceutical containers or vessels are used to hold any of the pharmaceutical formulations of the conjugates of the patent application. The containers are vials, bottles, pre-filled syringes, pre-filled syringes, or auto-injector syringes. Liquid formulations can be freeze-dried or drum-dried in the form of cake or powder in borosilicate vials or soda-lime glass vials. Solid powders may also be prepared by efficient spray drying and then packed into vials or pharmaceutical containers for storage and distribution.

[0247] In further embodiments, the present invention provides a method for preparing a formulation, comprising the following steps: (a) lyophilizing a formulation comprising a conjugate, excipients, and a buffer system; and (b) reconstituting the lyophilized mixture from step (a) in a reconstitution medium so that the reconstituted formulation is stable. The formulation from step (a) may further comprise a stabilizer and one or more excipients selected from the group comprising the above-mentioned bulking agents, salts, surfactants, and preservatives. As the reconstitution medium, several diluted organic acids or water, i.e., sterile water or bacteriostatic water for injection (BWFI), can be used. The reconstitution medium can be selected in an amount of about 10 to about 250 mM from the group consisting of water, i.e., sterile water, bacteriostatic water for injection (BWFI), or acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, acidic solutions of sodium chloride, acidic solutions of magnesium chloride, and acidic solutions of arginine.

[0248] The liquid pharmaceutical formulations of the conjugates of this application need to exhibit various predefined properties. Stability is one of the main concerns for liquid pharmaceuticals, as proteins / antibodies tend to form soluble and insoluble aggregates during manufacturing and storage. Furthermore, various chemical reactions (deamination, oxidation, clipping, isomerization, etc.) can occur in solution, potentially leading to increased levels of degradation products and loss of biological activity. Preferably, the conjugates in liquid or lyophilized formulations need to exhibit a shelf life of more than 6 months at 25°C. More preferably, the conjugates, in either liquid or lyophilized formulations, need to exhibit a shelf life of more than 12 months at 25°C. The most preferred liquid formulations should exhibit a shelf life of approximately 24–36 months at 2–8°C, and the lyophilized formulations should exhibit a shelf life of up to approximately 60 months at 2–8°C. Both liquid and lyophilized formulations need to exhibit a shelf life of at least 2 years at -20°C or -70°C.

[0249] In some embodiments, the formulation is stable after freezing (e.g., -20°C or -70°C) and thawing, for example, after one, two, or three freeze-thaw cycles. Stability can be qualitatively and / or quantitatively evaluated by various methods, such as: assessment of drug / antibody (protein) ratio and aggregate formation (e.g., using ultraviolet, size exclusion chromatography, turbidity measurement, and visual inspection); assessment of charge heterogeneity using electrofocusing or capillary zone electrophoresis such as cation exchange chromatography or image capillaries; sequence analysis of amino or carboxyl group ends; mass spectrometry, or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS), or HPLC-MS / MS; SDS-PAGE analysis for comparison of antibody reduction or integrity; peptide mapping (e.g., trypsin or lysine-carbon); and evaluation of the biological activity or antigen-binding function of the antibody. The instability may include one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), shear / hydrolysis / fracture (e.g., hinge fracture), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, and catabolic glycosylation.

[0250] The stable conjugate also needs to "maintain its biological activity" in the pharmaceutical formulation, for example, the biological activity of the conjugate at a specific point in time, e.g., 12 months, must be within approximately 20%, preferably within approximately 10% (within the assay error) of the biological activity determined by antigen-binding assays and / or in vitro cytotoxicity assays, etc., at the time the pharmaceutical formulation was prepared.

[0251] For in vivo clinical application, the bis-linked conjugates of the present invention may be provided in the form of a solution or as a lyophilized solid that can be redissolved in sterile water for injection. Examples of appropriate methods of administering the conjugates are as follows: The conjugates are administered intravenously as a bolus daily, weekly, every two weeks, every three weeks, every four weeks, or monthly for 8 to 54 weeks. The bolus dose is dissolved in 50 to 1000 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 4 to 54 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.

[0252] 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).

[0253] 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.

[0254] Generally speaking, the bis-linked conjugate of the present invention may be a parenteral formulation dissolved in physiological buffer 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, week, two weeks, three weeks, or month, and preferred dose ranges are 0.01 mg / kg body weight to 25 mg / kg body weight per week, two weeks, three weeks, or month. Preferred drug doses may appropriately depend on variables such as 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).

[0255] The conjugates of the present invention, via the linkers, can also be administered in unit doses, where "unit dose" refers to a single dose administered to one patient. These units can be used in simple and convenient packaging and maintain physical and chemical stability as either the active conjugate itself or 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, week, two weeks, three weeks, or month. Preferably, the unit dose is 1 mg to 500 mg administered 1 to 4 times per month, and more preferably, 1 mg to 100 mg administered once every week, two weeks, or three weeks. 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, spray, or skin patch.

[0256] In yet another embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a conjugate of formula (I) or formula (III), or any conjugate described herein, may be administered simultaneously with other therapeutic agents such as chemotherapeutic agents, radiotherapy agents, immunotherapeutic agents, autoimmune disease agents, anti-infective agents, or other conjugates for synergistically treating or preventing cancer, autoimmune diseases, or infectious diseases. The synergistic agent is preferably selected from one or more of the following agents: abatacept, abemaciclib, abiraterone acetate, Abraxane, acetaminophen / hydrocodone, acalabrutinib, aducanumab, adalimumab, ADXS31-142, ADXS-HER2, afatinib dimareate, aldesleukin, alectinib, alemtuzumab, alitretinoin, ad-truss Zumab emtansine, amphetamine / dextroamphetamine, anastrozole, aripiprazole, anthracycline, aripiprazole, atazanavir, atezolizumab, atorvastatin, avelumab, axicabtageneciloleucel, axitinib, bellinostat, live BCG, bevacizumab, bexarotene, blinatumomab, bortezomib, bosutinib, ble Ntuximab vedotin, brigatinib, budesonide, budesonide / formoterol, buprenorphine, cabazitaxel, cabozatinib, capmatinib, capecitabine, carfilzomib, chimeric antigen receptor-modified T (CAR-T) cells, celecoxib, ceritinib, cetuximab, pidamide, cyclosporine, cinacalcet, crizotinib, cobimetinib, cosentyx (Cose Ntyx), Crizotinib, Tisagenlecurecel, CTL019, Dabigatran, Dabrafenib, Dacarbazine, Dacrizumab, Dacomotinib, Daptomycin, Daratumumab, Darbepoetin α, Darunavir, Dasatinib, Denileukin difutitox, Denosumab, Depakote, Dexlansoprazole, Dexmethylphenidate, Dexamethasone, DigniCap Cooling System, Dinutuximab, Doxycycline, Duloxetine, Duvelisib, Durvalumab, Elotuzumab, Emtricitab / Rilpivirine / Tenofovir, Disoproxil fumarate, Emtricitab / Tenofovir / Efavirenz, Enoxaparin,Ensartinib, Enzalutamide, Epoetin α, Erlotinib, Esomeprazole, Eszopiclone, Etanercept, Everolimus, Exemestane, Everolimus, Exenatide ER, Ezetimibe, Ezetimibe / Simvastatin, Fenofibrate, Filgrastim, Fingolimod, Fluticasone propionate, Fluticasone / Salmeterol, Fulvestrant, Gazyva, Gefitinib, Glatiramer, Goserelin acetate, Icotinib, Imatinib, Ibritumomab tiuxetan, Ibrutinib, Idelalisib, Ifosf Imiquimod, Infliximab, Imiquimod, ImmuCyst, ImmunoBCG, Iniparib, Insulin Aspart, Insulin Detemir, Insulin Glargine, Insulin Lispro, Interferon α, Interferon α-1b, Interferon α-2a, Interferon α-2b, Interferon β, Interferon β1a, Interferon β1b, Interferon γ-1a, Lapatinib, Ipilimumab, Ipratropium bromide / salbutamol, Ixazomib, Kanuma, Lanreotide acetate, Lenalidomide, Re Nariomid, lenvatinib mesylate, letrozole, levothyroxine, lidocaine, linezolid, liraglutide, lisdexamfetamine, LN-144, lorlatinib, memantine, methylphenidate, metoprolol, mekinist, mericitabine / rilpivirine / tenofovir, modafinil, mometasone, Mycidac-C, necitumumab, neratinib, nilotinib, niraparib, nivolumab, ofatumumab, obinutuzumab, olaparib, olmesartan, olmesartan / hydrochlorothiazide, omalizumab, omega-3 fatty acids Ethyl ester, oncolin, oseltamivir, osimertinib, oxycodone, palbociclib, palivizumab, panitumumab, panobinostat, pazopanib, pembrolizumab, PD-1 antibody, PD-L1 antibody, pemetrexed, pertuzumab, pneumococcal conjugate vaccine, pomalidomide, pregabalin, ProscaVax, propranolol, quetiapine, rabeprazole, radium-223 chloride, raloxifene, raltegravir, ramucirumab, ranibizumab, regorafenib, ribociclib, rituximab, rivaroxaban, romidepsin,Rosuvastatin, ruxolitinib phosphate, salbutamol, savolitinib, semaglutide, sevelamer, sildenafil, siltuximab, ciplucel-T, sitagliptin, sitagliptin / metformin, solifenacin, solanezumab, sonidegib, sorafenib, sunitinib, tacrolimus, tacrimus, tadalafil, tamoxifen, Tafinlar, Talimogene laherparepvec, talazoparib, telaprevir, talazoparib, temozolimide, temsirolimus, tenofovir / emtricitabine, tenofovir disoproxil fumarate, testosterone gel, thalidomide, TICE BCG, tiotropium bromide, tisagenlecleucel, toremifene, trametinib, trastuzumab, ecteinascidin 743, trametinib, tremelimumab, trifluridine / tipiracil, Uro-BCG, ustekinumab, valsartan, veliparib, vandetanib, vemurafenib, venetoclax, vorinostat, ziv-aflibercept, and zostavax, as well as their analogues, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients for these, or combinations thereof.

[0257] The drug / cytotoxic agents used in the conjugation via the branched conjugates of the present invention may be analogs and / or derivatives of the amatoxins described herein. Those skilled in the art of drug / cytotoxic agents will readily understand that each of the amatoxins described herein may be modified such that the resulting compound still retains the specificity and / or activity of the starting compound. Those skilled in the art will also understand that many of these analog and derivative compounds can be used in place of the amatoxin analogs described herein. Accordingly, the amatoxin conjugates of the present invention include many analogs and derivatives of amatoxin compounds not described in detail herein.

[0258] All references cited herein and in the following examples are explicitly incorporated by reference in their entirety. [Examples]

[0259] The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the application. Unless otherwise specified, the cell lines described in the following examples were maintained in culture media based on conditions specified by the American Type Culture Collection (ATCC), the German Microbial Cell Culture Collection (DMSZ) in Braunschweig, Germany, or the Shanghai Institute of Cell Culture, Chinese Academy of Sciences. Unless otherwise specified, all cell culture reagents were supplied by Invitrogen. All anhydrous solvents were commercially available and stored in nitrogen-filled sealed 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 detected using a Bruker 500 MHz instrument. Chemical shifts (Δ) were expressed in parts per million (ppm), with tetramethylsilane as the standard at 0.00, and the unit of the binding constant (J) was Hz. Mass spectrometry data were acquired using Waters Xevo QTOF mass spectrometry equipped with a Waters Acquity UPLC separator and Acquity TUV detector. Generally, UPLC separation was performed on a C8 column using mobile phase A: 1% formic acid and phase B: 100% CH3CN.

[0260] Example 1; Fmoc-Hyp(O t Bu)-Ile-O t Synthesis of Bu(1-1) [ka]

[0261] At 0℃, H-Ile-O tTo a DMF solution (300 mL) of Bu·HCl (25.0 g, 0.11 mol), Fmoc-Hyp(OtBu)-OH (45.9 g, 1.0 equivalent), HOBt (16.7 g, 1.1 equivalents), EDC (23.7 g, 1.1 equivalents), and DIPEA (48.7 mL, 2.5 equivalents) were added in sequence. The reaction mixture was stirred at 10 - 25 °C for 4 hours, diluted with water (500 mL), and extracted with ethyl acetate (300 mL × 3). The combined organic phases were washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by silica gel column (petroleum ether / ethyl acetate from 10:1 to 1:1) to obtain 44.5 g of 1- as a white solid (yield 68.8%). ESI m / z C 34 H 47 N2O6[M+H]<000096]8>: Calculated: 578.33, Found: 578.35.

[0262] Example 2; H-Hyp(O t Bu)-Ile-O t Synthesis of Bu(2) ] [[ID=]7]]

Chemical Structure

[0263] To a DMF solution (200 mL) of Compound 1-1 (44.5 g, 76.9 mmol), piperidine (40 mL) was added, and the mixture was stirred at 10 - 25 °C for 1 hour. The DMF was removed under high vacuum. A solid crude product was obtained, which was purified by silica gel column (from 1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 27.2 g of the title compound as a colorless oil (yield 93.7%). ESI m / z C 19 H 37 N2O4[M+H] + : Calculated: 357.2753, Found: 357.2768.

[0264] Example 3; Fmoc-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(2-1)

Chemical Structure

[0265] At 0°C, iBuO2CCl (10.9 mL, 1.1 equivalent) was added dropwise to a THF solution (500 mL) of Fmoc-Asn(Trt)-OH (50 g, 1.1 equivalent) and NMM (27.2 mL, 3.0 equivalent). The mixture was stirred at 0°C for 30 minutes, then stirred at room temperature for 3 hours. Subsequently, compound 2 (27.2 g, 1.0 equivalent) was added dropwise to a THF solution (200 mL) while stirring at 0°C. After stirring at room temperature for 16 hours, water (500 mL) was added, and the mixture was extracted with ethyl acetate (300 mL x 3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, concentrated to a crude product, and purified by silica gel column (10:1~1:1 petroleum ether / ethyl acetate) to obtain 52.3 g of 2-1 as a white solid (yield 70.2%). ESI m / z C 57 H 67 N4O8[M+H] + : 935.4882, Measured value: 935.4895.

[0266] Example 4; H-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(3) [ka]

[0267] To a 100 mL solution of compound 2-1 (20 g, 21.4 mmol) in DMF, piperidine (20 mL) was added. The mixture was stirred at 10-25°C for 1 hour, concentrated under high vacuum to remove DMF, and a solid crude product was obtained. This was then analyzed using a silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 14.0 g of the title compound as a colorless oil (yield 92.3%). ESI m / z C 42 H 57 N4O6[M+H] + : 713.4279, measured value: 713.4285.

[0268] Example 5; Fmoc-Cys(Trt)-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(3-1) [ka]

[0269] A mixture of compound 3 (7.3 g, 10.2 mmol), Fmoc-Cys(Trt)-OH (6.0 g, 1 equivalent), and EDC (9.7 g, 5.0 equivalents) in dichloromethane (80 mL) was stirred at room temperature for 16 hours. Water (500 mL) was added, and the mixture was extracted with ethyl acetate (300 mL x 3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, concentrated to a crude product, and purified by silica gel column (10:1~1:1 petroleum ether / ethyl acetate) to obtain 9.8 g of 3-1 as a white foamy solid (yield 75.2%). ESI m / z C 78 H 84 N5O9S [M+H] + Calculated value: 1266.5990, Measured value: 1266.5980.

[0270] Example 6; H-Cys(Trt)-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(4) [ka]

[0271] To a 50 mL solution of compound 3-1 (9.0 g, 7.03 mmol) in DMF, piperidine (10 mL) was added, and the mixture was stirred at 10-25°C for 1 hour. After removing the DMF under high vacuum, the crude product was purified by silica gel column chromatography (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 7 g of the title compound as a colorless oil (yield 95.2%). ESI m / z C 64 H 76 N5O7S [M+H] + Calculated value: 1058.5466, Measured value: 1058.5460.

[0272] Example 7; Cbz-Ile-Gly-O t Synthesis of Bu(5-1) [ka]

[0273] At 0°C, add H-Gly-O to a 120 mL DMF solution of Cbz-L-Ile-OH (15 g, 57.1 mmol, 1.0 equivalent). t Bu·HCl (10.5 g, 1.0 equivalent), HOBt (9.3 g, 1.2 equivalents), EDC (13.2 g, 1.2 equivalents), and DIPEA (25 mL, 2.5 equivalents) were added. The reaction mixture was stirred at 10-25°C for 16 hours. Next, water (300 mL) was added, and the reaction mixture was extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was purified by silica gel column (10:1-1:1 petroleum ether / ethyl acetate) to obtain 19.5 g of 5-1 as a white solid (yield 90.1%). ESI m / z C 20 H 31 N2O5[M+H] + Calculated value: 379.2234, Measured value: 379.2248.

[0274] Example 8; H-Ile-Gly-O t Synthesis of Bu(6) [ka]

[0275] A mixture of compound 5-1 (19.5 g) in methanol was mixed with Pd / C (10 wt%, 2.0 g, containing 64.2% H2O). The mixture was stirred under a hydrogen balloon (1 atm) for 16 hours, then filtered, and the filtrate was concentrated to obtain compound 6 as a colorless oil (11.5 g, yield 93.2%). ESI m / z C 12 H 25 N2O3[M+H] + Calculated value: 245.1866, Measured value: 245.1860.

[0276] Example 9; Fmoc-Gly-Ile-Gly-O t Synthesis of Bu(6-1) [ka]

[0277] At 0°C, a 30 mL solution of compound 6 (3.0 g, 12.3 mmol, 1.0 equivalent) in DMF was mixed with Fmoc-Gly-OH (3.6 g, 1.0 equivalent), HOBt (1.99 g, 1.2 equivalents), EDC (2.82 g, 1.2 equivalents), and DIPEA (3.2 mL, 1.5 equivalents). The reaction mixture was stirred at 10-25°C for 2.5 hours. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, concentrated to a crude product, and purified by silica gel column chromatography (10:1-1:1 petroleum ether / ethyl acetate) to obtain 6.6 g of 6-1 as a waxy solid (100% yield). ESI m / z C 29 H 38 N3O6[M+H] + Calculated value: 524.2761, Measured value: 524.2778.

[0278] Example 10; Synthesis of Fmoc-Gly-Ile-Gly-OH (7a) [ka]

[0279] A 25 mL solution of compound 6-1 (6.6 g) in dichloromethane was stirred, and 25 mL of TFA was added. The reaction mixture was stirred at room temperature for 16 hours, then concentrated and evaporated three times with toluene. Compound 7a was then obtained as a waxy solid (8.2 g, crude). ESI m / z C 25 H 30 N3O6[M+H] + Calculated value: 468.2135, Measured value: 468.2147.

[0280] Example 11; Fmoc-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(7a-1) [ka]

[0281] At 0°C, compound 4 (9.0g, 1.0 equivalent), EDC (7.2g, 5.0 equivalents), and DIPEA (6.8mL, 3.5 equivalents) (pH 7.5) were added to a 100mL dichloromethane solution of compound 7a (8.2g, crude, 1.1 equivalents). The reaction mixture was stirred at 10-25°C for 2.5 hours. Water (500mL) was added, and the mixture was extracted with ethyl acetate (300mL x 3). The combined organic phase was washed with brine (300mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 6.8g of 7a-1 as a pale yellow oily substance (yield 53.1%). ESI m / z C 89 H 103 N8O 12 S [M+H] + Calculated value: 1507.7417, Measured value: 1507.7442.

[0282] Example 12; H-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(O t Bu)-Ile-O t Synthesis of Bu(9) [ka]

[0283] To a 30 mL solution of compound 7a-1 (6.8 g) in DMF, piperidine (6 mL) was added, and the mixture was stirred at 10-25°C for 1 hour. Next, the DMF was removed under high vacuum to obtain a solid crude product, which was purified using silica gel column chromatography (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 5.2 g of the title compound as a colorless oil (yield 91.2%). ESI m / z C 74 H 193 N8O 10 S [M+H] + Calculated value: 1285.6736, Measured value: 1285.6750.

[0284] Example 13; Synthesis of 6-nitro-D-tryptophan (10a) [ka]

[0285] To a 500 mL glacial acetic acid suspension of D-tryptophan (40.8 g, 0.20 mol) and urea (0.50 g), a 30 mL glacial acetic acid solution of fuming nitric acid (7.5 mL) was added while vigorously stirring. The solid dissolved, and the solution turned yellow. Stirring was continued, and the solution was returned to a suspension. At 10°C, an additional 17.5 mL of fuming nitric acid in 70 mL of glacial acetic acid was slowly added to the suspension. The solid dissolved, and the solution changed from yellow to brown. After the addition was complete, the solution was stirred at room temperature for 22 hours. The reaction mixture was concentrated to approximately 100 mL, then water (200 mL) was added to form a yellow precipitate, which was collected by filtration and washed with a small amount of water and ethyl acetate. The solid was dried outdoors and weighed 16.8 g. The filtrate was further concentrated to dryness and recrystallized with 5% HNO3 to obtain another product (10 g). ESI MS m / z C 11 H 12 N3O4[M+H] + Calculated value: 250.0829, Measured value: 250.0835.

[0286] Example 14; Synthesis of 6-nitro-N,N'-bis(tert-butyloxycarbonyl)-D-tryptophan (11) [ka]

[0287] At 0°C, a mixture of compound 10a (6.0 g, 24.1 mmol) in dichloromethane (50 mL) was mixed with NaOH (9.7 g, 10 equivalents) and Bu4NHSO4 (1.6 g, 0.2 equivalents). Subsequently, at 0°C, a dichloromethane solution of Boc2O (20.2 g, 3.5 equivalents) (30 mL) was added. The reaction mixture was stirred at 10-25°C for 16 hours, then water (60 mL) was added, and the mixture was extracted with dichloromethane (300 mL x 3). The combined organic phase was washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 7.5 g of the title compound as a pale yellow oily substance (yield 69.4%). ESI MS m / z C 21 H 28 N3O8[M+H] + Calculated value: 450.0908, Measured value: 450.0930.

[0288] Example 15; Synthesis of 6-nitro-N,N'-bis(tert-butyloxycarbonyl)-D-tryptophan benzyl ester (12) [ka]

[0289] A mixture of compound 11 (7.1 g, 15.8 g, 1.0 equivalent), K2CO3 (4.3 g, 2.0 equivalent), and BnBr (3.5 mL, 1.1 equivalent) in acetone (80 mL) was refluxed for 2.5 hours, then cooled to room temperature, water (30 mL) was added, and the mixture was extracted with dichloromethane (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. This was purified using a silica gel column (10:1-3:1 petroleum ether / ethyl acetate) to obtain 8.1 g of compound 12 as a pale red oily substance (yield 95.2%). ESI MS m / z C 28 H 34 N3O8[M+H] + Calculated value: 540.2347, Measured value: 540.2360.

[0290] Example 16; Synthesis of 2-benzyl 1,8-di-tert-butyl 3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (13) [ka]

[0291] Preparation of DMDO solution: Distilled water (20 mL), acetone (30 mL), and NaHCO3 (24 g, 0.285 mol) were placed in a 1 L round-bottom flask and cooled in an ice / water bath with a magnetic stirrer. After 20 minutes, stirring was stopped and oxone (25 g, 0.0406 mol) was added all at once. The flask was loosely covered and the slurry was immersed in the ice bath and stirred vigorously for 15 minutes. Next, the flask containing the reaction slurry was attached to a rotary evaporator equipped with a bath at room temperature. A bump valve (250 mL) was cooled in a dry ice / acetone bath and a vacuum of 165 mtor was applied via a benchtop diaphragm pump. After 15 minutes, the bath temperature was raised to 40°C over 10 minutes. When the bath reached 40°C, the vacuum was released and the distillation was immediately stopped by lifting the flask out of the warm water bath. The pale yellow acetone solution of DMDO was decanted directly into a graduated cylinder via a bump valve to measure the total volume of the solution (approximately 25 mL), and then the solution was dried with sodium sulfate.

[0292] Sodium sulfate was removed by filtration, and the solution was rinsed with 5 mL of acetone. The titration of the resulting DMDO solution could be performed according to the procedure of Adam et al. (Adam, W.; Chan, YY; Cremer, D.; Gauss, J.; Scheutzow, D.; Scheutzow, D.; Schindler, MJ Org. Chem. 1987, 52, 2800-2803). The results consistently showed 2.1–2.3 mmol of DMDO in the solution. The DMDO solution was used immediately after titration.

[0293] The above cold DMDO solution was added dropwise to a mixture of compound 12 (0.60 g, 1.11 mmol) in acetone while stirring at 0°C. The mixture was stirred at 0°C for 2 hours, then stirred at room temperature for 16 hours. The mixture was concentrated to obtain the crude product. Water (20 mL) was added, and the resulting solution was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (10:1 to 3:1 petroleum ether / ethyl acetate) to obtain 0.41 g of compound 13 as a gray solid. ESI MS m / z C 28 H 34 N3O9[M+H] + Calculated value: 556.2296, Measured value: 556.2320.

[0294] Example 17; Synthesis of 1,8-bis(tert-butoxycarbonyl)-3a-hydroxy-6-nitro-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (14) [ka]

[0295] A mixture of compound 13 (0.31 g, 0.56 mmol) and THF (15 mL) was mixed with an aqueous solution of NaOH (0.089 g, 4.0 equivalents) (8 mL). The mixture was stirred at 40°C for 16 hours. The mixture was then concentrated to remove the THF, the residue was diluted with water (15 mL), and washed with ethyl acetate (30 mL x 2). The resulting aqueous phase was adjusted to pH 3 with 1 N HCl and extracted with ethyl acetate (20 mL x 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain 0.22 g of compound 14 as a colorless oil (yield 83.8%). ESI MS m / z C 21 H 28 N3O9[M+H] + Calculated value: 466.1826, Measured value: 466.1840.

[0296] Example 18; Synthesis of N-phenylselenophthalimide (15) [ka]

[0297] Under nitrogen, a mixture of potassium phthalimide (4.03 g, 21.8 mmol) and phenylselenyl chloride (5.00 g, 26.1 mmol) was mixed with dry hexane (20 mL). After stirring at room temperature for 3 hours, dry dichloromethane (100 mL) was added, and the pale red solution was filtered to remove solid material. The filtrate was concentrated to approximately 20 mL and diluted with dry hexane (80 mL). The resulting precipitate was collected by filtration, washed with dry hexane, and dried under vacuum. A pale yellow solid was obtained (4.55 g, yield 69%). ESI MS m / z C 14 H 10 NO 2 Se [M+H] + : 303.9878, Measured value: 303.9890.

[0298] Example 19; Synthesis of 6-nitro-D-tryptophan methyl ester (16) [ka]

[0299] To a methanol solution of 10a (2.00 g, 0.803 mmol), thionyl chloride (0.58 mL, 8.03 mmol) was added dropwise. The mixture was then heated under reflux and stirred for 2 hours. After cooling to room temperature, the reaction mixture was diluted with water (60 mL), pH adjusted to 8.0 using 10% sodium hydroxide, and then extracted with ethyl acetate (60 mL x 3). The combined organic phase was dried over sodium sulfate, filtered, and concentrated to obtain a pale red solid (1.50 g, 71.4%). ESI MS m / z C 12 H 14 N3O4[M+H ]+ : 264.0985, Measured value: 264.0942.

[0300] Example 20; Synthesis of 6-nitro-N,N'-bis(tert-butyloxycarbonyl)-D-tryptophan methyl ester (17) [ka]

[0301] At 0°C, a mixture of compound 16 (720 mg, 2.74 mmol) in dichloromethane (20 mL) was mixed with NaOH (1.09 g, 10 equivalents) and Bu4NHSO4 (183 mg, 0.2 equivalents). Subsequently, at 0°C, a 5 mL solution of Boc2O (2.3 mL, 3.5 equivalents) in dichloromethane was added. The reaction mixture was stirred at 10-25°C for 8 hours, then water (30 mL) was added, and the mixture was extracted with dichloromethane (300 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. This was purified by silica gel column chromatography (10:1-3:1 petroleum ether / ethyl acetate) to obtain 750 mg of the title compound as a pale yellow oily substance (yield 64.2%). ESI MS m / z C 22 H 30 N3O8[M+H] + Calculated value: 464.2034, Measured value: 464.2058.

[0302] Example 21; Synthesis of (2S)-1,8-di-tert-butyl2-methyl6-nitro-3a-(phenylceranil)-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (18) [ka]

[0303] To a 20 mL solution of compound 17 (700 mg, 1.51 mmol) in dichloromethane, sodium sulfate (1.8 g), pyridinium 4-toluenesulfonate (68 mg, 0.2 equivalents), and N-phenylselenophthalimide (715 mg, 1.5 equivalents) were added. The reaction mixture was stirred overnight at room temperature. The mixture was then diluted in an eater (30 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. This was purified by silica gel column chromatography (10:1-3:1 petroleum ether / ethyl acetate) to obtain 400 mg of the title compound as a pale yellow oil (yield 43.0%), as well as several recovered starting materials. ESI MS m / z C 28 H 34 N3O8[M+H] + Calculated value: 620.1512, Measured value: 620.1545.

[0304] Example 22; Synthesis of (2S)-1,8-di-tert-butyl2-methyl3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,2,8(2H,8aH)-tricarboxylate (19) [ka]

[0305] At 0°C, a 30 mmol solution of compound 18 (1.80 g, 2.90 mmol) in dichloromethane was mixed with K2CO3 (2.00 g, 5.0 equivalents) and m-CPBA (85%, 1.48 g, 2.5 equivalents). The reaction mixture was warmed to room temperature, stirred overnight, then diluted with dichloromethane (100 mL) and filtered. The filtrate was washed with saturated NaHSO3 (30 mL x 3) and brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column (20:1-4:1 petroleum ether / ethyl acetate) to obtain 0.94 g of the title compound as a white foam (yield 67.7%). ESI MS m / z C 22 H 30 N3O9[M+H] +Calculated value: 479.1983, Measured value: 479.1995.

[0306] Example 23; Synthesis of 1,8-bis(tert-butoxycarbonyl)-3a-hydroxy-6-nitro-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (14) [ka]

[0307] To a THF / methanol / water (2:2:1) solution of compound 19 (0.90 g, 1.88 mmol), LiOH (0.23 g, 5.0 equivalents) was added. The solution was stirred overnight at room temperature, then concentrated and purified by silica gel column (methanol / dichloromethane, 1:20-1:3) to obtain the title compound (0.85 g, 97.3% yield). ESI MS m / z C 21 H 28 N3O9[M+H] + Calculated value: 466.1826, Measured value: 466.1845.

[0308] Example 24; Synthesis of (S)-3-(1H-indole-2-yl)-2-(tritylamino)propanoic acid (32) [ka]

[0309] At room temperature, chlorotrimethylsilane (3.4 mL, 26.9 mmol) was slowly added to a methylene chloride suspension (40 mL) of L-tryptophan (5.00 g, 24.5 mL). The mixture was continuously stirred for 4.5 hours, then triethylamine (6.8 mL, 49.0 mmol) was added, followed by a methylene chloride solution (20 mL) of triphenylmethyl chloride (7.17 g, 25.7 mmol). The mixture was stirred at room temperature for 20 hours, then quenched with methanol (25 mL). The reaction mixture was concentrated to almost dryness, redissolved in methylene chloride, and washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was further dissolved in methylene chloride, filtered through a Celite pad, and the filtrate was concentrated to obtain a pale white foam (11.8 g), which was used directly in the next step. ESI MS m / z 446.30([M+N] + ).

[0310] Example 25; Synthesis of (S)-methyl 2-(3-(1H-indole-2-yl)-2-(tritylamino)propanamide) acetate (33) [ka]

[0311] To a 30 mL THF solution of Acid 32 (9.27 g, 30.7 mmol), glycine methyl hydrochloride (2.85 g, 22.8 mmol) and HOBt (3.08 g, 22.8 mmol) were added. The mixture was cooled to 0°C, triethylamine (7.4 mL, 51.9 mmol) was added, followed by the gradual addition of EDC·HCl (4.38 g, 22.8 mmol). The mixture was warmed to room temperature, stirred for 20 hours, then concentrated, redissolved in methylene chloride, and washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was ground over ethyl acetate, and the white solid was collected by filtration (6.46 g, 65% yield in two steps). ESI MS m / z 518.20 ([M+N] + ).

[0312] Example 26; Synthesis of methyl 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamide)acetate (34) [ka]

[0313] At -78°C, a 20 mL solution of methylene chloride containing Trt-Trp-Gly-OMe (0.80 g, 1.54 mmol) was added to a 2.25 mmol solution of acetone containing DMDO. After 1 hour, the mixture was concentrated to dryness under reduced pressure at room temperature. The crude substance was purified by column chromatography (hexane / ethyl acetate / Et3N 70:30:1 to 30:70:1) to obtain a pale yellow foamy substance, a mixture of the two diastereomers (0.58 g, 70% yield). ESI MS m / z 534.22 ([M+N] + ).

[0314] Example 27; Synthesis of 2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamide)acetic acid (35) [ka]

[0315] To a solution of Tr-Hpi-Gly-OMe (a mixture of diastereomers) (0.80 g, 1.50 mmol) in dioxane / water (30 mL, v / v 2:1), LiOH (0.63 g, 15.0 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes (after consumption of the starting material by TLC (CH2Cl2 / methanol, 9:1)). The reaction mixture was concentrated to dryness, and the residue was purified by eluting with dichloromethane / methanol / triethylamine (90:10:1) using a short silica gel plug. The fractions were combined to obtain a pale yellow solid as the triethylamine salts of the two diastereomers (0.89 g, 95% yield).

[0316] Example 28; Synthesis of (2S)-di-tert-butyl 2-(((5S,8R,14S)-5-((2S,4R)-4-(tert-butoxy)-2-(((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-14-((S)-sec-butyl)-3,7,10,13,16-pentaoxo-1,1,1-triphenyl-8-((tritylthio)methyl)-2,6,9,12,15-pentazaheptadecan-17-yl)carbamoyl)-3a-hydroxy-6-nitro-3,3a-dihydropyrrolo[2,3-b]indole-1,8(2H,8aH)-dicarboxylate (36) [ka]

[0317] At 0°C, compound 9 (0.75g, 1.5 equivalents), EDC (0.17g, 1.5 equivalents), HOBt (0.12g, 1.5 equivalents), and DIPEA (0.25mL, 2.5 equivalents) were added to a 5mL DMF solution of compound 14 (0.27g, 0.58 mmol). The reaction mixture was stirred at 10-25°C for 16 hours. Water (20mL) was added, and the mixture was extracted with ethyl acetate (30mL x 3). The combined organic phase was washed with brine (30mL), dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was purified by silica gel column (1:1 petroleum ether / ethyl acetate to 10:1 dichloromethane / methanol) to obtain 0.5g of compound 36 as a pale yellow oily substance (yield 52.3%). ESI MS m / z C 95 H 118 N 11 O 18 S [M+H] + : 1732.8378, Measured value: 1732.8405.

[0318] Example 29; Synthesis of (2S,3S)-2-((2S,4R)-1-((S)-4-amino-2-((3R,9S,15S)-15-amino-9-((S)-sec-butyl)-19-nitro-5,8,11,14-tetraoxo-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,21-hexadecahydro-[1,4,7,10,13]thiatetraazacyclooctadecino[18,17-b]indole-3-carboxamide)-4-oxobutanoyl)-4-hydroxypyrrolidine-2-carboxamide)-3-methylpentanoic acid (37) [ka]

[0319] To a 1.0 mL solution of compound 36 (200 mg) in dichloromethane, TFA (2.0 mL) was added. The reaction mixture was stirred at room temperature for 16 hours, then concentrated and evaporated three times with toluene. The crude product was purified by preparative HPLC (acetonitrile / water) to obtain compound 37 as a pale yellow oil (50 mg, yield 47.1%). ESI MS m / z 918.40 ([M+N] + ).

[0320] Example 30; Synthesis of Compound 38 [ka]

[0321] At 0°C, EDC (124.0 mg, 4.0 equivalents), HOBt (88.0 mg, 4.0 equivalents), and DIPEA (128 μL, 4 equivalents) were added to a DMF solution (20 mL) of compound 37 (150 mg, 0.16 mmol). The reaction mixture was stirred at 10-25°C for 16 hours, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to obtain crude product 38 as a yellow oil (92.0 mg, yield 64.6%), which was used in the next step without further purification. ESI MS m / z 900.36 ([M+N] +).

[0322] Example 31; Synthesis of Compound 39 [ka]

[0323] Compound 38 (50.0 mg, 55.5 μmol) was mixed with methanol (50 mL) and Pd / C (10 wt%, 200 mg, containing 64.2% H2O). The mixture was stirred for 2 hours under a hydrogen gas balloon (1 atm), then filtered, and the filtrate was concentrated. Compound 39 was obtained as a colorless oil (45.0 mg, yield 93.3%). ESI MS m / z 870.39 ([M+N] + ).

[0324] Example 32; Synthesis of Compound 40 [ka]

[0325] At 0°C, m-CPBA (85%, 24 mg, 1.2 equivalents) was added to a 20 mL solution of compound 38 (90.0 mg, 100.0 μmol) in dichloromethane. The reaction mixture was then warmed to room temperature and stirred for 2 hours. The reaction mixture was diluted with dichloromethane (50 mL), quenched with saturated NaHSO3 (30 mL), and washed with saturated NaHCO3 (30 mL) and brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative HPLC (acetonitrile / water) to obtain the title compound as a white foam (57.1 mg, yield 62%). ESI MS m / z 916.30 ([M+N] + ).

[0326] Example 33; Synthesis of Compound 42 [ka]

[0327] Compound 40 (57.0 mg, 62.3 μmol) was mixed with methanol (50 mL) and Pd / C (10 wt%, 200 mg, containing 64.2% H2O). The mixture was stirred under a hydrogen gas balloon (1 atm) for 2 hours, then filtered, and the filtrate was concentrated. Compound 42 was obtained as a white foam (50.0 mg, 90.7% yield). ESI MS m / z 886.56 ([M+N] + ).

[0328] Example 34: Solid-phase synthesis of monocyclic octapeptide 63 Fmoc-Ile-OH was attached to 2-chlorotrityl chloride resin according to the following protocol.

[0329] Fmoc-Ile-OH (0.35 g, 1.0 mmol) and DIPEA (0.70 mL, 4.0 mmol) were dissolved in dry methylene chloride (10 mL). The resulting solution was added to chlorotrityl resin (1.0 g, 0.911 mmol / g, GLBiochem), and the mixture was shaken under nitrogen for 1.5 hours. Methanol (2 mL) was then added, and shaking was continued for 30 minutes. The liquid was drained under vacuum, and the resin was washed with methylene chloride (15 mL), DMF (10 mL), and methanol (10 mL), and dried under vacuum.

[0330] The coupling was performed according to the following protocol:

[0331] The resin was placed in a column and swollen with DMF (10 mL) for 30 minutes. The solvent was removed under vacuum, and the N-terminal Fmoc protecting group was cleaved by shaking with 20% piperidine in DMF for 30 minutes. Following deprotection, the resin was washed with DMF (3 × 10 mL), followed by CH2Cl2 (3 × 10 mL), and then again with DMF (3 × 10 mL). The following Fmoc-protecting amino acid (Fmoc-Xaa-OH, 5 equivalents) was coupled to the resin with a DMF solution (10 mL) of coupling reagents HBTU (5 equivalents) and DIPEA (10 equivalents) while shaking for 2 hours. Next, the resin was thoroughly washed with DMF (3 × 10 mL), followed by CH2Cl2 (3 × 10 mL) and DMF (3 × 10 mL). Small samples were taken and treated with a hexafluoroisopropanol (HFIP) CH2Cl2 solution for 5 minutes to cleave the peptide from the resin, which was then confirmed by mass spectrometry. For coupling of non-commercial amino acids such as Trt-Hpi-Gly-OH, smaller equivalents (3 equivalents) and longer treatment times (3 hours) were used.

[0332] After all coupling was complete, the resin-bound peptide was transferred to a round-bottom flask, 10 mL of TFA was added, and the mixture was stirred at room temperature for 5 hours. Acid-unstable protecting groups were simultaneously removed during the TFA treatment. The resin was filtered and washed with 10 mL of CH2Cl2 and 10 mL of methanol. The filtrate was concentrated and partitioned into water and ethyl acetate. The aqueous layer was purified by preparative HPLC (water / MeCN) to obtain monocyclic octapeptide 63 as a white solid (40.3 mg, yield 5%). ESI MS m / z 888.38 ([M+N] + ).

[0333] Example 35; Ile 3 Synthesis of -S-deoxo-amanitin (64) [ka]

[0334] To a 50 mL solution of a monocyclic octapeptide (257 mg, 0.289 mmol) in dried DMF, EDC·HCl (277 mg, 1.45 mmol), HOBt (390 mg, 2.89 mmol), and DIPEA (0.25 mL, 1.45 mmol) were added. The reaction mixture was stirred at room temperature for 20 hours, then concentrated and purified by preparative HPLC (H2O / MeCN) to obtain a white solid compound 64 (90.1 mg, yield 36%). ESI MS m / z 870.40 ([M+N] + ).

[0335] Example 36; Synthesis of Compound 65 [ka]

[0336] At 0°C, t-BuONO (70 μL, 0.575 mmol) was added to a 10 mL THF solution of compound 64 (50.0 mg, 0.0575 mmol, 1.0 equivalent). The reaction mixture was stirred at 0°C for 1 hour, then at room temperature for 20 hours. After adding water (50 mL), the reaction mixture was concentrated and purified by preparative HPLC (H2O / MeCN) to obtain a white solid (26.2 mg, 50% yield). ESI MS m / z 915.38 ([M+N] + ).

[0337] Example 37; Synthesis of Compound 68 [ka]

[0338] A mixture of a nitro compound (26 mg, 0.0284 mmol) and Pd / C (10 wt%, 100 mg) in methanol (20 mL) was hydrogenated at room temperature for 1 hour (1 atm H2), and then filtered through Celite (filtration aid). The filtrate was concentrated to obtain a white solid (25 mg, 99% yield). ESI MS m / z 885.38 ([M+N]) + ).

[0339] Example 38; Synthesis of Compound 69 [ka]

[0340] In an ice bath, a solution of compound 68 (25 mg, 0.0282 mmol) in THF / methanol / water (2:2:1, 50.0 mL) was mixed with LiOH (3.40 mg, 5 equivalents). The reaction was stirred at 0°C for 30 minutes. The mixture was then concentrated and purified using a short silica gel column (0-10% methanol / dichloromethane) to obtain a white foamy substance (15.2 mg, yield 61%). ESI MS m / z 871.38 ([M+N] + ).

[0341] Example 39; Synthesis of Compound 71 [ka]

[0342] To a 20 mL solution of compound 3-N,N-(2''-maleimidoethyl)(2',5',8',11',14',17',20',23',26'-nonoxaoctacosane-28'sulfin)aminopropanoic acid (70) (14.1 mg, 0.0206 mmol) in dichloromethane, NHS (2.8 mg, 0.0248 mmol) and EDC·HCl (4.9 mg, 0.0258 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then diluted with dichloromethane (100 mL), and washed with water (20 mL) and brine (20 mL). The organic phase was concentrated to obtain the crude product, which was used directly without further purification.

[0343] The crude product and compound 69 (15 mg, 0.0172 mmol) were dissolved in DMF (10 mL), and DIPEA (15 μL, 5 equivalents) was added. The reaction mixture was stirred overnight at room temperature, then concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (12.0 mg, 36% yield). ESI MS m / z 1523.68 ([M+N] + ).

[0344] Example 40; Synthesis of tert-butyl 2,5,8,11,14,17,20,23,26-nonoxaoctacosane-28-oate (136) [ka]

[0345] To a 1.0 L THF solution of 2,5,8,11,14,17,20,23-octaoxapentacosan-25-ol (38.4 g, 100 mmol), NaH (60%, 8.0 g, 200 mmol) was added. After stirring at room temperature for 30 minutes, 2-tert-butyl bromoacetate (48.8 g, 250 mmol) was added to the mixture and stirred at room temperature for 1 hour. Next, the mixture was poured into ice water and extracted with dichloromethane. The organic layer was washed with brine and dried over anhydrous sodium sulfate. Compound 136 was obtained as a yellow oil (27.6 g, yield 59%) by purification by column chromatography (0% to 5% methanol / dichloromethane). ESI MS m / z 499.40 ([M+N] + ).

[0346] Example 41; Synthesis of 2,5,8,11,14,17,20,23,26-nonoxaoctacosane-28-acid (137) [ka]

[0347] Compound 136 (35.6 g, 73.8 mmol) was dissolved in dichloromethane (400 mL), and then formic acid (600 mL) was added. The resulting solution was stirred overnight at 25°C. All volatile substances were removed under vacuum, and the title product was obtained as a yellow oily substance (31.0 g, theoretical yield). ESI MS m / z 443.45 ([M+N] + ).

[0348] Example 42: Synthesis of 2,5,8,11,14,17,20,23,26-nonoxaoctacosane-28-oil chloride (138) [ka]

[0349] Compound 137 (31.0 g, 73.8 mmol) was dissolved in dichloromethane (600 mL), to which (COCl)2 (100 mL) and DMF (52 g, 0.74 mmol) were added. The resulting solution was stirred at room temperature for 4 hours, and all volatile substances were removed under vacuum to obtain the title product (32.8 g) as a yellow oily substance. ESI MS m / z 461.38 ([M+N] + ).

[0350] Example 43: Synthesis of (S)-34-(((benzyloxy)carbonyl)amino)-28-oxo-2,5,8,11,14,17,20,23,26-nonaoxa-29-azapentatriacontan-35-acid(139) [ka]

[0351] ZL-Lys-OH (41.4 g, 147.6 mmol), Na2CO3 (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 THF solution of compound 138 (32.8 g, 73.8 mmol) (20 mL) was added. The resulting mixture was stirred at room temperature for 1 hour. The THF was removed under vacuum, and concentrated HCl was added to the aqueous solution under ice cooling until the pH reached 3. After extraction with dichloromethane, the organic layer was washed with brine, dried over sodium sulfate, and concentrated to obtain the title product as a yellow oil (50.0 g, 99% yield). ESI MS m / z 705.30 ([M+N] + ).

[0352] Example 44: Synthesis of (S)-tert-butyl 34-(((benzyloxy)carbonyl)amino)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate(140) [ka]

[0353] A mixture of tert-butyl 4-aminobutanoate (1.03 g, 6.12 mmol) and compound 139 (3.91 g, 5.56 mmol) in DMF (18 mL) was cooled to 0°C, and HATU (2.32 g, 6.12 mmol) and TEA (1.2 mL, 8.34 mmol) were added in sequence. 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 compound 140 (5.10 g, 99% yield). ESI MS m / z 846.50 ([M+N] + ).

[0354] Example 45: Synthesis of (S)-tert-butyl34-amino-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate (141) [ka]

[0355] In a hydrogenation bottle, a methanol solution (50 mL) of compound 140 (1.0 g, 1.18 mmol) was added to Pd / C (10 wt%, 0.10 g). The mixture was shaken for 2 hours, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 141 (0.93 g, yield >100%). ESI MS m / z 712.50 ([M+N] + ).

[0356] Example 46; Synthesis of (S)-tert-butyl34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate (142) [ka]

[0357] At room temperature, a 95% EtOH solution (50 mL) of compound 141 (0.93 g, 1.18 mmol) and N-succinimidyl 4-maleimide butyrate (0.50 g, 1.77 mmol, 1.5 equivalents) was added to a NaH2PO4 solution (0.1 M, pH 5.0, 10 mL). The mixture was stirred overnight, 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 oily substance (0.82 g, 80%). ESI MS m / z 877.52 ([M+N] + ).

[0358] Example 47; Synthesis of (S)-34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-acid (143) [ka]

[0359] Compound 144 (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 143 (0.80 g, crude product). ESI MS m / z 820.45 ([M+N] + ).

[0360] Example 48; Synthesis of (S)-2,5-dioxopyrrolidine-1-yl34-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)butanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate(144) [ka]

[0361] To a DMA solution (5.0 mL) of compound 143 (0.80 g, crude, 0.94 mmol), NHS (0.12 g, 1.03 mmol) and EDC hydrochloric acid (0.27 g, 1.41 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours, then 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 oil (0.67 g, yield 78%). ESI MS m / z 918.55 ([M+N] + ).

[0362] Example 49; Synthesis of Compound 145 [ka]

[0363] To an ethanol solution (10 mL) of compound 42 (50 mg, 0.0565 mmol) and compound 144 (77 mg, 1.5 equivalents), 0.1 M sodium dihydrogen phosphate (10 mL) was added and the mixture was stirred for 30 minutes. The reaction product was concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (44 mg, yield 46%). ESI MS m / z 1689.10 ([M+N] + ).

[0364] Example 50; Synthesis of tert-butyl(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)carbamate (147) [ka]

[0365] 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 partially added. 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, yield 87%). ESI MS m / z 241.12 ([M+N] + ).

[0366] Example 51; Synthesis of tert-butyl(2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)carbamate (148) [ka]

[0367] In a high-pressure tube, compound 147 (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. During the reaction, the colorless solution turned yellow. Next, the mixture was cooled to room temperature and concentrated. The resulting white solid was pulverized with ethyl ether to obtain compound 148 (6.5 g, yield 84%). ESI MS m / z 309.13 ([M+N] + ).

[0368] Example 52; Synthesis of 2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione hydrochloride (149) [ka]

[0369] A 15 mL solution of compound 148 (9.93 g, 32.2 mmol) in dioxane was treated with concentrated hydrochloric acid (15 mL) at room temperature for 3 hours. The reaction product was concentrated, the resulting solid was collected by filtration, and the filter cake was washed with ethyl acetate. The solid was dried overnight in an oven (50°C) to obtain compound (149) (6.94 g, 88% yield). ESI MS m / z 206.05 ([M+N] + ).

[0370] Example 53; Synthesis of Compound 151 [ka]

[0371] At -10°C, POCl3 (0.47 mL, 5 mmol) was added to a THF solution (10 mL) of compound 149 (1.22 g, 5 mmol). After stirring for 10 minutes, 2,5,8,11,14,17,20,23,26-nonoxaoctacosane-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 716.29 ([M+N] + ).

[0372] Example 54; Synthesis of Compound 111 [ka]

[0373] A 10.0 mL solution of compound 42 (1.50 g, 1.69 mmol), 4-(((benzyloxy)carbonyl)amino)butanoic acid N-succinimidyl ester (0.67 g, 1.2 equivalents), and DIPEA (0.44 mL, 1.5 equivalents) in DMF was stirred overnight at room temperature, then concentrated and purified by short silica gel column (10-85% ethyl acetate / petroleum ether) to obtain a colorless oil (1.58 g, yield 85%). ESI MS m / z 1105.47([M+N] +The resulting oily substance was dissolved in THF (5.0 mL), stirred with Pd / C (10 wt%, containing 62% water, 50 mg) under a hydrogen gas balloon for 1 hour, then filtered through Celite and concentrated to obtain a colorless oily substance (1.40 g, 100% yield), which was used without further purification. ESI MS m / z 971.43 ([M+N] + ).

[0374] Example 55; Synthesis of Compound 152 [ka]

[0375] At 0°C, one-fifth of the above solution of compound 151 (1.0 mmol) was mixed with DIPEA (0.17 mL, 1.0 mmol), followed by the addition of a dichloromethane solution of compound 111 (0.97 g, 1.0 mmol) (1.0 mL). The reaction mixture was stirred at 0°C for 2.5 hours, then at 30°C overnight. After concentration, the residue was purified by silica gel column chromatography (1-50% ethyl acetate / petroleum ether and 0-10% methanol / dichloromethane) to obtain the title product 152 as a colorless oil (0.99 g, 60%). ESI MS m / z 1650.74 ([M+N] + ).

[0376] Example 56; Synthesis of Compound 153 [ka]

[0377] A toluene / DMA solution (1:1, 2.0 mL) of compound 152 (0.99 g, 0.0006 mmol) was heated at 100°C for 2 hours. The solution was then concentrated and purified using a silica gel column (0-10% methanol / dichloromethane) to obtain the title compound as a white foam (0.48 g, 52% yield). ESI MS m / z 1582.90 ([M+N] + ).

[0378] Example 57; Synthesis of methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (156) [ka]

[0379] 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 242.42 [M+Na] +

[0380] Example 58; Synthesis of 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate methyl(157) [ka]

[0381] To a 350 ml solution of anhydrous pyridine containing methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (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 was used in the next step without further purification. ESI MS m / z 376.06 ([M+N] + ).

[0382] Example 59; Synthesis of 3,6-endoxo-Δ-tetrahydrophthalimide (159) [ka]

[0383] 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 in a 1 L autoclave vessel at 100°C for 8 hours. The vessel was cooled to room temperature, the solid was rinsed with methanol, concentrated, and crystallized in ethyl acetate / hexane 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] + 188.04.

[0384] Example 60; Synthesis of methyl 4-((2-((3aR,4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)(2-((4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-2(3H)-yl)ethyl)amino)-4-oxobutanoate (160) [ka]

[0385] To a solution of methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (157, freshly prepared, 90% purity, 8.5 g, approximately 20 mmol) in DMA (350 ml), 3,6-endoxo-Δ-tetrahydrophthalimide (10.2 g, 61.8 mmol), sodium carbonate (8.0 g, 75.5 mmol), and sodium iodide (0.3 g, 2.0 mmol) were added. The mixture was stirred overnight at room temperature, concentrated, diluted with ethyl acetate (350 ml), and washed with saturated NaHCO3 solution (300 ml), saturated NaCl solution (300 ml), and 1 M sodium dihydrogen phosphate (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 / hexane to obtain the title compound (7.9 g, 77% yield). ESI MS m / z [M+Na] + 536.4.

[0386] Example 61; Synthesis of 4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanoic acid (161) [ka]

[0387] Compound 160 (3.0 g, 5.8 mmol) and trimethylstannanol (4.8 g, 26.4 mmol) were refluxed at 80°C for 8 hours in 150 ml of 1,2-dichloroethane. The mixture was then cooled to room temperature, and the residue was eluted with dichloromethane / methanol using a short silica gel column to remove excess trimethyltin hydroxide. The pooled fractions were then combined, concentrated, diluted with DMA and toluene, and heated to 120°C with overnight stirring. 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] + 386.2.

[0388] Example 62; Synthesis of (S)-tert-butyl34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate(163) [ka]

[0389] EDC·HCl (0.81 g, 4.20 mmol) was added to a DMA solution (20 mL) of compound 161 (1.62 g, 4.20 mmol) and compound 141 (2.71 g, 3.82 mmol). The reaction mixture was stirred overnight at room temperature. The mixture was 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 1057.85 ([M+N]) + ).

[0390] Example 63; Synthesis of (S)-34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-acid (164) [ka]

[0391] A 10 mL solution of compound 163 (3.20 g, 3.03 mmol) in formic acid was stirred overnight at room temperature. The solution was then concentrated and evaporated three times with toluene to obtain a colorless oily substance (3.00 g, crude), which was used without further purification. ESI MS m / z 1001.50 ([M+N]) + ).

[0392] Example 64; Synthesis of (S)-2,5-dioxopyrrolidine-1-yl34-(4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)amino)-4-oxobutanamide)-28,35-dioxo-2,5,8,11,14,17,20,23,26-nonaoxa-29,36-diazatetracontane-40-oate(165) [ka]

[0393] To a DMA solution (15.0 mL) of compound 164 (3.00 g, crude, 3.03 mmol), NHS (0.38 g, 3.33 mmol) and EDC·HCl (0.87 g, 4.55 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 a colorless oil (2.90 g, yield 90%). ESI MS m / z 1098.50 ([M+N] + ).

[0394] Example 65; Synthesis of Compound 166 [ka]

[0395] To an ethanol solution (10 mL) of compound 42 (50 mg, 0.0565 mmol) and compound 165 (93 mg, 1.5 equivalents), 0.1 M NaH2PO4 (10 mL) was added and the mixture was stirred for 30 minutes. The reaction product was concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (63 mg, yield 60%). ESI MS m / z 1868.80 ([M+N] + ).

[0396] Example 66; Synthesis of 14-(benzyloxy)-14-oxotetradecanoic acid (183) [ka]

[0397] To a 30 mL DMF solution of tetradecanedioic acid (2.06 g, 8 mmol), K2CO3 (1.1 g, 8 mmol) and BnBr (1.36 g, 8 mmol) were added. The mixture was stirred overnight at room temperature. It was then concentrated and purified by column chromatography (ethyl acetate / petroleum ether) to obtain the title compound 183 (1.2 g, 45% yield). ESI MS m / z 349.23 ([M+N]) + ).

[0398] Example 67; Synthesis of tert-butyl(185) 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate [ka]

[0399] To a solution of 2,2'-(ethane-1,2-diyrbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 equivalents) in anhydrous THF (200 mL), sodium (0.1 g) was added. 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 THF was removed using a rotary evaporator, brine (300 mL) was added, and the resulting mixture was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oil (30.20 g, yield 79.0%), which was used without further purification. MS ESI m / z 278.17 ([M+H] + ).

[0400] Example 68; Synthesis of tert-butyl(186) 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate [ka]

[0401] At 0°C, 30.20 g, 108.5 mmol, 1.0 equivalent of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 equivalent) and 41.37 g, 217.0 mmol, 2.0 equivalents of TsCl (220 mL) were added to a solution of DCM (30.0 mL, 217.0 mmol, 2.0 equivalents). 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 Ricagel column chromatography (3:1 hexane / ethyl acetate) to obtain a colorless oil (39.4 g, yield 84.0%). MS ESI m / z 433.28 ([M+H] + ).

[0402] Example 69; Synthesis of tert-butyl (187) 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate [ka]

[0403] 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) tert-butyl propanoate (39.4 g, 91.1 mmol, 1.0 equivalent) was dissolved in anhydrous DMF solution (100 mL) and NaN3 (20.67 g, 316.6 mmol, 3.5 equivalents) was added. The mixture was stirred overnight at room temperature. Water (500 mL) was added and extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with water (3 × 900 mL) and brine (900 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (5:1 hexane / ethyl acetate) to obtain a pale yellow oily substance (23.8 g, yield 85.53%). MS ESI m / z 326.2 ([M+Na] + ).

[0404] Example 70; Synthesis of tert-butyl(188) 3-(2-(2-(2-azinoethoxy)ethoxy)ethoxy)propanoate [ka]

[0405] Raney nickel (7.5 g, suspended in water) was washed with water (3 times) and isopropyl alcohol (3 times), and mixed with compound 187 (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under an H2 balloon at room temperature for 16 hours, and then washed with isopropyl alcohol on a Celite pad. The filtrate was concentrated and purified by column chromatography (5-25% methanol / DCM) to obtain a pale yellow oily substance (2.60 g, yield 57%). MS ESI m / z 279.19 ([M+H] + ).

[0406] Example 71; Synthesis of 27-benzyl 1-tert-butyl 14-oxo-4,7,10-trioxa-13-azaheptacosan-1,27-diode (189) [ka]

[0407] To a 50 mL dichloromethane solution of compound 188 (2.60 g, 9.35 mmol) and compound 183 (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. It was 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 189 (4.94 g, 87% yield). ESI m / z 608.40 ([M+H] + ).

[0408] Example 72; Synthesis of 3,16-dioxo-1-phenyl-2,20,23,26-tetraoxa-17-azanonacosane-29-acid(190) [ka]

[0409] To a 20 mL solution of compound 189 (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, and both the compound and dichloromethane were evaporated twice. The residue was then placed in a pump to obtain compound 190 (4.50 g, crude product). ESI MS m / z 552.35 ([M+N] + ).

[0410] Example 73; Synthesis of 40-benzyl 1-tert-butyl 14,27-dioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontane-1,40-dioete (191) [ka]

[0411] To a 50 mL dichloromethane solution of compound 190 (4.50 g, crude, 8.14 mmol) and compound 188 (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 191 (5.22 g, 92% yield). ESI m / z 811.52 ([M+H] + ).

[0412] Example 74; Synthesis of 3,16,29-trioxo-1-phenyl-2,20,23,26,33,36,39-heptaoxa-17,30-diazadotetracontane-42-acid(192) [ka]

[0413] To a 15 mL solution of compound 191 (5.22 g, 6.44 mmol) in dichloromethane, 15 mL of TFA was added. The reaction mixture was stirred at room temperature for 1 hour, then concentrated to dryness, and both the compound and dichloromethane were evaporated twice. The residue was then placed in a pump to obtain compound 192 (4.90 g, crude product). ESI MS m / z 755.46 ([M+N] + ).

[0414] Example 75; Synthesis of 40-benzyl 1-(2,5-dioxopyrrolidine-1-yl)14,27-dioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontane-1,40-dioete (193) [ka]

[0415] To a 30 mL solution of compound 192 (4.90 g, crude, 6.44 mmol) in dichloromethane, NHS (0.81 g, 7.08 mmol) and EDC salt·HCl (1.85 g, 9.66 mmol) were added, followed by DIPEA (2.8 mL, 16.1 mmol). 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 a colorless oil (4.90 g, 90% yield). ESI MS m / z 852.48 ([M+N]) + ).

[0416] Example 76; Synthesis of 1-((2,5-dioxopyrrolidine-1-yl)oxy)-1,14,27-trioxo-4,7,10,17,20,23-hexaoxa-13,26-diazatetracontane-40-acid (194) [ka]

[0417] In a hydrogenation bottle, a methanol solution (20 mL) of compound 193 (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 H2, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 194 (4.50 g, yield >100%). ESI MS m / z 762.44 ([M+N] + ).

[0418] Example 77; Synthesis of Compound 195 [ka]

[0419] To an ethanol solution (10 mL) of compound 56 (40 mg, 0.0454 mmol) and compound 144 (50 mg, 0.0545 mmol), 0.1 M NaH2PO4 (10 mL) was added and the mixture was stirred for 30 minutes. The reaction product was concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (44 mg, yield 40%). ESI MS m / z 1704.80 ([M+N] + ).

[0420] Example 78; Synthesis of Compound 196 [ka]

[0421] A mixture of compound 195 (31 mg, 0.0182 mmol) and compound 194 (17 mg, 0.0218 mmol) in DMA (10 mL) was mixed with DIPEA (5 μL, 0.0273 mmol). The reaction mixture was stirred overnight at room temperature, then concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (26 mg, 61% yield). ESI MS m / z 2351.60 ([M+N]) + ).

[0422] Example 79; Synthesis of (6S,13S)-di-tert-butyl9,10-bis(((benzyloxy)carbonyl)amino)-5,8,11,14-tetraoxo-6,13-bis(4-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioete(209) [ka]

[0423] To a DMA solution (60 mL) of (S)-tert-butyl-12-amino-2,2-dimethyl-6,13-dioxo-5-oxa-7,14-diaza-2-silaheptadecane-17-oate (6.02 g, 14.4 mmol) and 2,3-bis(((benzyloxy)carbonyl)amino)succinic acid (5.00 g, 12.0 mmol), EDC hydrochloric acid (2.76 g, 14.4 mmol) and DIPEA (4.7 mL, 26.4 mmol) were added. The reaction mixture was stirred overnight at room temperature. It was then diluted with 150 mL of dichloromethane and poured into a separatory funnel containing 100 mL of water. The organic phase was separated, washed with brine (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (10-80% ethyl acetate / petroleum ether) to obtain the title compound 209 (12.4 g, yield 85%). ESI MS m / z 1215.63([M+N] + ).

[0424] Example 80; Synthesis of (6S,13S)-di-tert-butyl9,10-diamino-5,8,11,14-tetraoxo-6,13-bis(4-(((2-(trimethylsilyl)ethoxy)carbonyl)amino))butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioete(210) [ka]

[0425] In a hydrogenation bottle, a methanol solution (50 mL) of compound 209 (12.4 g, 10.2 mmol) was mixed with Pd / C (10 wt%, 0.10 g). The mixture was shaken for 2 hours, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 210 as a colorless oil (9.47 g, 98% yield). ESI MS m / z 947.56 ([M+N] + ).

[0426] Example 81; Synthesis of (6S,13S)-di-tert-butyl9,10-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-5,8,11,14-tetraoxo-6,13-bis(4-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-dioete(211) [ka]

[0427] To a 50 mL solution of compound 210 (9.47 g, 10.0 mmol) in dichloromethane, NHS (1.39 g, 12.0 mmol) and EDC·HCl (2.30 g, 12.0 mmol) were added, followed by DIPEA (3.8 mL, 22.0 mmol). 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 (10-80% ethyl acetate / petroleum ether) to obtain a colorless oil (9.49 g, yield 76%). ESI MS m / z 1249.72 ([M+N] + ).

[0428] Example 82; Synthesis of (6S,13S)-9,10-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)-5,8,11,14-tetraoxo-6,13-bis(4-(((2-(trimethylsilyl)ethoxy)carbonyl)amino)butyl)-4,7,12,15-tetraazaoctadecane-1,18-diacid(212) [ka]

[0429] A solution of compound 211 (9.49 g, 7.60 mmol) in THF (15 mL) was treated with 4N HCl (2 mL) at 0°C for 30 minutes, then concentrated and loaded onto a short silica gel column. Elution was performed with 0-15% methanol / dichloromethane to obtain a colorless oily substance (8.50 g, 90% yield). ESI MS m / z 1249.72 ([M+N] + ).

[0430] Example 83; Synthesis of Compound 213 [ka]

[0431] To a 10 mL DMF solution of compound 212 (139.0 mg, 0.111 mmol) and compound 52 (50.0 mg, 0.0555 mmol), TBTU (35.6 mg, 0.111 mmol) and DIPEA (20.0 μL, 0.111 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After removing the DMF under high vacuum, the residue was purified by preparative HPLC (acetonitrile / water) to obtain a colorless oil (140.1 mg, yield 63%). ESI MS m / z 2002.84([M+N] + ).

[0432] Example 84; Synthesis of Compound 214 [ka]

[0433] At 0°C, a 10 mL solution of compound 213 (140 mg, 0.0699 mmol) in THF was treated with TBAF (1.0 M in THF, 350 μL) for 30 minutes, then concentrated. A colorless oil was obtained by short silica gel column (0-10% methanol / dichloromethane) (100.3 mg, yield 88%). ESI MS m / z 1714.72 ([M+N] + ).

[0434] Example 85; Synthesis of Compound 215 [ka]

[0435] Compound 214 (99.8 mg, 0.0583 mmol) and Compound 194 (110.2 mg, 0.146 mmol) were mixed in THF (10 mL) and phosphate buffer (10 mL, 0.5 M, pH 7.7), stirred overnight at room temperature, then concentrated, and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (79.2 mg, 45% yield). ESI MS m / z 3007.56 ([M+N]) + ).

[0436] Example 86; Synthesis of 2-(1,3-dioxoisoindorin-2-yl)acetyl chloride (223) [ka]

[0437] To a 100 mL solution of N-phthaloylglycine (10.0 g, 48.7 mmol) in dichloromethane, oxalyl chloride (6.3 mL, 73.1 mmol) was added at room temperature, followed by dropwise addition of DMF. The reaction mixture was stirred for 2 hours, then concentrated to obtain compound 223 (10.8 g) as a yellow solid.

[0438] Example 87; Synthesis of tert-butyl 2-(2-(1,3-dioxoisoindolin-2-yl)acetyl)hydrazine carboxylate (224) [ka]

[0439] At 0°C, Et3N (13.5 mL, 97.4 mmol) was added to a 200 mL solution of Boc-hydrazine (7.08 g, 53.5 mmol) in dichloromethane, followed by compound 223 (10.8 g, 48.7 mmol). The reaction mixture was stirred at room temperature for 30 minutes, then poured into 100 mL of ice water and extracted with dichloromethane (3 × 100 mL). The combined organic phase was washed with water (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid (15.5 g, 100% yield). ESI MS m / z 320.12([M+N]) + ).

[0440] Example 88; Synthesis of 2-(1,3-dioxoisoindorin-2-yl)acetohydrazide (225) [ka]

[0441] Compound 224 (15.5 g, 48.7 mmol) was dissolved in dichloromethane (150 mL) and treated with TFA (50 mL) at room temperature for 1 hour. The mixture was then concentrated to obtain a white solid (10.6 g, 100% yield). ESI MS m / z 220.06 ([M+N] + ).

[0442] Example 89; Synthesis of 2-(1,3-dioxoiisoindolin-2-yl)-N'-(2-(1,3-dioxoiisoindolin-2-yl)acetyl)acetohydrazide (226) [ka]

[0443] At 0°C, Et3N (13.5 mL, 97.4 mmol) and compound 223 (10.8 g, 48.7 mmol) were added to a 200 mL dichloromethane solution of compound 225 (10.6 g, 48.7 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The precipitate was collected by filtration, suspended in water (100 mL), and stirred for 20 minutes. The mixture was filtered again, and a white solid was collected (15.7 g, 80% yield). ESI MS m / z 407.09 ([M+N]) + ).

[0444] Example 90; Synthesis of di-tert-butyl 2,2'-(1,2-bis(2-(1,3-dioxoisoindoline-2-yl)acetyl)hydrazine-1,2-diyl) diacetate (227) [ka]

[0445] At 0°C, NaH (0.5g, 12.3 mmol) was gradually added to a DMF solution (40 mL) of compound 226 (2.0 g, 4.92 mmol). The mixture was warmed to room temperature and stirred for 3 hours. Then, tert-butyl bromoacetate (2.0 g, 10.3 mmol) was added, and the reaction was stirred overnight. After that, the mixture was poured into ice water (100 mL) and extracted with dichloromethane (3 × 50 mL). The combined organic phase was washed with water (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel chromatography to obtain a white solid (1.5 g, yield 50%). ESI MS m / z 635.23 ([M+N] + ).

[0446] Example 91; Synthesis of di-tert-butyl 2,2'-(1,2-bis(2-aminoacetyl)hydrazine-1,2-diyl)diacetate (228) [ka]

[0447] A mixture of compound 227 (1.5 g, 2.36 mmol) and hydrazine (442 mg, 7.08 mmol) in ethanol (30 mL) was refluxed for 1 hour, then cooled to room temperature and filtered. The filtrate was concentrated, transferred to ethyl acetate (20 mL), and filtered again. The filtrate was concentrated to obtain a white solid (750 mg, yield 85%). ESI MS m / z 375.22 ([M+N] + ).

[0448] Example 92; Synthesis of di-tert-butyl 2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl)hydrazine-1,2-diyl) diacetate (229) [ka]

[0449] To a THF solution (2 mL) of compound 228 (750 mg, 2 mmol), 30 mL of saturated NaHCO3 aqueous solution was added, then cooled to 0°C, and N-methoxycarbonylmaleimide (622 mg, 4 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour. A white solid was collected by filtration (854 mg, yield 80%). ESI MS m / z 535.20 ([M+N] + ).

[0450] Example 93; Synthesis of 2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl)hydrazine-1,2-diyl)diacetic acid (230) [ka]

[0451] Compound 229 (854 mg, 1.6 mmol) was dissolved in dichloromethane (3 mL) and treated with TFA (3 mL) at room temperature for 2 hours. The reaction mixture was then concentrated to obtain compound 230 (675 mg, 100% yield). ESI MS m / z 423.07 ([M+N] + ).

[0452] Example 94; Synthesis of di-tert-butyl 4,4'-((2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl)hydrazine-1,2-diyl)bis(acetyl))bis(azandiyl))dibutanoate (231) [ka]

[0453] At 0°C, tert-butyl 4-aminobutanoate (158 mg, 0.99 mmol) and EDC (189.7 mg, 0.99 mmol) were added to a 5 mL DMF solution of compound 230 (200 mg, 0.47 mmol). The reaction mixture was warmed to room temperature, stirred overnight, poured into ice water, and extracted with dichloromethane (3 × 10 mL). The combined organic phase was washed with 1 N HCl (5 mL), water (5 mL), and brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid (330 mg, yield 100%).

[0454] Example 95; Synthesis of bis(2,5-dioxopyrrolidine-1-yl)4,4'-((2,2'-(1,2-bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)acetyl)hydrazine-1,2-diyl)bis(acetyl))bis(azandiyl))dibutanoate (233) [ka]

[0455] Compound 231 (330 mg, 0.47 mmol) was dissolved in dichloromethane (3 mL) and treated with TFA (3 mL) at room temperature for 2 hours. The reaction mixture was concentrated, redissolved in DMF (5 mL), cooled to 0°C, and NHS (113 mg, 0.98 mmol) and EDC (189 mg, 0.98 mmol) were added in sequence. The reaction mixture was warmed to room temperature, stirred overnight, poured into ice water, and extracted with dichloromethane (3 × 20 mL). The combined organic phase was washed with water (5 mL) and brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid (369 mg, 100% yield). ESI MS m / z 787.21 ([M+N] + ).

[0456] Example 96; Synthesis of (S)-48-(((benzyloxy)carbonyl)amino)-3,16,29,42-tetraoxo-1-phenyl-2,20,23,26,33,36,39-heptaoxa-17,30,43-triazanonatetracontan-49-acid(235) [ka]

[0457] At 0°C, HATU (0.50 g, 1.32 mmol) and TEA (0.06 mL, 1.32 mmol) were added to a 10 mL dichloromethane solution of compound 192 (1.00 g, 1.32 mmol). The reaction mixture was stirred at 0°C for 30 minutes, and then Z-Lys-OH (0.40 g, 1.43 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, then diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 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 a colorless oil (1.28 g, yield 95%). ESI MS m / z 1017.60 ([M+N] + ).

[0458] Example 97; Synthesis of (S)-47-benzyl 1-(2,5-dioxopyrrolidine-1-yl)2-(((benzyloxy)carbonyl)amino)-8,21,34-trioxo-11,14,17,24,27,30-hexaoxa-7,20,33-triazaheptatetracontan-1,47-dioete(236) [ka]

[0459] To a 10 mL solution of compound 235 (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 TEA (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 a colorless oil (1.28 g, yield 91%). ESI MS m / z 1114.62 ([M+N] + ).

[0460] Example 98; Synthesis of Compound 237 [ka]

[0461] To a 10 mL DMF solution of compound 56 (50.2 mg, 0.0555 mmol) and compound 236 (136.1 mg, 0.122 mmol), DIPEA (20 μL, 0.122 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After removing the DMF under high vacuum, the residue was purified by preparative HPLC (acetonitrile / water) to obtain a colorless oil (70.3 mg, yield 44%). ESI MS m / z 2899.80 ([M+N]) + ).

[0462] Example 99; Synthesis of Compound 238 [ka]

[0463] In a hydrogenation bottle, a methanol solution (50 mL) of compound 237 (70.0 mg, 0.0241 mmol) was added to Pd / C (10 wt%, 145 mg). The mixture was stirred with hydrogen gas at 1 atm for 2 hours, filtered through Celite (filtration aid), and the filtrate was concentrated to obtain compound 238 (65 mg, >100% yield). ESI MS m / z 2631.63 ([M+N] + ).

[0464] Example 100; Synthesis of Compound 239 [ka]

[0465] Compound 238 (65 mg, 0.0247 mmol) and Compound 233 (29 mg, 0.0371 mmol) were mixed in EtOH (10 mL) and phosphate buffer (10 mL, 0.5 M, pH 7.7) and stirred overnight at room temperature. The mixture was then concentrated and purified by preparative HPLC (acetonitrile / water) to obtain a white foamy substance (28.3 mg, yield 36%). ESI MS m / z 3187.60 ([M+N] + ).

[0466] Example 101; Synthesis of di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate [ka]

[0467] To a 150 ml DMF solution of di-tert-butylhydrazine-1,2-dicarboxylate (8.01 g, 34.4 mmol), NaH (60% in oil, 2.76 g, 68.8 mmol) was added. After stirring at room temperature for 30 minutes, tert-butyl 2-bromoacetate (14.01 g, 72.1 mmol) was added. The mixture was stirred overnight, quenched with methanol (3 ml), concentrated, diluted with SiO2 (100 ml) and water (100 ml), separated, and the aqueous layer was extracted with SiO2 (2 × 50 ml). The organic layers were combined, dried over MgSO4, filtered, concentrated, and purified by SiO2 column chromatography (SiO2 / hexane 1:5~1:3) to obtain the title compound as a colorless oil (12.98 g, yield 82%). MS ESI m / z C 22 H 41 N2O8[M+H] + Calculated value: 461.28, measured value: 461.40.

[0468] Example 102; Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0469] Di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) was dissolved in a 1,4-dioxane solution (40 ml) with HCl (12 M, 10 ml). The mixture was stirred for 30 minutes, diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and co-evaporated with dioxane (20 ml) and toluene (40 ml) to dry. No further production was obtained, and the crude title product for the next step was obtained (2.15 g, yield 103%, ~93% purity). MS ESI m / z C4H9N2O4[M+H] + Calculated value: 149.05, Measured value: 149.40.

[0470] Example 103; Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid [ka]

[0471] (E)-3-bromoacryloyl bromide (5.01 g, 23.60 mmol) was added to 2,2'-(hydrazine-1,2-diyl)diacetic acid (1.10 g, 7.43 mmol) in a mixture of THF (50 ml) and NaH2PO4 (0.1 M, 80 ml, pH 6.0). The mixture was stirred for 6 hours, concentrated, and purified by silica gel column elution with water / CH3CN (1:9) containing 3% formic acid to obtain the title compound (2.35 g, yield 77%, approximately 93% purity). MS ESI m / z C 10 H 11 Br2N2O6[M+H] + Calculated value: 412.89, Measured value: 413.50.

[0472] Example 104; Synthesis of 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetyl chloride [ka]

[0473] 2,2'-(1,2-bis((E)-3-bromoacryloyl)hydrazine-1,2-diyl)diacetic acid (210 mg, 0.509 mmol) was added to dichloroethane (15 ml) with (COCl)2 (505 mg, 4.01 mmol), followed by 0.040 ml of DMF. After stirring at room temperature for 2 hours, the mixture was concentrate...

Claims

1. Side-chain conjugated compounds of formula (III): 【Transformation 8】 During the ceremony, 【Chemistry 2】 represents a single bond; n is between 1 and 30; T is a cell-binding agent / molecule selected from the group consisting of antibodies, single-chain antibodies, antibody fragments that bind to target cells, monoclonal antibodies, single-chain monoclonal antibodies, monoclonal antibody fragments that bind to target cells, chimeric antibodies, chimeric antibody fragments that bind to target cells, domain antibodies, domain antibody fragments that bind to target cells, antibody-mimicking adnectin, DARPins, cell-binding ligands, and cell receptor agonists. L １ and L ２ is CH; W is an amide bond; w is 1 or 2; V １ and V ２ are each independently a spacer unit selected from the following structures; v １ and v ２ are each independently 0 or 1, provided that v １ and v ２ are not both 0 at the same time; v １ or v ２ is 0, meaning that one of the side chain Q １ or Q ２ fragments is absent; 【Chemistry 14】 Q １ and Q ２ These two can be expressed independently by equation (I - q1): 【Transformation 3】 During the ceremony, 【Chemistry 4】 is, L １ or L ２ This is the site where it connects; G １ and G ２ CH is independent. ２ is; G ３ is OR Ａ or NHC(O)(CH ２ ) ｑ１’ It is C(O)OH; X １ and X ２ is O; R Ａ is H or C １ ~C ８ It is alkyl; Y ２ NH, NR １ It is; p １ It is 0; p ２ and p ３ Each is independently between 1 and 100; q １ , q １’ and q ２ They are independently 1 to 24; D is the amanita toxin of formula (II) below, or an isotope of a chemical element, or a pharmaceutically acceptable salt, hydrate, or hydrated salt thereof; or its optical isomer, racemate, diastereomer, or enantiomer: 【Transformation 5】 During the ceremony, 【Transformation 6】 This is an independent linking site that connects with W; The single bond on the aromatic (indole) ring is R １０ and R １１ One of them means that the bond is at the 5th carbon position of the indole ring, and the other means that the bond is at the 6th carbon position of the indole ring; 【Transformation 7】 The symbol () can optionally indicate a single bond or the absence of a bond; R １ and R ２ These are independently H, OH, and CH ２ OH, CH(OH)CH ２ OH, CH(CH ３ )CH ２ OH, CH(OH)CH ３ , and C １ -C ８ Selected from alkyl groups; R ３ is either H or OH; R ４ H, OH, -OR １２ , and -OCOR １２ Selected from; R ５ OH, NH ２ , -OR １２ , and -NHR １２ Selected from; R ６ H, OH, CH ２ OH, CH(OH)CH ２ OH, CH(CH ２ OH) ２ , CH (CH ３ )OH, CH ２ CH ２ Selected from OH, ProOH, and BuOH; R ７ is selected from H, CH ３ , CH(CH ３ ), ２ , CH(CH ３ )CH ２ CH ３ , CH ２ OH, CH(CH ３ )OH, CH(OH)CH ２ OH, CH ２ CH(OH)CH ２ OH, CH(CH ２ OH) ２ , CH ２ C(OH)(CH ２ OH) ２ , CH ２ C(OH)(CH ３ )(CH ２ OH), CH ２ C(OH)(CH(CH ３ ))(CH ２ OH), CH ２ CH ２ CH ２ OH, PrOH, BuOH, and C １ ~C ８ alkyl; R ８ is selected from H, CH ３ , CH(CH ３ ), ２ , CH(CH ３ ), CH ２ CH ３ , and C １ to C ８ alkyl; R ９ H and C １ ~C ８ Selected from alkyl groups; If not bonded with W, R １０ and R １１ H, NH ２ , or OH; when bonded with W, R １０ and R １１ These are independent of -OCO- and -OCOR. １２’ , -OCOCH ２ , -NR １２ CO-, or -NR １２ COR １２’ It is; In the formula, R １２ is H or C １ -C ８ It is alkyl, R １２’ C １ -C ８ It is alkylene; X is S, SO, or SO ２ It is; m' is either 0 or 1.

2. Side-chain linked compounds of formula (V): 【Chemistry 11】 During the ceremony, D, W, w, L １ , L ２ Q １ Q ２ , v １ , and v ２ This is the same definition as in claim 1; V １’ and V ２’ V is a structure selected from the following, and in equation (V), V １’ and V ２’ Both may be the same or different: 【Chemistry 25】 In the above formula, Lv is selected from the following: 【Chemistry 10】 In the formula, Lv ３ The leaving group is selected from F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, and an anhydride that is self-formed or formed with other anhydrides.

3. D (amanita toxin structure) is a side-chain conjugated compound according to claim 1 or a side-chain linked compound according to claim 2, selected from IIa, IIb, IIc, II-01, II-02, II-03, II-04, II-05, II-06, II-07, II-08, II-09, II-10, II-11, II-12, II-13, or II-14, or one or more chemical elements thereof, isotopes, pharmaceutically acceptable salts, hydrates, hydrated salts; polymorphic crystals of these compounds; or optical isomers, racemates, diastereomers or enantiomers: 【Chemistry 13】 【change】 【change】 【change】 During the ceremony, Z ２ is oxygen or a lone pair of electrons; R １５ is H or C １ -C ８ It is a linear or branched alkyl group; Z １ R １２’ It is; X is S; X １ These are independently O, NH, or N(R) １２ ) is; R １ , R ２ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ , R １１ , R １２ , and R １２’ This is the same definition as in claim 1.

4. A side-chain conjugated compound of formula (III) as described in claim 1, having any of the following structures, or isotopes, pharmaceutically acceptable salts, hydrates, or hydrated salts thereof of one or more chemical elements; or polymorphic crystals of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: [Chemistry 18] 【change】 In the formula, mAb is a monoclonal antibody.

5. A side-chain linked compound of formula (V) according to claim 2 having any of the following structures, or isotopes, pharmaceutically acceptable salts, hydrates, or hydrated salts thereof of one or more chemical elements; or polymorphic crystals of these compounds; or optical isomers, racemates, diastereomers, or enantiomers: 【Chemistry 20】 【change】

6. The cell binding agent / molecule T targets tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, autoimmune disease cells, activated tumor cells, bone marrow cells, activated T cells, affected B cells, melanocytes, or any cell expressing any one of the following antigens or receptors, according to claim 1 or 4: CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD1 1b, CD11c, CD11d, CD12w, CD14, CD15, CD16, CD16a, CD16b, CDw17, CD18, CD1 9, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, C D32, CD32a, CD32b, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, C D42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49b , CD49c, CD49c, CD49d, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD 57, CD58, CD59, CD60, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63 , CD64, CD65, CD65s, CD66, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67 , CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD76, CD77, CD78, C D79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD85a, CD85b, CD85 c, 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、CD120、CD120a、CD120b、CD121、CD121a、CD121b、CD122、CD123、CD123a、CD124、CD125、CD126、CD127、CD128、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CDw145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156、CD156a、CD156b、CD156c、CD156d、CD157、CD158、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f2、CD158g、CD158h、CD158i、CD158j、CD158k、CD159、CD159a、CD159b、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CDw186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CDw198、CDw199、CD200、CD201、CD202、CD202(a,b)、CD203、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210a、CDw210b、CD211、CD212、CD213、CD213a1、CD213a2、CD214、CD215、CD216、CD217、CD218、CD218a、CD218、CD21b9、CD220、CD221、CD222、 CD223、CD224、CD225、CD226、CD227、C D228、CD229、CD230、CD231、CD232、CD233、CD234、CD235、CD235b、C D236、CD237、CD238、CD239、CD240、CD240ce、CD240d、CD241、CD242、CD243、 CD244、CD245、CD246、CD247、CD248、CD249、CD250、CD251、CD252、CD253、CD2 54, CAD255, CAD256, CAD257, CAD258, CAD259, CAD260, CAD261, CAD262, CAD263, CAD264 、CD265、CD266、CD267、CD268、CD269、 CD270、CD271、CD272、CD273、CD274、CD 275、CD276、CD277、CD278、CD279、CD2 81、CD282、CD283、CD284、CD285、CD28 6, CDD287, CDD288, CDD289, CDD290, CDD291, CDD292, CDD293, CDD294, CDD295, CDD296 D297、CD298、CD299、CD300、CD300a、C D300b、CD300c、CD301、CD302、CD303、 CD304、CD305、CD306、CD307、CD307a、 CD307b、CD307c、CD307d、CD307e、CD30 7f、CD308、CD309、CD310、CD311、CD31 2、CD313、CD314、CD315、CD316、CD317 、CD318、CD319、CD320、CD321、CD322、 CD323、CD324、CD325、CD326、CD327、CD 328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD33 8、CD339、CD340、CD341、CD342、CD343 、CD344、CD345、CD346、CD347、CD348、C D349、CD350、CD351、CD352、CD353、CD354、CD355、CD356、CD357、CD358、CD3 59、CD360、CD361、CD362、CD363、CD36 4、CD365、CD366、CD367、CD368、CD369、CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD379, C D381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CRIPTO, CR IPTO, CR, CR1, CRGF, CRIPTO, CXCR5, LY64, TDGF1, 4-1BB, APO2, ASLG659, B MPR1B, 4-1BB, 5AC, 5T4 (chorionic glycoprotein, TPBG, 5T4, Wnt activation inhibitory factor 1 or WAIF1), adenocarcinoma anti- Original, AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, α-integrin, αvβ6, aminopeptidase N, amyloid β, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, Bacillus anthrax toxin protective antigen, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthrax, BAFF (B-cell activator), BCMA, B-lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16) CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canine IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (C-C motif chemokine 11), CCR4 (CC chemokine receptor type 4), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (claudin-18), clamping factor A, cMet, CRIPTO, FCSF1R (colony-stimulating factor 1 receptor), CS F2 (Colony-stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), CSP4, CTLA4 (Cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4, CXC chemokine receptor type 4, cADP ribose hydrolase, cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4), DR5 (death receptor 5), Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, ED-B,EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, epicyalin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), Escherichia coli, ETV6-AML, FAP (fibroblast-activating protein α), FCGR1, α-fetoprotein, fibrin II, β-chain, fibronectin external domain B, FOLR (folate receptor), folate receptor α, folate hydrolase, Fos-related antigen 1F, RSV F protein, Frizzled receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycol Lunoiraminic acid, GM3, GMCSF receptor α chain, growth differentiation factor 8, GP100, GPNMB (transmembrane protein NMB), GUCY2C (guanylate cyclase 2C, guanylate cyclase C (GC-C), enteric guanylate cyclase, guanylate cyclase-C receptor, heat-stable enterotoxin receptor (hSTAR)), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B Virus, HER1 (Human Epidermal Growth Factor Receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (Stem Cell Growth Factor / Cell Dispersion Factor), HHGFR, HIV-1, Histone Complex, HLA-DA (Human Leukocyte Antigen), HLA-DR10, HLA-DRB, HMWMAA, Human Chorionic Gonadotropin, HGGF, Human Cell Scattering Factor Receptor Kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, interleukin (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-27, or IL-28), IL-31RA, ILGF2 (insulin-like growth factor 2),Integrins (α4, α, ＩＩＩｂ β ３ , αvβ3, α ４ β ７ α5β1, α6β4, α7β7, αIIβ3, α5β5, αvβ5), interferon-gamma-inducing protein, ITAGA2, ITGB2, KIR2D, LCK, Le, Regmine, Lewis -Y antigen, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGEA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (macrophage migration inhibitor or glycosylation inhibitor (GIF)), MS4A1 (transmembrane 4-domain subfamily A member 1), MSLN (Mesothelin), MUC1 (Mucin 1, cell surface-associated (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (Monocyte chemotactic protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (Transmembrane 4-domain subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (Granulocyte antigen), Nectin-4 (ASG-22ME), NG F, neuronal apoptosis regulatory proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, Page4, PAP, anti(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein 1), PDGF-R α, (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate cotransporter, PMEL17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI)), rhesus factor (Rhesusfactor), RANKL, PhoC, Ras variant, RG55, ROBO4, RS virus, RON, sarcoma metastasis breakpoint, SART3, sclerostin, SLAMF7 (SLAM family member 7), selectin P, SDC1 (syndecane 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEA P1 (prostate 1 6-transmembrane epithelial antigen), STEAP2, STn, TAG-22 (tumor-associated glycoprotein 72), Survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor epithelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor β2), Tie (CD202b ), Tie2, TIM-1 (CDX-014), TN, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophotrophic membrane glycoprotein), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1), TRAILR2 (cell death receptor 5 (DR 5)) Cells expressing major related calcium signaling transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2, VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or any insulin growth factor receptor, or any epidermal growth factor receptor.

7. The side-chain conjugated compound according to claim 6, wherein the tumor cells are selected from the group consisting of lymphoma cells, myeloma cells, renal cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, non-small cell lung cancer cells, testicular cancer cells, or malignant cells.

8. A pharmaceutical composition for the treatment or prevention of cancer, autoimmune diseases, or infectious diseases, comprising a therapeutically effective amount of a side-chain conjugated compound according to claim 1 or 4, and a pharmaceutically acceptable salt, carrier, diluent, or excipient, or combination of the conjugate.

9. By weight, 0.01% to 99% of one or more side-chain conjugated compounds according to claim 1 or 4; One or more polyols in an amount of 0.0% to 20.0%; One or more surfactants in an amount of 0.0% to 2.0%; One or more preservatives in a concentration of 0.0% to 5.0%; One or more amino acids in a concentration of 0.0% to 30%; One or more antioxidants in an amount of 0.0% to 5.0%; One or more metal chelating agents in an amount of 0.0% to 0.3%; One or more buffer salts in an amount of 0.0% to 30.0% to adjust the pH of the formulation to 4.5 to 7.5; When reconstituted for administration to a patient, one or more isotonic agents should be added in an amount of 0.0% to 30.0% to adjust the osmotic pressure between 250 and 350 mOsm; Includes, The polyol is selected from fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melegitose, raffinose, mannitol, xylitol, erythritol, maltitol, lactitol, erythritol, treitol, sorbitol, glycerol, or L-gluconate or its metal salt; The surfactants mentioned above include polysorbate 20, polysorbate 40, polysorbate 65, polysorbate 80, polysorbate 81, or polysorbate 85, poloxamer, poly(ethylene oxide)-poly(propylene oxide), polyethylene-polypropylene, triton; sodium dodecyl sulfate (SDS), sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl -, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methylcocoyl taurate or disodium methyloleyl taurate; dodecyl betaine, dodecyldimethylamine oxide, cocamidopropyl betaine and cocoamphoglycinate; or isostearylethylimonium ethosulfate; selected from polyethyl glycol, polypropylene glycol and copolymers of ethylene and propylene glycol; The preservative is selected from benzyl alcohol, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol; The aforementioned amino acid is selected from arginine, cystine, glycine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycyltamic acid, or aspartic acid; The antioxidant is selected from ascorbic acid, glutathione, cystine, or methionine; The chelating agent is selected from EDTA or EGTA; The buffer salt is selected from sodium, potassium, ammonium, or trihydroxyethylamino salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris or tromethamine hydrochloride, phosphate, or sulfate; or arginine, glycine, glycylglycine, or histidine and anionic acetate, chloride, phosphate, sulfate, or succinate; The pharmaceutical composition according to claim 8, wherein the isotonic agent is selected from mannitol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate, trisodium citrate, or sodium chloride, and is either a liquid formulation or a prepared freeze-dried solid / powder.

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