Conjugation of cytotoxic drug with bis-linkage

Abstract

To provide a bis-linkage method for producing a conjugate of a cell-binding agent and a cytotoxic drug molecule in a specific manner.SOLUTION: A method for producing a conjugate compound of Formula (I-v) or Formula (I-w), wherein two or more residues of a cell-binding agent / molecule are simultaneously or sequentially reacted with a readily reactive bis-linker compound.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the conjugation of cell-binding molecules and cytotoxic agents via bis-linkers (double linkers). Cytotoxic agents / molecules, especially amino acids, hydroxyl groups, diamino, aminohydroxyl, dihybrid Contains two functional groups: hydroxyl, carboxyl, hydrazine, aldehyde, and thiol. The present invention also relates to a bis-linking method for the conjugation of drugs. The present invention relates to a method for producing a combination drug (cytotoxic agent). [Background technology]

[0002] Brentuximab vedotin (Adcetris) for relapsed / refractory Hodgkin lymphoma Non-patent literature 1 and 2) and ado-trastuzumab emta for recurrent HER2+ breast cancer As evidenced by the clinical success of vasodilators (Non-Patent Documents 3 and 4), antibody-drug conjugates have Antibody-derived cancer cells (ADCs) have become a promising targeted therapy for cancer. They have three key components: , monoclonal antibodies, cytotoxic payloads, and All ADC conditional linkers, including the linker and cytotoxic moiety linking moiety, are ADC However, the drug being conjugated must have specific reactive groups. Contains functional groups to ensure circulation stability and facilitate drug release upon antigen binding and cellular uptake Once the linked volume components are in circulation and off-target, The scope of the technology for the coupling body remains the same, as it must not cause serious harm to normal tissues. However, the number of such methods is limited (Non-Patent Documents 5 to 8).

[0003] In early ADCs, the linkers used were unstable, especially for ADCs targeting liquid tumors. This resulted in the release of free drug in the circulation and consequent off-target toxicity (Non-Patent Document 9 In the current generation of ADCs, the conjugates are more stable and the cytotoxic agents are significantly more potent. (Non-Patent Document 10). However, off-target toxicity has been a major issue in the development of ADC drugs. This remains a major challenge in the development of HIV (Non-Patent Document 11). Ado-trastuzumab etantansine (T-DM1) using a (non-cleavable) MCC linker , Kadcyla®) in patients with HER2-positive metastatic breast cancer (mBC) Patients with HER2 tumors or those already treated for mBC or within 6 months of adjuvant therapy It has shown significant benefits for patients who have developed recurrent tumors (Non-Patent Documents 12-14). T-DM1 has shown little benefit to patients when comparing efficacy with side effects, so HE As first-line treatment for patients with R-2 positive unresectable locally advanced or metastatic breast cancer, and It has failed in clinical trials as a second-line treatment for ER-2-positive advanced gastric cancer (Non-patent Document 15 ~18).

[0004] To address the issue of off-target toxicity, research and development into ADC chemistry and design is Currently, beyond the efficacy of a single payload, there is a range of linker-payload moieties and conjugation chemistries Expanding, specifically addressing the activity of ADC conjugate-payloads against targets / diseases (Non-Patent Documents 19 and 20). Today, many drug developers and academic institutions are Longer circulating half-life, higher potency, as well as improved batch-to-batch consistency in ADC manufacturing efficacy, reduced potential off-target toxicity, and narrow in vivo range of ADCs New confidence for site-specific ADC conjugates with likely pharmacokinetic (PK) properties There is a strong focus on establishing highly reliable methods (Non-Patent Documents 21-25). These specific conjugation methods reported previously involve the use of designed cysteine ​​residues (Non-Patent Document 1). 26 and 27, Patent Documents 1 to 5), selenocysteine ​​(Non-Patent Documents 28 to 29, Patent Documents 6), cysteine-containing tags using perfluoroaromatic reagents (Non-Patent Document 30), thiols Fucose (Non-Patent Document 31), unnatural amino acids (Non-Patent Documents 32 to 35, Patent Documents 7 to 1 7), conjugation to reduced intermolecular disulfides by re-crosslinking of dibromomaleimide (Non-Patent Document 36), bissulfone reagents (Non-patent document 37, Patent documents 18-19), dibromopyridin Zindione (Non-patent Document 38), galactosyltransferase and sialyltransferase (Non-patent Document 39, Patent Document 20), formylglycine synthase (FGE) (Non-Patent Document 40, Patent Document 21 ~25), phosphopantetheinyl transferases (PPTases) (Non-patent Document 4 1), sortase A (Non-patent document 42), Streptoverticillium mobaraense Transglutaminase (Non-Patent Documents 43-44, Patent Document 26) or microbial transglutaminase Genetically engineered by taminomethyltransferase (MTGase) (Non-patent documents 45-46, Patent documents 27-28) The mTG (metastatic glutamine tag) is an isopeptide bond formed outside the protein backbone. It contains enzymes / bacteria that form peptide bonds (Non-patent documents 47-49).

[0005] We have developed bromomaleimide and dibromomaleimide linkers (Patent Document 29), 2,3 -disubstituted succinic acid / 2-monosubstituted / 2,3-disubstituted fumaric or maleic acid linkers (Patent Document Patent Documents 30-31 (WO2016056228), acetylenedicarboxylic acid conjugates (Patent Documents 32-33), Alternatively, a hydrazine conjugate (Patent Document 34) may be used to convert the interchain disulfide bonds of natural antibodies. Several conjugation methods have been disclosed to re-bridge the reduced thiol pairs. The ADCs made by this method are not linked to conventional non-selective cysteine ​​or lysine residues on the antibody. It demonstrated a superior therapeutic index window than the conjugate. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 8,309,300 [Patent Document 2] U.S. Patent No. 7,855,275 [Patent Document 3] U.S. Patent No. 7,521,541 [Patent Document 4] U.S. Patent No. 7,723,485 [Patent Document 5] International Publication No. WO2008 / 141044 [Patent Document 6] U.S. Patent No. 8,916,159 [Patent Document 7] U.S. Patent 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. Patent No. 7,332,571 [Patent Document 13] U.S. Patent No. 7,696,312 [Patent Document 14] U.S. Patent No. 7,638,299 [Patent Document 15] International Publication No. WO2007 / 130453 [Patent Document 16] U.S. Patent No. 7,632,492 [Patent Document 17] U.S. Patent No. 7,829,659 [Patent Document 18] International Publication No. WO2013 / 190272 [Patent Document 19] International Publication No. WO2014 / 064424 [Patent Document 20] U.S. Patent Application No. 20140294867 [Patent Document 21] U.S. Patent No. 7,985,783 [Patent Document 22] U.S. Patent No. 8,097,701 [Patent Document 23] U.S. Patent No. 8,349,910 [Patent Document 24] U.S. Patent Application No. 20140141025 [Patent Document 25] U.S. Patent Application No. 20100210543 [Patent Document 26] U.S. Patent No. 8,871,908 [Patent Document 27] U.S. Patent Application No. 20130189287 [Patent Document 28] U.S. Patent No. 7,893,019 [Patent Document 29] International Publication No. WO2014 / 009774 [Patent Document 30] International Publication No. WO2015 / 155753 [Patent Document 31] International Publication No. WO2016 / 056228 [Patent Document 32] International Publication No. WO2015 / 151080 [Patent Document 33] International Publication No. WO2016 / 0596228 [Patent Document 34] International Publication No. WO2015 / 151081

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[0008] Herein, we present a method for the preparation of bis-linked and cytotoxic molecules, particularly diamino, aminohydroxyl , dihydroxyl, carboxyl, aldehyde, and thiol dual groups This paper describes a method for conjugating cytotoxic agents with bis-linked cyclohexyl esters (C-C) prepared by bis-linkage. The immunoconjugates have an extended half-life during targeted delivery and do not penetrate non-target cells, tissues, or blood circulation. Organ exposure was minimized, resulting in reduced off-target toxicity. [Means for solving the problem]

[0009] The present invention relates to antibodies and cytotoxic agents, particularly those containing amino, hydroxyl, diamino, or amino - Two functional groups: hydroxyl, dihydroxyl, carboxyl, hydrazine, or thiol The present invention provides a bis-linkage to a cytotoxic agent having a functional group, and also provides a method for binding cell-binding molecules to cells in a specific manner. A bis-linker is provided for attachment to a cytotoxic molecule.

[0010] In one embodiment of the present invention, the bis-linkage is represented by formula (I): [ka]

[0011] During the ceremony, [ka] represents a single bond; [ka] is optionally either a single or double bond or absent; n and m1 are independently 1 to 20.

[0012] The cell-binding molecules in parentheses linked to Z1 and Z2 are currently known or have been identified. bind to a portion of a cell population for which therapeutic or other biological modification is desired, and Preferably, the cell binding agent is a molecule / drug that forms a complex with or reacts with the cell binding agent. The molecule may be an immunotherapeutic protein, an antibody, an antibody fragment, or a peptide having four or more amino acids. It is Do.

[0013] The cytotoxic molecules / agents in parentheses are therapeutic agents, immunotherapeutic proteins / molecules, or cytotoxic agents. Functional molecules for enhancing binding or stabilization of cell-binding agents, or cell surface receptor binding ligands and for inhibiting cell proliferation.

[0014] X and Y are independently the same or different and are disulfides, thioethers, or thioesters. , peptides, hydrazones, ethers, esters, carbamates, carbonates, amines ( secondary, tertiary, or quaternary), imines, cycloheteroalkanes, heteroaromatic, alkane represents a functional group that links to a cytotoxic drug via an oxime or amide bond; preferably X and Y are independently NH; NHNH; N(R1); N(R1)N(R2); O; S; S- S, O-NH, ON(R1), CH2-NH, CH2-N(R1), CH=NH, CH =N(R1), S(O), S(O2), P(O)(OH), S(O)NH, S(O2)N H, P(O)(OH)NH, NHS(O)NH, NHS(O2)NH, NHP(O)(O H)NH, N(R1)S(O)N(R2), N(R1)S(O2)N(R2), N(R1 )P(O)(OH)N(R2), OS(O)NH, OS(O2)NH, OP(O)(OH )NH, C(O), C(NH), C(NR1), C(O)NH, C(NH)NH, C(N R1)NH, OC(O)NH, OC(NH)NH;OC(NR1)NH, NHC(O)N H;NHC(NH)NH;NHC(NR1)NH, C(O)NH, C(NH)NH, C( NR1)NH, OC(O)N(R1), OC(NH)N(R1), OC(NR1)N(R 1), NHC(O)N(R1), NHC(NH)N(R1), NHC(NR1)N(R1 ), N(R1)C(O)N(R1), N(R1)C(NH)N(R1), N(R1)C( NR1)N(R1); or C1-C6 alkyl, C2-C8 alkenyl, heteroalkyl , alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar -Alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkyl carbonyl, or heteroaryl;

[0015] Z1 and Z2 are independently the same or different disulfides attached to cell-binding molecules, Ethers, esters, thioethers, thioesters, peptides, hydrazones, carbamate esters, carbonates, amines (secondary, tertiary, or quaternary), imines, cycloheteroalkanes, are functional groups that form heteroaromatic, alkyl oxime, or amide bonds; Z and Z 2 independently have the following structures: C(O)CH, C(O)C, C(O)CH2, ArC H2, C(O), NH;NHNH;N(R1);N(R1)N(R2);O;S;SS , O-NH, ON(R1), CH2-NH, CH2-N(R1), CH=NH, CH= N(R1), S(O), S(O2), P(O)(OH), S(O)NH, S(O2)NH , P(O)(OH)NH, NHS(O)NH, NHS(O2)NH, NHP(O)(OH )NH, N(R1)S(O)N(R2), N(R1)S(O2)N(R2), N(R1) P(O)(OH)N(R2), OS(O)NH, OS(O2)NH, OP(O)(OH) NH, C(O), C(NH), C(NR1), C(O)NH, C(NH)NH, C(NR 1)NH, OC(O)NH, OC(NH)NH; OC(NR1)NH, NHC(O)NH ;NHC(NH)NH;NHC(NR1)NH, C(O)NH, C(NH)NH, C(N R1)NH, OC(O)N(R1), OC(NH)N(R1), OC(NR1)N(R1 ), NHC(O)N(R1), NHC(NH)N(R1), NHC(NR1)N(R1) , N(R1)C(O)N(R1), N(R1)C(NH)N(R1), N(R1)C(N R1)N(R1); or C1-C8 alkyl, C2-C8 heteroalkyl, alkylcyclo C3-C8 aryl, Ar-alkyl, or heterocycloalkyl heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, Or heteroaryl.

[0016] Z1 and Z2 are preferably attached to a thiol pair of the cell binding agent / molecule. The thiol is preferably dithiothreitol (DTT), dithioerythritol (DTE) ), L-glutathione (GSH), tris(2-carboxyethyl)phosphine (TCE P), 2-mercaptoethylamine (β-MEA), or / and β-mercaptoethanol (β-ME, 2-ME) to reduce the interchain disulfides of the cell-binding agent. The bond is a pair of reduced sulfur atoms.

[0017] L1 and L2 are chains of atoms selected from C, N, O, S, Si, and P, and are preferably or 0 to 500 atoms, and is covalently bonded to X and Z1, and Y and Z2. The atoms used to form L2 are alkylene, alkenylene, alkynylene, ether, Polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazo Amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxyamines , urethane, amino acid, peptide, acyloxyamine, or hydroxamic acid, or They may be linked in any chemically relevant manner to form combinations thereof. Preferably, L1 and L2 are independently the same or different and are selected from the group consisting of O; NH; S; NHNH; N (R3);N(R3)N(R3');Formula (OCH2CH2) p OR3, or (OCH2C H(CH3) p OR3, or NH(CH2CH2O) p R3, or NH(CH2CH( CH3)O) p R3, or N[(CH2CH2O) p R3]-[(CH2CH2O) p ' R3'], or (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3, wherein p and p' are independently 0 to about an integer selected from 1000, or a combination thereof; C1-C8 alkyl; C2 -C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C 8 Aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcyclo It is alkyl, alkylcarbonyl, or heteroaryl.

[0018] wherein R1, R2, R3, R4, and R3' are independently H; C1-C8 alkyl; C 2-C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C3- C8 aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcyclo alkyl, alkylcarbonyl, or heteroaryl; or C1-C8 ester, aryl or a terephthalate, or an amide; or 1 to 8 amino acids; or a group of the formula (OCH2CH2) p or (OCH2CH(CH3)) p wherein p is from 0 to about 5000, or any combination thereof.

[0019] L1 and L2 are optionally 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PAB), 4- Thio-pentanoic acid ester (SPP), 4-(N-maleimidomethyl)cyclohexane- 1-carboxylic acid ester (MCC), (4-acetyl)aminobenzoic acid (SIAB), 4 -Thio-butyric acid ester (SPDB), 4-thio-2-hydroxysulfonyl-butyric acid ester (2-sulfo-SPDB), or natural amino acids having 1 to 8 natural or unnatural amino acids Alternatively, the natural amino acid may comprise one or more of the non-natural peptide linker components. Aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, Onine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenyl The amino acid sequence is selected from the group consisting of arginine, glycine, proline, tryptophan, and alanine.

[0020] Additionally, L1 and L2 may independently comprise one of the following hydrophilic structures: [ka]

[0021] During the ceremony, [ka] are binding sites; X2, X3, X4, X5, and X6 are independently NH; NHNH; N (R3); N(R3)N(R3'); O; S; C1-C6 alkyl; C2-C6 heteroaromatic alkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, A r-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, ar alkylcarbonyl, or heteroaryl; or 1 to 8 amino acids, wherein R3 and R3' are independently H; C1-C8 alkyl; C2-C8 heteroalkyl , alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar- alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl carbonyl, or heteroaryl; or C2-C8 ester, ether, or amide; or formula (OCH2CH2) p or (OCH2CH(CH3)) p Polyethylene having a hydroxyl group, where p is an integer from 0 to about 5000, or It is a combination.

[0022] X1 and Y1 are independently O, NH, CH2, N(CH3), NHNH, S, C(O)O , C(O)NH; m1=1-20.

[0023] Furthermore, L1, L2, X, Y, Z1, and Z2 may not exist independently, but may exist in combination with L1 and Z1 or L2 and Z2 cannot exist simultaneously.

[0024] In another aspect, the present invention provides a labile bis-linker of formula (II): wherein two or more residues of the cell-binding molecule are simultaneously or sequentially linked to form formula (I): You can react with it:

[0025] [ka]

[0026] During the ceremony: [ka] represents a single bond; [ka] is optionally either a single bond, a double bond, or a triple bond, or is optionally not present It doesn't have to be; [ka] When represents a triple bond, both Lv1 and Lv2 are absent;

[0027] The cytotoxic molecules in parentheses, m1, X, Y, L1, L2, Z1, and Z2, are of formula (I) and It is the same definition;

[0028] Lv1 and Lv2 are the same or different thiols, amines, carboxylates, and carboxyl groups on cell-binding molecules. Represents a leaving group capable of reacting with a carboxylic acid, selenol, phenol, or hydroxyl group. The leaving group may be, but is not limited to, a halide (fluoride, chloride, bromide, Iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), N-succinimide Niimidyloxy (NHS), phenoxyl; dinitrophenoxyl; pentafluoro Phenoxyl, tetrafluorophenoxyl, difluorophenoxyl, monofluorophenoxyl Noxyl, Pentachlorophenoxyl, 1H-imidazol-1-yl, Chlorphenoxy Sil, dichlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazole- yl)oxyl, 2-ethyl-5-phenylisoxazolium-3'-sulfonate, Phenyloxadiazole-sulfonyl (-sulfone-ODA), 2-ethyl-5- Phenylisoxazolium-yl, phenyloxadiazol-yl (ODA), oxa Diazolyl, unsaturated carbon (carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur yellow-nitrogen, phosphorus-nitrogen, oxygen-nitrogen, or carbon-oxygen double or triple bonds), or The intermediate molecule generated by the condensing reagent for the knob reaction, or one of the following structures: .

[0029] [ka] JPEG2025138629000012.jpg90166

[0030] During the ceremony, X1' is F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 is independently H, aromatic, heteroaromatic, or one or more H atoms are -R1, - Halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O) an aromatic group substituted independently by 2R1, or -COOR1; Level 3: F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NH S); Phenol; Dinitrophenol; Pentafluorophenol; Tetrafluorophenol phenol; difluorophenol; monochlorophenol; pentachlorophenol; Imidazole;Dichlorophenol;Tetrachlorophenol;1-Hydroxy Tosylate;Mesylate;2-Ethyl-5-phenylisoxazol benzotriazole-3'-sulfonate, its own or other anhydrides (e.g., acetic anhydride, acetic anhydride, anhydride formed with methyl methyl ether, a condensation reagent for peptide bond reaction or Mitsunobu reaction is a leaving group selected from an intermediate molecule produced by the drug; R1 and R2 are H, C1-C8 alkyl, C2-C8 alkenyl, heteroalkyl, Aryl, cycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl Heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbocycle aryl, or heteroaryl, or C2-C8 ester, ether, or amide; or is a peptide containing 1 to 8 amino acids; or a peptide of the formula (OCH2CH2) p Or (OCH2 CH(CH3)) p where p is from 0 to about 500. It is an integer up to 0, or a combination of the above groups.

[0031] In another aspect, the present invention provides a labile bis-linker of formula (III): wherein two or more functional groups of the cytotoxic molecule are simultaneously or sequentially linked to form formula (I): You can react to it automatically:

[0032] [ka]

[0033] In the formula, m1, n, cell-binding agent / molecule, L1, L2, and Z2 have the same definitions as in formula (I). X' and Y' are independently reacted with the residue group of a cytotoxic agent simultaneously or sequentially to form a cytotoxic agent; are functional groups capable of forming X and Y, respectively, wherein X and Y are the groups in formula (I) It is defined as:

[0034] Preferably, X' and Y' are N-hydroxysuccinimide esters, p-nitrophenyl esters, phenyl ester, dinitrophenyl ester, pentafluorophenyl ester, pyridinyl Nitropyridyl disulfide, nitropyridyl disulfide, maleimide, hydrazide, haloacetate carboxylic acid halide, acetylenedicarboxylic acid group, or carboxylic acid halide.

[0035] Preferably, X and Y have one of the following structures: [ka] JPEG2025138629000015.jpg49170

[0036] During the ceremony, X1' is F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 and R5 are independently H, R1, aromatic, heteroaromatic, one or several H atoms -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, an aromatic group substituted with -S(O)R or -COOR; Level 3 is methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoro Trifluoromethyl-sulfonyl(triflate), trifluoromethylsulfonate, nitrophen Noxyl, N-succinimidyloxy (NHS), phenoxyl; dinitrophenoxy pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl Sil, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl , 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl chlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy Sil, 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole- Generated using condensation reagents for the oxadiazol-yl (ODA), oxadiazol-yl, or Mitsunobu reaction is a leaving group selected from the intermediate molecule, wherein R and R are defined above. There is something.

[0037] In another aspect, the present invention provides a labile bis-linker of formula (IV): The cytotoxic molecule and the cell-binding molecule can react with each other independently, simultaneously, or sequentially. to form formula (I).

[0038] [ka]

[0039] In the formula, m1, L1, L2, Z1, and Z2 have the same definitions as in formula (I); Lv1 and Lv2 has the same definition as in formula (II), and X' and Y' have the same definition as in formula (III). be.

[0040] n is 1 to 20; and T has the same definition as in formula (I) above.

[0041] The present invention further relates to a method for preparing the cell-binding molecule-drug conjugate of formula (I). [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 shows a general synthesis of the bis-linked conjugates of this patent, doubly linked via attachment of a phenyldiamine, phenyldiol, or aminophenol group of a drug at one end to a pair of thiols on a cell-binding molecule at the other end, where the wavy line represents the linking moiety of the remaining or absent drug (not shown here). [Figure 2] We present the synthesis of tyrosine (Tyr) and tubtyrosine (Tut) analogs bearing amino or nitro groups on the benzene ring for bis-linking to cell-binding molecules. [Figure 3] 1 shows the synthesis of components of tubulysin analogs. [Figure 4] 1 shows the synthesis of components of tubulysin analogs. [Figure 5] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 6] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 7] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 8] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 9] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 10] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 11] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 12] The synthesis of the components of the bis-linker and the bis-linking of a tubulysin (Tup) analogue, which is a component of tubulysin, are shown. [Figure 13] The synthesis of tubulysin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 14] The synthesis of tubulysin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 15] The synthesis of tubulysin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 16]The synthesis of tubulysin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 17] The synthesis of conjugates of tubulysin analogs containing bis-linkers to antibodies via a pair of thiols on the antibody, and the synthesis of tubuphenylaline (Tup) analogs with bis-linkers bearing double amide bonds are shown. [Figure 18] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 19] The synthesis of conjugates of tubulysin analogs containing bis-linkers to antibodies via a pair of thiols in the antibody, and the synthesis of tubuphenylaline (Tup) analogs with bis-linkers bearing double amide bonds are shown. [Figure 20] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 21] 1 shows the synthesis of a tubulysin analog containing a bis-linker and its attachment to an antibody via a pair of thiols in the antibody. [Figure 22] 1 shows the synthesis of components of dimethyl auristatin analogs. [Figure 23] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 24] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 25] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 26] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 27] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 28] 1 shows the synthesis of dimethyl auristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 29] The synthesis of amatoxin analogues having a diamino group on the aromatic ring is shown. [Figure 30] The synthesis of amatoxin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 31] 1 shows the synthesis of a bis-linker and the attachment of the linker to an amatoxin analog. [Figure 32] The synthesis of amatoxin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 33] The synthesis of amatoxin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 34] The synthesis of amatoxin analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 35] The synthesis of amatoxin analogs and dimethylauristatin F analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are presented. [Figure 36] The synthesis of tubulysin analogs and CBI dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are presented. [Figure 37] 1 shows the synthesis of CBI dimeric analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 38] 1 shows the synthesis of CBI dimeric analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 39] 1 shows the synthesis of CBI dimeric analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 40]1 shows the synthesis of CBI dimeric analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody. [Figure 41] 1 shows the synthesis of PBD dimer analogs containing bis-linkers. [Figure 42] The synthesis of PBD dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 43] The synthesis of PBD dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 44] The synthesis of PBD dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 45] The synthesis of PBD dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 46] The synthesis of PBD dimer analogs containing bis-linkers and their attachment to antibodies via a pair of thiols in the antibody are shown. [Figure 47]The antitumor effects of conjugates A-3a, B-6a, B-12a, B-15a, B-18a, B-20a, B-21a, B-24a, B-28a, C-3a, D-2a, and T-DM1 were compared with PBS (control) in a human gastric tumor N87 cell model. Conjugates A-3a, B-6a, B-12a, B-15a, B-18a, B-20a, B-21a, B-24a, B-28a, and T-DM1 were administered intravenously at a dose of 3 mg / kg, and conjugates C-3a and D-1a were administered intravenously at a dose of 1 mg / kg. All 12 conjugates shown here demonstrated antitumor activity. Animals in the groups treated with conjugates B-24a, C-3a, B-20a, B-21a, and D-20a showed superior antitumor activity to T-DM1. However, animals in the groups treated with conjugates B-18a, B-15a, A-3a, B-6a, B-28a, and B-12a showed inferior antitumor activity to T-DM1. T-DM1 inhibited tumor growth at a dose of 3 mg / kg for 28 days but failed to eliminate tumors during the study. In contrast, conjugates B-20a, B-21a, and D-20a eradicated tumors in some animals from days 15 to 43. [Figure 48] Photographs showing in vitro study animals and excised tumors after sacrifice in the PBS, A-3a, B-15a, B-21a, and T-DM1 groups. Five of eight animals in the conjugate B-21a group had no tumors (indicated as "absent"). Five of eight animals in the conjugate B-15a group died on day 43 due to tumor overgrowth (indicated as "fat"). [Figure 49] Figure 1 shows a stability study of conjugate B-21a in mouse serum compared to the regular mono-linked conjugates T-1a and T-DM1. The results show that the conjugates with bis-linkers are more stable in mouse serum than the regular conjugates containing mono-linkers. DETAILED DESCRIPTION OF THE INVENTION

[0043] Definition:

[0044] "Alkyl" means an alkyl group formed by removing one or two hydrogen atoms from a carbon atom. It refers to an aliphatic hydrocarbon group or monovalent group derived from a carboxylic acid. The term "branched" means that the linear alkyl group has one or more carbon atoms. A group having multiple lower C alkyl groups, such as methyl, ethyl, or propyl groups, attached thereto. Specific examples of alkyl groups include methyl, ethyl, n-propyl, and i-propyl. , n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, Cyclopentyl, cyclohexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2 ,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2, 4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4 -Dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isohexyl C1-C8 alkyl groups include unsubstituted butyl, n-octyl, and isooctyl. and may be substituted with one or more substituents (but not limited to the following substituents). The substituents 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)2 R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N (R')2 and -CN, where R' is independently C1-C8 alkyl and aryl.

[0045] "Halogen" refers to a fluorine, chlorine, bromine, or iodine atom, and includes fluorine and chlorine atoms. is preferred.

[0046] "Heteroalkyl" means a group of 1 to 4 carbon atoms independently selected from the group consisting of O, S, and N. "C2-C8 alkyl substituted at a heteroatom selected from:

[0047] "Carbocycle" means a monocyclic ring system having 3 to 8 carbon atoms or a monocyclic ring system having 7 to 13 carbon atoms. refers to a bicyclic saturated or unsaturated ring. Monocyclic carbocyclic rings have 3 to 6 rings, more typically 5 or 6 Bicyclic carbocycles have 7 to 12 ring atoms and are bicyclic [4,5], [ Arranged as [5,5], [5,6], or [6,6], or 9-10 ring atoms and arranged as a bicyclic ring system [5,6] or [6,6]. Rings (C3~C8 carbocycles) include -cyclopropyl, -cyclobutyl , -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl , -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cycloheptatrienyl Includes, but is not limited to, -cyclooctyl, and -cyclooctadienyl.

[0048] C3-C8 carbocycles can have 3, 4, 5, 6, 7, or refers to an 8-membered saturated or unsaturated non-aromatic carbocyclic compound. C3-C8 carbocyclic rings are unsubstituted It may be substituted with one or more substituents, such as Examples include, but are not limited to, -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C (O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -S(O)R ', -S(O)2R', -OH, -halogen, -N3, -NH2, -NH(R'), -N (R')2, and -CN, where each R' is independently C1 to C8 alkyl and and aryl.

[0049] "Alkenyl" refers to a straight-chain alkyl group having 2 to 8 carbon atoms in the chain and containing a carbon-carbon double bond. Alkenyl groups include, for example, ethenyl, Propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl These include hexyl, hexylenyl, heptenyl, and octenyl.

[0050] "Alkynyl" refers to a straight-chain alkyl group having 2 to 8 carbon atoms in the chain and containing a carbon-carbon triple bond. The alkynyl group refers to an aliphatic hydrocarbon group which may be cyclic or branched. Examples of the alkynyl group include ethynyl, Propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, 5-pentynyl, n- Includes pentynyl, hexynyl, heptynyl, and octynyl.

[0051] An "alkylene" is a group formed by attaching two hydrogen atoms to the same or two different carbon atoms of a parent alkane. a saturated, C1-18, monovalent radical center derived by removing It refers to a straight or branched chain or cyclic hydrocarbon group. Typical alkylene groups include methylene ( -CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH 2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), etc. Not limited to:

[0052] "Alkenylene" refers to a group that has two hydrogen atoms attached to the same or two different carbon atoms of a parent alkene. Unsaturated carbon atoms having 2 to 18 carbon atoms and two monovalent radical centers derived by removing a carbon atom Typical alkenylene groups include 1, Including, but not limited to, 2-ethylene (-CH=CH-).

[0053] An "alkynylene" is an alkylene group that is formed by attaching two hydrogen atoms to the same or two different carbon atoms of a parent alkyne. Unsaturated carbon atoms having 2 to 18 carbon atoms and two monovalent radical centers derived by removing a carbon atom Typical alkynylene groups include acetonitrile, ... Examples include, but are not limited to, ethylene, propargyl, and 4-pentynyl.

[0054] "Aryl" or "Ar" refers to an aryl group having 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. "Heteroaromatic group" refers to an aromatic or heteroaromatic group consisting of one or several rings, including a ring containing a cyclic group. The term "aromatic group" refers to a group in which several carbons, preferably 1, 2, 3, or 4 carbon atoms, on the aromatic group are bonded together. , O, N, Si, Se, P, or S, preferably O, S, and N The term aryl or Ar also refers to a group in which one or more H atoms are independently replaced by -R', -halogen. N, -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''), - Replaced by P(O)(OR')(OR'') or -OP(O)(OR')(OR'') The R' and R'' are independently H, alkyl, alkenyl, alkyn ... quinyl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt thereof. do.

[0055] A "heterocycle" is a ring in which 1 to 4 ring carbon atoms are independently selected from O, N, It refers to ring systems substituted with heteroatoms from the group of S, Se, B, Si, and P. The most common heteroatoms are O, N, and S. Heterocycles are described in The Handbook of Chemistry and Ph , 78th Edition, CRC Press, Inc., 1997-1998, pp. 225-226, and the disclosure thereof are incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to: However, epoxy, aziridinyl, thiranyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl Lysinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranilinyl Imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydropyranyl dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyriminyl dihydrothiopyranyl, dihydrothiopyranyl, azepanyl, and condensation resulting from condensation with a phenyl group The system is included.

[0056] The term "heteroaryl" or aromatic heterocycle refers to a 3- to 14-membered, preferably 5- to 10-membered, aromatic heterocycle. Aromatic heterocyclic, monocyclic, bicyclic, or polycyclic rings, examples of which include pyrrolyl, pyridyl, and the like. , pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quino Linyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl , benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl pyrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl benzimidazolyl, isoxazolyl, pyridyl-N-oxide, and phenyl groups This includes condensed systems resulting from the condensation of

[0057] "Alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl" "Heteroaryl", "heterocyclic" etc. have two hydrogen atoms. The corresponding "alkylene," "cycloalkylene," and "cycloalkylene" groups are formed by removing atoms. ", "alkenylene", "alkynylene", "arylene", "heteroarylene", " It also refers to heterocyclic groups, etc.

[0058] "Arylalkyl" refers to an alkyl group consisting of a carbon atom, typically a terminal or sp 3 Water bonded to a carbon atom It refers to an acyclic alkyl group in which one of the carbon atoms is substituted with an aryl group. The alkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl , 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2 -naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, etc. Includes:

[0059] "Heteroarylalkyl" refers to a heteroaryl group consisting of a carbon atom, typically a terminal or sp 3 Attached to a carbon atom It refers to an acyclic alkyl group in which one of the hydrogen atoms is replaced by a heteroaryl group. Typical heteroarylalkyl groups include 2-benzimidazolylmethyl, 2-furylethy Contains rules.

[0060] Examples of "hydroxy protecting groups" include methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether silyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether , acetate esters, substituted acetate esters, pivaloates, benzoates, methanesulfonates , and p-toluenesulfonate.

[0061] A "leaving group" refers to a functional group that can be displaced by another functional group. Suitable leaving groups are well known in the art and include, for example, halides (e.g., chloride). , bromide, and iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (thiazolinone), trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonyl Preferred leaving groups include nitrophenol; N-hydroxysuccinate; Cinimide (NHS); Phenol; Dinitrophenol; Pentafluorophenol; Tetrafluorophenol; Difluorophenol; Monofluorophenol; Pentak Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol 1-Hydroxybenzotriazole;Tosylate;Mesylate;2-Ethyl-5 -phenylisoxazolium-3'-sulfonate, formed with itself or with other acid anhydrides acid anhydrides (e.g., acetic anhydride, formic anhydride); or for peptide coupling reactions or an intermediate formed by a condensation reagent for the Mitsunobu reaction.

[0062] The following abbreviations may be used herein and have the definitions indicated below: Boc, tert-Butoxycarbonyl; BroP, Bromotrispyrrolizinophosphonium hexa Fluorophosphate; CDI, 1,1'-carbonyldiimidazole; DCC, dicyclohexyl Dihexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane ;DIAD, diisopropyl azodicarboxylate;DIBAL-H, hydrogenated diisobutyl aldehyde Aluminum; DIPEA, diisopropylethylamine; DEPC, diethylphosphoric acid didiate; DMA, N,N-dimethylacetamide; DMAP, 4-(N,N-dimethyl (amino)pyridine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide dithioester; DTT, dithioester; EDC, 1-(3-dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU , O-(7-azabenzotriazol-1-yl)-N,N,N'-N'-tetramethyl Uronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; H PLC, high-pressure liquid chromatography; NHS, N-hydroxysuccinimide; MMP , 4-methylmorpholine; PAB, p-aminobenzyl; PBS, phosphate-buffered saline (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography TCEP, tris(2-carboxyethyl)phosphine; TFA, triflate Fluoroacetic acid; THF, tetrahydrofuran; Val, valine.

[0063] "Amino acids" can be natural and / or unnatural 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, Styridine, isoleucine, leucine, lysine, methionine, phenylalanine, proline , serine, threonine, tyrosine, tryptophan, and valine. are derived forms of protein-forming amino acids, e.g., hydroxyproline, lanthionine 2-aminoisobutyric acid, dehydroalanine, γ-aminobutyric acid (neurotransmitter), ornithin Citrulline, β-alanine (3-aminopropanoic acid), γ-carboxyglutamate Selenocysteine ​​(present as in most eukaryotes, but not directly linked to DNA) (not present in some archaea and one bacterium), pyrrolysine (only present in some archaea and one bacterium), N-phosphatase methylmethionine (often the first amino acid in proteins in bacteria, mitochondria, and chloroplasts) amino acid), 5-hydroxytryptophan, L-dihydroxyphenylalanine, triiodothyronine Dothyronine, L-3,4-dihydroxyphenylalanine (DOPA), and O-phosphoric acid The term amino acid also includes amino acid analogs and mimetics. is the same as that of a natural amino acid, except that the R group is not one found in a natural amino acid. These are compounds with the same general structure: H2N(R)CHCO2H. Examples of analogs include Moselin, norleucine, methionine sulfoxide, and methionine methyl sulfonium Preferably, the amino acid mimetic has a structure different from the general chemical structure of an α-amino acid. The term "unnatural amino acid" refers to a compound that has the structure, but functions similarly to, It is intended to represent the "D" stereochemical form, whereas naturally occurring amino acids are in the "L" form. When 1 to 8 amino acids are used in the present application, the amino acid sequence is selected from the cleavage recognition sequences of proteases. Many cleavage recognition sequences are known in the art, for example, Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 25 4 (1994);Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymo l. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994);Smith et al. Me th. Enzymol. 244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995) See, e.g., AA-Val, the disclosure of which is incorporated herein by reference. In particular, the sequence , 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.

[0064] A "glycoside" is a compound in which a sugar group is bonded to another group via its anomeric carbon through a glycosidic bond. Glycosides are molecules that are O-(O-glycosides), N-(glycosylamines), ), S-(thioglycoside), or C-(C-glycoside) glycosidic bonds The core empirical formula is C m (H2O) n (where m is different from n, m and n are <36), where glycosides include glucose (dextrose ), fructose (levulose), allose, altrose, mannose, gulose, yogurt Dose, galactose, talose, galactosamine, glucosamine, sialic acid, N-acetylglucosamine Glucosamine, sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose) sucrose), ribose, arabinose, xylose, lyxose, sorbitol, mannitol , sucrose, lactose, maltose, trehalose, maltodextrin, raffinose It contains stachyose, glucuronic acid (glucuronide), and stachyose. L-form, 5-atom furanose-form, 6-atom cyclic pyranose-form, or acyclic, α-isomer (-OH of the anomeric carbon below the plane of the carbon atoms in the Haworth projection), or β-isomer (Haworth It can be the -OH of the anomeric carbon above the plane of the Worth projection. The term "ol" is used herein to refer to an oligosaccharide containing 3 to 6 sugar units.

[0065] "Pharmaceutically" or "pharmaceutically acceptable" means that a substance can be administered to an animal or human in an appropriate manner. "Inactive Ingredients" refers to molecules and compositions that do not cause adverse, allergic or other untoward reactions in the context of the intended use.

[0066] A "pharmaceutically acceptable solvate" or "solvate" is disclosed as a compound containing one or more solvent molecules. Examples of solvents that form pharmacologically acceptable solvates include water. , isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanol These include, but are not limited to, phenolamines.

[0067] "Pharmaceutically acceptable excipients" include preservatives or antioxidants, fillers, disintegrants, wetting agents, milk Solubilizing agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents For pharmaceutical active substances, this includes any carrier, diluent, adjuvant, or vehicle, such as an agent. The use of such media and agents is well known in the art. Except in the event that such agent is incompatible with the active ingredient, its use in therapeutic compositions is not contemplated. Supplementary active ingredients can also be incorporated into the compositions as suitable therapeutic combinations. can be done.

[0068] As used herein, a "pharmaceutical salt" refers to a compound in which the parent compound is converted into an acid or base salt thereof. Derivatives of the disclosed compounds are those that have been modified by making them pharmaceutically acceptable. The salts can be any of the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include quaternary ammonium salts of hydrochloric acid, hydrobromic acid, Salts derived from inorganic acids such as sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, etc.; acetic acid, propionic acid, etc. Acid, succinic acid, tartaric acid, citric acid, methanesulfone, benzenesulfone, glucuronic acid , glutamic, benzoic, salicylic acid, toluene sulfonic, oxalic, fumaric, Salts prepared from organic acids such as maleic acid and lactic acid are also included. Ammonium salts of thiamine, meglumine, epolamine, etc., sodium, potassium, calcium Examples of suitable salts include metal salts of zinc, magnesium, etc.

[0069] In the present invention, pharmaceutical salts are prepared by conventional chemical methods containing acidic or basic residues. Generally, these salts can be prepared from the parent compound by dissolving the salt in water, an organic solvent, or both. In a mixed solvent of the free acid or free base form of these compounds and a stoichiometric amount of an appropriate salt It can be obtained by reaction with a base or an acid. Ethers are generally used as non-aqueous reaction solvents. ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. For a list of salts, see Remington's Pharmaceutical Sciences iences”, 17th edition. Mack Publishing Company, Eas ton, PA, 1985, p. 1418, the disclosure of which is incorporated by reference. .

[0070] "Administering" or "Administration" ion) means any manner of transferring, delivering, introducing or transporting medicines or other drugs to a subject. Such modes include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, lesional, nasal, and the like. Also contemplated by the present invention are intrathecal, intravenous, or intracavitary administration. Such devices may utilize active or passive transport. The delivery device may be a slow release or a fast release delivery device.

[0071] The novel conjugates disclosed herein utilize bridge linkers. Examples of bridge links and their synthesis are shown in Figures 1-34.

[0072] Cell-binding agent-cytotoxic molecule conjugates via bis-linkages

[0073] The bis-linkage of the conjugate is represented by formula (I): [ka]

[0074] During the ceremony, [ka] represents a single bond; [ka] is optionally either a single or double bond, or absent; n and m1 are independently The numbers range from 1 to 20.

[0075] The cell-binding molecules in parentheses linked to Z1 and Z2 are currently known or have been identified. bind to a portion of a cell population for which therapeutic or other biological modification is desired, and Preferably, the cell binding agent is a molecule / drug that forms a complex with or reacts with the cell binding agent. / Molecules include immunotherapeutic proteins, antibodies, single-chain antibodies; antibody fragments that bind to target cells; monoclonal antibodies; clonal antibody; single-chain monoclonal antibody; or monoclonal antibody that binds to target cells Chimeric antibody fragments; chimeric antibody fragments that bind to target cells; domain antibodies; Binding domain antibody cross-sections; antibody-mimetic adnectins; DARPins; lymphokines ; hormones; vitamins; growth factors; colony-stimulating factors; or nutrient transport molecules; transferrins or a protein, or an antibody, or an albumin; amines, polymers, dendrimers, liposomes, nanoparticles, vesicles, or (viral) capsids The small cell binding molecules or binding ligands that bind to the ATP-binding domain are small cell binding molecules or binding ligands.

[0076] The cytotoxic molecules in parentheses are drugs / molecules / drugs for treatment or targets for immunotherapy. functional molecules to enhance the binding or stabilization of proteins / molecules or cell-binding agents; Monitoring cell surface receptor binding ligands, or inhibition of cell proliferation, or cell-associated molecule activity for detection, detection, or research; or analogs or prodrugs thereof, or pharmaceutical acceptable salts, hydrates or hydrated salts, or crystal structures, or optical isomers, racemates diastereomeric immunotherapeutic compounds, enantiomers or enantiomers of chemotherapy compounds an anantiomer, an antibody (probody) or an antibody (probody) fragment; or an siRNA or or a DNA molecule; or a cell surface-binding ligand.

[0077] Preferably, the cytotoxic molecule is any of a number of small molecule drugs, including Examples of suitable steroids include, but are not limited to, tubulysins, calicheamicins, auristatins, meiotic steroids, and the like. Tansinoids, CC-1065 analogs, morpholinos, doxorubicins, taxanes , cryptophycins, amatoxins (amanitins), epothilones, eribulin, Geldanamycins, duocarmycins, daunoma Isines, methotrexate, vindesines, vincristine and benzodiazepine dimers (e.g., pyrrolobenzodiazepines (PBDs), tomaize mycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinone It contains dimers of zolidinobenzodiazepines.

[0078] X and Y are independently the same or different and are disulfides, thioethers, or thioesters. , peptides, hydrazones, ethers, esters, carbamates, carbonates, amines ( secondary, tertiary, or quaternary), imines, cycloheteroalkanes, heteroaromatic, alkane represents a functional group that links to a cytotoxic drug via an oxime or amide bond; preferably X and Y are independently NH; NHNH; N(R1); N(R1)N(R2); O; S; S- S, O-NH, ON(R1), CH2-NH, CH2-N(R1), CH=NH, CH =N(R1), S(O), S(O2), P(O)(OH), S(O)NH, S(O2)N H, P(O)(OH)NH, NHS(O)NH, NHS(O2)NH, NHP(O)(O H)NH, N(R1)S(O)N(R2), N(R1)S(O2)N(R2), N(R1 )P(O)(OH)N(R2), OS(O)NH, OS(O2)NH, OP(O)(OH )NH, C(O), C(NH), C(NR1), C(O)NH, C(NH)NH, C(N R1)NH, OC(O)NH, OC(NH)NH;OC(NR1)NH, NHC(O)N H;NHC(NH)NH;NHC(NR1)NH, C(O)NH, C(NH)NH, C( NR1)NH, OC(O)N(R1), OC(NH)N(R1), OC(NR1)N(R 1), NHC(O)N(R1), NHC(NH)N(R1), NHC(NR1)N(R1 ), N(R1)C(O)N(R1), N(R1)C(NH)N(R1), N(R1)C( NR1)N(R1); or C1-C6 alkyl, C2-C8 alkenyl, heteroalkyl , alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar -Alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkyl aryl, arylcarbonyl, or heteroaryl.

[0079] Z1 and Z2 are independently the same or different disulfides attached to cell-binding molecules, Ethers, esters, thioethers, thioesters, peptides, hydrazones, carbamate esters, carbonates, amines (secondary, tertiary, or quaternary), imines, cycloheteroalkanes, is a heteroaromatic, alkyl oxime, or amide bond-forming functional group; preferably , Z1 and Z2 independently have the following structures: C(O)CH, C(O)C, C(O)C H2, ArCH2, C(O), NH;NHNH;N(R1);N(R1)N(R2);O ;S;SS, O-NH, ON(R1), CH2-NH, CH2-N(R1), CH= NH, CH=N(R1), S(O), S(O2), P(O)(OH), S(O)NH, S (O2)NH, P(O)(OH)NH, NHS(O)NH, NHS(O2)NH, NHP (O)(OH)NH, N(R1)S(O)N(R2), N(R1)S(O2)N(R2) , N(R1)P(O)(OH)N(R2), OS(O)NH, OS(O2)NH, OP( O)(OH)NH, C(O), C(NH), C(NR1), C(O)NH, C(NH)N H, C(NR1)NH, OC(O)NH, OC(NH)NH;OC(NR1)NH, NH C(O)NH;NHC(NH)NH;NHC(NR1)NH,C(O)NH,C(NH) NH, C(NR1)NH, OC(O)N(R1), OC(NH)N(R1), OC(NR 1)N(R1), NHC(O)N(R1), NHC(NH)N(R1), NHC(NR1 )N(R1), N(R1)C(O)N(R1), N(R1)C(NH)N(R1), N( R1)C(NR1)N(R1); or C1-C8 alkyl, C2-C8 heteroalkyl, Alkylcycloalkyl or heterocycloalkyl; C3-C8 aryl, Ar- Alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkyl carbonyl, or heteroaryl.

[0080] Preferably, Z1 and Z2 are attached to a thiol pair of the cell binding agent / molecule. The thiol is preferably dithiothreitol (DTT), dithioerythritol (D TE), dithiolbutylamine (DTBA), L-glutathione (GSH), Tris( 2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-M EA), or / and β-mercaptoethanol (β-ME, 2-ME), The agent reduces the interchain disulfides of the cell-binding agent to a pair of sulfur atoms.

[0081] L1 and L2 each have 0 to 500 atoms selected from C, N, O, S, Si, and P. and is a chain of atoms covalently bonded to X and Z1, and to Y and Z2. Formation of L1 and L2 The atoms used in may be bonded in any chemically relevant way, preferably C1 -C 20 Alkylene, alkenylene, and alkynylene, ether, polyoxyalkylene amines, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, Semicarbazide, carbazide, alkoxyamine, alkoxylamine, urethane, amino acid, peptide, acyloxyamine, or hydroxamic acid, or a combination thereof More preferably, L1 and L2 are independently the same or different and are O, NH, S , NHNH, N(R3), N(R3)N(R3'), C1-C8 alkyl, amide, amino Imines, hydrazines, hydrazones; C2-C8 heteroalkyl, alkylcycloalkenyl Alkyl, ether, ester, hydrazone, urea, semicarbazide, carbazide, alkoxy Thiamine, alkoxylamine, urethane, amino acid, peptide, acyloxylamine, Hydroxamic acid, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, Heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl; p OR3, or (OCH2CH(CH3) ) p OR3, or NH(CH2CH2O) p R3, or NH(CH2CH(CH3)O) p R3, or N[(CH2CH2O) p R3]-[(CH2CH2O) p 'R3'], also is (OCH2CH2) p COOR3, or CH2CH2(OCH2CH2) p COOR3 In the formula, p and p' are independently from 0 to about 5000. or a combination thereof, wherein R3 and R3' are independently selected integers. Specifically, H; C1-C8 alkyl; C2-C8 heteroalkyl, alkylcycloalkyl , or heterocycloalkyl; C3-C8 aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl or C2-C8 ester, ether, or amide; or 1 to 8 amino acids; or the formula (OCH2CH2) p or (OCH2CH(CH3)) p Polyethylene O oxy units, where p is an integer from 0 to about 5000, or a combination of the above. is.

[0082] L1 and L2 are optionally 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PAB), 4- Thio-pentanoic acid ester (SPP), 4-(N-maleimidomethyl)cyclohexane- 1-carboxylic acid ester (MCC), (4-acetyl)aminobenzoic acid (SIAB), 4 -Thio-butyric acid ester (SPDB), 4-thio-2-hydroxysulfonyl-butyric acid ester (2-sulfo-SPDB), or natural amino acids having 1 to 8 natural or unnatural amino acids Alternatively, the natural amino acid may comprise one or more of the non-natural peptide linker components. Aspartic acid, glutamic acid, arginine, histidine, lysine, serine, threonine, Onine, asparagine, glutamine, cysteine, selenocysteine, tyrosine, phenyl The amino acid sequence is selected from the group consisting of arginine, glycine, proline, tryptophan, and alanine.

[0083] L1 and L2 are independently a self-immolative or non-self-immolative moiety, a peptide unit, a hydrazoline It also includes amine bonds, disulfide, ester, oxime, amide, or thioether bonds. The self-immolative unit may be, but is not limited to, para-aminobenzylcarbamoyl. Moyl (PAB) group, 2-aminoimidazole-5-methanol derivatives, heterocyclic PABs The derivatives, β-glucuronides, and o- or p-aminobenzyl acetals are electronically similar. This includes aromatic compounds.

[0084] The self-immolative linker moiety preferably has one of the following structures: [ka]

[0085] In the formula, ( * ) atoms labeled with an additional spacer or releasable linker unit, cell Attachment points for toxic agents and / or cell adhesion molecules (CBAs); X 1 , Y 1 , Z 2 , and Z 3 are independently NH, O or S; Z 1 are independently H, NHR1, OR1, SR1, COX1R1, where X1 and R1 are as defined above; v is 0 or 1. ru;U 1 are independently H, OH, C1-C6 alkyl, (OCH2CH2) n , F, Cl, Br, I, OR5, SR5, NR5R5', N=NR5, N=R5, NR5R5', NO 2, SOR5R5', SO2R5, SO3R5, OSO3R5, PR5R5', POR5 R5', PO2R5R5', OPO(OR5)(OR5'), or OCH2PO(OR5 )(OR5'), wherein R5 and R5' are each independently H, C1-C8 alkyl, alkyl; C2-C8 alkenyl, alkynyl, heteroalkyl, or amino acid; C3- C8 aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroara alkyl, alkylcarbonyl, or glycoside group; or a medicinal cation salt.

[0086] The non-self-immolative linker component is one of the following structures: [ka] JPEG2025138629000022.jpg130169

[0087] In the formula, ( * ) atoms labeled with an additional spacer or releasable linker unit, cell Attachment points for toxic agents and / or cell adhesion molecules (CBAs); X 1 , Y 1 , U 1 , R5 The definitions of R5' and R5' are as above; r is 0 to 100; m and n are each independently The numbers range from 0 to 6.

[0088] More preferably, L1 and L2 may independently be releasable links. The terms pH labile, acid labile, base labile, oxidative labile, metabolic labile At least one bond that can be cleaved under physiological conditions, such as a biochemically unstable or enzyme labile bond. The physiological conditions that result in bond disruption are not necessarily It is understood that the present invention does not involve any biological or metabolic processes, but instead involves hydrolysis or substitution reactions. Standard chemical reactions such as endosomal reactions, e.g., endosomes, which have a lower pH than the cytoplasmic pH, and and / or disulfide with intracellular thiols such as glutathione, which are abundant in millimolar concentrations in malignant cells. This includes bond exchange reactions.

[0089] Examples of releasable linkers L1 or L2 include, but are not limited to:-(C R5R6) m (Aa) r (CR7R8) n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (Aa) r (OCH2CH2) t -, -(Aa) r -(CR5R6) m (CR7R8) n (OCH2CH2) t -, -(CR5R6) m (CR7R8) n (OC H2CH2) r -(Aa) t -、-(CR5R6) m (CR7=R8)(CR9R 10 ) n (Aa) t (OCH2CH2) r -、-(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-(CR5R6) m (Aa) t (NR1 1CO)(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCO)( Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-(CR5R6) m (OCNR 7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (CO )(Aa) t -(CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -、-(CR5R6) m (OCNR7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -,-(CR5R 6) m (CO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -,-(CR5R 6) m -phenyl-CO(Aa) t (CR7R8) n -, -(CR5R6) m -Frills- CO(Aa) t (CR7R8) n -, -(CR5R6) m -oxazolyl-CO(Aa) t (CR7R8) n -, -(CR5R6) m -Thiazolyl-CO(Aa) t (CR7R8 ) n -, -(CR5R6) m -Thienyl-CO(CR7R8) n -, -(CR5R6) t -Imidazolyl-CO-(CR7R8) n -, -(CR5R6) t -morpholino-CO( Aa) t -(CR7R8) n -, -(CR5R6) t -Piperazino-CO(Aa) t -( CR7R8) n -, -(CR5R6) t -N-Methylpiperazino-CO(Aa) t -(C R7R8) n -, -(CR5R) m -(Aa) t Phenyl-, -(CR5R6) m -(A a) tFrill-,-(CR5R6) m -oxazolyl (Aa) t -, -(CR5R6) m -Thiazolyl (Aa) t -, -(CR5R6) m -Thienyl-(Aa ) t -, -(CR5R6) m -Imidazolyl (Aa) t -, -(CR5R6) m -Morpho Rino (Aa) t -, -(CR5R6) m -Piperazino-(Aa) t -,-(CR5R6 ) m -N-methylpiperazino-(Aa) t -, -K(CR5R6) m (Aa) r (CR7 R8) n (OCH2CH2) t -, -K(CR5R6) m (CR7R8) n (Aa) r ( OCH2CH2) t -, -K(Aa) r -(CR5R6) m (CR7R8) n (OCH2 CH2) t -, -K(CR5R6) m (CR7R8) n (OCH2CH2) r (Aa) t -, -K(CR5R6) m -(CR7=R8)(CR9R 10 ) n (Aa) t (OCH2 CH2) r -, -K(CR5R6) m (NR 11 CO)(Aa) t (CR9R 10 ) n ( OCH2CH2) r -, -K(CR5R6) m (Aa) t (NR 11 CO)(CR9R1 0) n (OCH2CH2) r -, -K(CR5R6) m (OCO)(Aa) t (CR9R 10 ) n -(OCH2CH2) r -, -K(CR5R6) m (OCNR7)(Aa) t ( CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (CO)(Aa) t - (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (NR 11 CO)( Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (OCO) (Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m (OCN R7)(Aa) t (CR9R 10 ) n (OCH2CH2) r -、-K-(CR5R6) m (CO)(Aa) t (CR9R 10 ) n (OCH2CH2) r -, -K(CR5R6) m -フェニル-CO(Aa)t (CR7R8) n -, -K-(CR5R6) m -Frill-C O(Aa) t -(CR7R8) n -, -K(CR5R6) m -oxazolyl-CO(Aa ) t (CR7R8) n -, -K(CR5R6) m -Thiazolyl-CO(Aa) t (CR7 R8) n -, -K(CR5R6) m -Thienyl-CO(CR7R8) n -, -K(CR5 R6) t -Imidazolyl-CO-(CR7R8) n -, -K(CR5R6) t -Morpholi No-CO(Aa) t -(CR7R8) n -, -K(CR5R6) t -piperazino-CO( Aa) t -(CR7R8) n -, -K(CR5R6) t -N-methylpiperazino-CO( Aa) t -(CR7R8) n -, -K(CR5R) m -(Aa) t Phenyl-, -K-( CR5R6) m -(Aa) t Frill-, -K(CR5R6) m -oxazolyl (Aa) t -, -K(CR5R6) m -Thiazolyl (Aa) t -, -K(CR5R6) m -Thienyl -(Aa) t -, -K(CR5R6) m -Imidazolyl (Aa) t -, -K(CR5R6 ) m -Morpholino-(Aa) t -, -K(CR5R6) m -Piperazino-(Aa) t G- , -K(CR5R6) m -N-methylpiperazino (Aa) t -.

[0090] In the formula, m, Aa, m, and n are as defined above; t and r are independently 0 to 100. R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of H, halide, C1-C 8 alkyl; C2-C8 aryl, alkenyl, alkynyl, ether, ester, amino and optionally one or more halides, CN, NR1R2, CF 3, OR1, aryl, heterocycle, S(O)R1, SO2R1, -CO2H, -SO3H, -OR1, -CO2R1, -CONR1, -PO2R1R2, -PO3H, or P(O) Substituted by R1R2R3; K is NR1, -SS-, -C(=O)-, -C(=O)NH- , -C(=O)O-, -C=NH-O-, -C=N-NH-, -C(=O)NH-NH- , O, S, Se, B, Het (heterocyclic or heteroaromatic ring having C3-C8), or 1-2 It is a peptide containing 0 amino acids.

[0091] Additionally, L1 and L2 may independently comprise one of the following hydrophilic structures: [ka]

[0092] During the ceremony, [ka] is a binding site; X2, X3, X4, X5, or X6 are independently NH; NHNH; N (R3); N(R3)N(R3'); O; S; C1-C6 alkyl; C2-C6 heteroaromatic alkyl, alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, A r-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, ar alkylcarbonyl, or heteroaryl; or 1 to 8 amino acids, wherein R3 and R3' are independently H; C1-C8 alkyl; C2-C8 heteroalkyl , alkylcycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar- alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl carbonyl, or heteroaryl; or C2-C8 ester, ether, or amide; or formula (OCH2CH2) p or (OCH2CH(CH3)) p Polyethylene having a hydroxyl unit, where p is an integer from 0 to about 5000, or It's a combination.

[0093] More preferably, R1, L1, or L2 is independently a chain alkyl having 1 to 6 carbon atoms, or is the formula (OCH2CH2) p polyethyleneoxy units (p=1 to 5000), or 1 to 4 A peptide containing 10 amino acid units (L or D) or a combination of the above.

[0094] Furthermore, X, Y, L1, L2, Z1, or Z2 is independently one or more of the following components: It can be configured as: [ka] JPEG2025138629000026.jpg248168JPEG2025138629000027.jpg156167 and L- or D-, natural or non-natural peptides containing 1 to 20 amino acids;

[0095] In the formula, the bond at the center of the atom can bond to any of the adjacent carbon atom bonds. the wavy lines are where you can connect within another bond.

[0096] Alternatively, X, Y, L1, L2, Z1, or Z2 may independently be absent, but L 1 and Z1, or L2 and Z2 cannot exist at the same time.

[0097] Preferably, the bis-linkage of the conjugate further comprises the following formula (Ia), (Ib), (Ic): , (Id), (Ie), (If), (Ig), (Ih), (Ii), (I- j), (Ik), (Im), (In), (Io), (Ip), (Iq), ( Ir), (Is), (It), (Iu), (Iv), and (Iw) R:

[0098] [ka] JPEG2025138629000029.jpg197116JPEG2025138629000030.jpg236141JPEG2025138629000031.jpg87157

[0099] In the formula, X7 and Y7 are independently CH, CH2, NH, O, S, NHNH, N(R1) and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. It means that they can be concatenated; [ka] , X, Y, R1, n, L1, and L2 are the same as above; and the cytotoxic agent is a cytotoxic agent that It is the same as a toxic molecule.

[0100] More preferably, X and Y are independently selected from amino, hydroxyl, diamino, Aminohydroxyl, dihydroxyl, carboxyl, aldehyde, hydrazine, thiol The alkyl group is a hydroxyl, phosphate, or sulfonyl group.

[0101] Preparation of conjugates of cell-binding molecules and drugs via bis-linkage

[0102] Preparation of conjugates of cell-binding molecules and drugs of the present invention and methods for producing conjugates via bis-linkage The synthetic routes are shown in Figures 1 to 46.

[0103] In one aspect, the present invention provides a labile bis(2-hydroxybenzoate) compound containing a cytotoxic molecule of formula (II): A conjugate compound is provided, wherein two or more residues of the cell-binding molecule form a compound of formula (I): It can react with it simultaneously or sequentially to obtain:

[0104] [ka]

[0105] During the ceremony: [ka] represents a single bond; [ka] is optionally either a single bond, a double bond, or a triple bond, or is optionally not present It doesn't have to be; [ka] When represents a triple bond, both Lv1 and Lv2 are absent; cytotoxic molecules in parentheses, m1, X, Y, L1, L2, Z1, and Z2 have the same definitions as in formula (I).

[0106] Lv1 and Lv2 are the same or different thiols, amines, carboxylates, and carboxyl groups on cell-binding molecules. Represents a leaving group capable of reacting with a carboxylic acid, selenol, phenol, or hydroxyl group. Lv1 and Lv2 are OH; F; Cl; Br; I; nitrophenol; N-hydroxyl Thiosuccinimide (NHS); phenol, dinitrophenol, pentafluorophenol phenol, tetrafluorophenol; difluorophenol; monofluorophenol; Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol 1-Hydroxybenzotriazole;Tosylate;Mesylate;2-Ethyl 5-phenyl-3'-oxazolium-3'-sulfonate, with itself or with other anhydrides Anhydrides formed: acetic anhydride or formyl anhydride; or EDC (N-(3-dimethylethyl) (N'-ethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl- N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl N-(2-morpholinoethyl)carbodiimide meso-p-toluenesulfonate ( CMC, or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TB TU(O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronate tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzyl (benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoride (Benzotriazol-1-yloxy)tripyrrolidine phosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate DEPC, chloro-N,N,N',N'-tetramethylformamidinium hexachloride Fluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3- Triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (H ATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3] Triazolo[4,5-b]pyridin-1-ium-3-oxide hexafluorophosphine 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (HDMA), phosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamide Dinium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl Thiuronium hexafluorophosphate, S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate -(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) (carbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexamethyl Trifluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyl) (dimethylaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphine O-(benzotriazol-1-yl)-N,N,N',N'-bis ... (Tetramethylene)uronium hexafluorophosphate (HBPyU), N-Benzyl N-cyclohexylcarbodiimide (with or without polymer linkage), dipyrrolidone Lysino(N-succinimidyloxy)-carbenium hexafluorophosphate (H SPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCIU) ), 2-chloro-1,3-dimethylimidazolinium tetrafluoroborate (CIB) , (benzotriazol-1-yloxy)dipiperidinocarbenium hexafluorophosphate sulfate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N, N,N',N'-Tetramethyluronium tetrafluoroborate (TCTU), bromo Tris(dimethylamino)phosphonium hexafluorophosphate (BroP), pro Pyruvic anhydride (PPACA, T3P®), 2-morpholinoethyl isopropyl Cyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl) )Uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridyl Dinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyl] phenylamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate ( TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl Methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethylmorpholinium chloride thyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N ,N,N',N'-Tetramethyluronium tetrafluoroborate (TDBTU), 1 ,1'-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl)a Di-tert-butyl azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate ( DBAD), diisopropyl azodicarboxylate (DIAD), or diethyl Peptide bond reaction selected from azodicarboxylate (DEAD) or Mitsunobu are selected from intermediate molecules generated by condensing reagents for the reaction; and further, Lv1 and Lv2 are anhydrides formed by itself or with other C1-C8 acid anhydrides. do.

[0107] Preferably, Lv1 and Lv2 are independently a halide (fluoride, chloride, bromide, , iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), triflate Trifluoromethylsulfonyl(triflate), trifluoromethylsulfonate, nitrof Phenoxyl, N-succinimidyl hydroxyl (NHS), Phenoxyl; Dinitrophenoxyl pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl Noxyl, difluorophenixyl, monochlorophenixyl, pentachlorophenoxy 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl Chlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy Sil, 2-ethyl-5-phenyloxazolium-3'-sulfonyl, phenyloxadia 2-ethyl-5-phenylisoxazolidinium Phenyloxadiazol-yl (ODA), oxadiazol-yl, unsaturated Sumocarbon (carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, oxygen-nitrogen or carbon-oxygen double or triple bond), or from one of the following structures: You can choose.

[0108] [ka] JPEG2025138629000038.jpg94165

[0109] During the ceremony, X1' is F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 is independently H, aromatic, heteroaromatic, or one or more H atoms are -R1, - Halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, -S(O) an aromatic group substituted independently by 2R1, or -COOR1; Level 3: F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NH S); Phenol; Dinitrophenol; Pentafluorophenol; Tetrafluorophenol phenol; difluorophenol; monochlorophenol; pentachlorophenol; Imidazole;Dichlorophenol;Tetrachlorophenol;1-Hydroxy Tosylate;Mesylate;2-Ethyl-5-phenylisoxazol benzoylsulfonate, formed from itself or from other anhydrides, e.g. anhydrous acetic acid Anhydrides formed with acids, formyl anhydride; for peptide bond reactions or Mitsunobu reactions is a leaving group selected from the intermediate molecule generated using a condensation reagent for

[0110] R1 and R2 are H, C1-C8 alkyl, C2-C8 alkenyl, heteroalkyl, Aryl, cycloalkyl, or heterocycloalkyl; C3-C8 aryl, Ar-alkyl Heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbocycle aryl, or heteroaryl, or C2-C8 ester, ether, or amide; or is a peptide containing 1 to 8 amino acids; or a peptide of the formula (OCH2CH2) p Or (OCH2 CH(CH3)) p where p is from 0 to about 500. It can be an integer up to 0, or a combination thereof.

[0111] In addition, the functional group X or Y that allows for the attachment of a drug or cytotoxic agent is preferably Disulfides, thioethers, thioesters, peptides, hydrazones, esters, carbamates The compound may contain groups that allow for attachment via mate, carbonate, alkoxide, or amide bonds. Such functional groups include thiol, disulfide, amino, carboxyl, and alkyl groups. Dehydes, ketones, maleimides, haloacetyls, hydrazines, alkoxyaminos, and / or Examples include, but are not limited to, hydroxy.

[0112] Preferably, the bis-linkage of the conjugate further comprises the formula (II-a), (II-b), (II-c) , (II-d), (II-e), (II-f), (II-g), (II-h), (II- i), (II-j), (II-k), (II-m), (II-n), (II-o), (I Iq), (II-r), (II-s), (II-t), (II-u), (II-v), (II-w), (II-x), (II-y), (II-z), (II-a1), (II- a2), (II-a3), and (II-a4):

[0113] [ka] JPEG2025138629000040.jpg253169JPEG2025138629000041.jpg34150

[0114] During the ceremony, X7 and Y7 are independently CH, CH2, NH, O, S, NHNH, N(R1), and N; X, Y, R1, n, [ka] , L1, and L2 are the same as above; the chemical bond between two atoms is means that any two atoms can be linked; R1, X, Y, n, L1, L2, Lv Level 1 and Level 2 are the same as above.

[0115] Preferably, Lv1 and Lv2 are independently Cl, Br, I, methanesulfonyl (methanyl), toluenesulfonyl (tosyl), trifluoromethyl-sulfonyl (triflate ), trifluoromethylsulfonate, and nitrophenoxyl do.

[0116] In another aspect, the present invention provides a labile compound conjugated to a cell binding agent / molecule of formula (III): and a reactive bis-linker, wherein two or more functional groups of the cytotoxic molecule form a group of formula (I): It can react with it simultaneously or sequentially to obtain:

[0117] [ka]

[0118] During the ceremony, m1, n, [ka] , cell binding agent / molecule, L1, L2, and Z2 have the same definitions as in formula (I); X' and Y' are independently reacted with the residue groups of a cytotoxic drug simultaneously or sequentially to form and a functional group capable of forming X and Y, wherein X and Y are Defined; X' and Y' are preferably disulfide substituents, maleimide, haloacetyl, alkoxy, amine, azide, ketone, aldehyde, hydrazine, amino, hydroxyl, carbo xylates, imidazoles, thiols, or alkynes; or N-hydroxysuccinic acid Imidoester, p-nitrophenyl ester, dinitrophenyl ester, pentafluoro Fluorophenyl esters, pentachlorophenyl esters, tetrafluorophenyl esters difluorophenyl ester; monofluorophenyl ester; pentachlorophenyl ester, dichlorophenyl ester, tetrachlorophenyl ester, or 1- Hydroxybenzotriazole esters; triflates, mesylates, or tosylates 2-Ethyl-5-phenylisoxazolium-3'-sulfonate;Pyridyldis sulfides, or nitropyridyl disulfides; maleimides, haloacetates, acetyl Dicarboxylic acid group, carboxylic acid halide (fluoride, chloride, bromide, or iodide) Preferably, X and Y have one of the following structures: do:

[0119] [ka] JPEG2025138629000046.jpg34170

[0120] During the ceremony, X1' is F, Cl, Br, I, or Lv3; X2' is O, NH, N(R1), or CH2; R3 and R5 are independently H, R1, aromatic, heteroaromatic, one or several H atoms -R1, -halogen, -OR1, -SR1, -NR1R2, -NO2, -S(O)R1, an aromatic group substituted with -S(O)R or -COOR; Level 3 is methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoro Trifluoromethyl-sulfonyl(triflate), trifluoromethylsulfonate, nitrophen Noxyl, N-succinimidyloxy (NHS), phenoxyl; dinitrophenoxy pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl Sil, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl , 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl chlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy Sil, 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole- Generated using condensation reagents for the oxadiazol-yl (ODA), oxadiazol-yl, or Mitsunobu reaction is a leaving group selected from the intermediate molecule, wherein R and R are defined above. There is something.

[0121] Preferably, the bis-linker compound for preparing the conjugate further comprises the following formula (III-a ), (III-b), (III-c), (III-d), (III-e), (III-f ), (III-g), (III-h), (III-i), (III-j), (III-k ), (III-l), (III-m), (III-n), (III-o), (III-p ), (III-r), (III-s), (III-t), (III-u), (III-v ), and (III-w):

[0122] [ka] JPEG2025138629000048.jpg247161JPEG2025138629000049.jpg255150

[0123] In the formula, X7 and Y7 are independently CH, CH2, NH, O, S, NHNH, N(R1) and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. R1, X', Y', n, L1, and L2 are the same as above.

[0124] In another aspect, the present invention provides a labile bis-linker of formula (IV): The cytotoxic molecule and the cell-binding molecule can react with each other independently, simultaneously, or sequentially. to form formula (I):

[0125] [ka]

[0126] During the ceremony, [ka] , m1, L1, L2, Z1, and Z2 have the same definitions as in claim 1; Lv1 and Lv2 has the same definition as in formula (II), and X' and Y' have the same definition as in formula (III).

[0127] Preferably, the bis-linker for preparing the conjugate further has the following formula (IV-a): Vb), (IV-c), (IV-d), (IV-e), (IV-f), (IV-g), (IV-h), (IV-i), (IV-j), (IV-k), (IV-m), (IV-n ), (IV-o), (IV-p), (IV-q), (IV-r), and (IV-s) To be:

[0128] [ka] JPEG2025138629000053.jpg240161

[0129] During the ceremony, X7 and Y7 are independently CH, CH2, NH, O, S, NHNH, N(R1), and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. This means that [ka] , R1, X', Y', n, L1, and L2 are the same as above.

[0130] Functional groups X' and Y' capable of reacting with the amine or hydroxyl terminal groups of the drug / cytotoxic agent Examples of hydroxysuccinimide esters include, but are not limited to, N-hydroxysuccinimide esters, p- Nitrophenyl ester, dinitrophenyl ester, pentafluorophenyl ester , carboxylic acid chloride, or carboxylic acid anhydride. Possible examples include, but are not limited to, pyridyl disulfide, nitropyridyl disulfides, maleimides, haloacetic acids, methylsulfonephenyloxadiazoles (O DA), carboxylic acid chlorides, and carboxylic acid anhydrides. Ketones or alkane Examples of compounds that can react with the aldehyde end include, but are not limited to, amines, alkoxy groups, and the like. The azide terminal and the hydroxylamine terminal can be hydroxylamine, hydrazine, or acyloxylamine. The reaction can be, but is not limited to, an alkyne.

[0131] Preparation of conjugates

[0132] The conjugate of formula (I) can be prepared by reacting an intermediate compound of formula (II), (III), or (IV), respectively: Some preparations of formula (II) are structurally shown in Figures 1-40. Generally, to synthesize the conjugate of formula (I), first, 0 In an aqueous medium containing 1 to 99.5% organic solvent or in a 100% aqueous medium, The two functional groups on the cytotoxic molecule and the X' and Y' groups of the linker of formula (IV) are sequentially or simultaneously linked. The compound of formula (II) is then reacted with the methyl group to form a compound of formula (II). Alternatively, they can be immediately, simultaneously, or sequentially separated by mixing with water at 0 to 60°C. Compatible (miscible) organic solvents (DMA, DMF, ethanol, methanol, acetone, Nitriles, THF, isopropanol, dioxane, propylene glycol, or ethylene The cells were incubated in an aqueous medium of pH 5-9 with or without the addition of 0-30% of benzoyl alcohol, etc. through reduction of two or more residues on the binding molecule, preferably a disulfide bond of the cell-binding molecule The resulting pair of free thiols can be reacted to give compounds of formula (I).

[0133] Alternatively, the conjugate of formula (I) may be first heated at 0-60° C. in a water-miscible organic solvent. In an aqueous medium of pH 5 to 9 to which 0 to 30% of and reducing two or more residues on the cell-binding molecule, preferably a disulfide bond of the cell-binding molecule. and a pair of free thiols generated through the reaction to form a modified cell of formula (III). The thiol pair is preferably dissolved in a water-miscible organic solvent. In an aqueous medium of pH 4-9 with or without the addition of 0-30% dithiothreitol ( DTT), dithioerythritol (DTE), L-glutathione (GSH), Tris(2 -carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine (β-ME A), or / and β-mercaptoethanol (β-ME, 2-ME). Disulfide bonds reduced from the interchain disulfide bonds of the cell-binding agent by a reducing agent capable of Then, to obtain the conjugate of formula (I) after column purification or dialysis, The reactive groups X' and Y' (independently, disulfide, thiol, thioester, maleimide, Do, haloacetyl, azide, 1-yne, ketone, aldehyde, alkoxyamino, triflate ester, carbonylimidazole, tosylate, mesylate, 2-ethyl-5-phenyl Isoxazolium-3'-sulfonate, or nitrophenol, N-hydroxysuccinimide carboxylic ester of phenol; dinitrophenol, penta Fluorophenols, tetrafluorophenols, difluorophenols, monofluorophenols Phenol, pentachlorophenol, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, anhydride, or hydrazide groups, or other The acid ester derivative can be used.) is miscible with water at 0 to 60°C and in an organic solvent. Drug / cytotoxic agent in aqueous medium at pH 4-9.5 with or without 0-30% added solvent. The reactive groups on the drug / cytotoxic agent are accordingly capable of reacting with two groups on the The modified cell-binding molecules react in different ways with each other. For example, the cell-binding agents of formula (I) The linkage containing a disulfide bond in the drug conjugate is the same as the disulfide bond in the modified cell-binding agent of formula (III). This is achieved by disulfide exchange between the sulfide bond and a drug containing a free thiol group. The linkage containing a thioether bond in the cell-binding agent-drug conjugate of formula (I) may be a maleimide or The compound of formula (III) with a haloacetyl or ethylsulfonyl modified cell-binding agent and a free thiol This is achieved by the reaction between the drug containing the hydroxyl group and the acid-labile hydrazone in the conjugate. The linkage can be effected by methods known in the art, such as by carboxylation of the drug or the compound of formula (III), as appropriate. The compound is obtained by reacting the hydroxyl group with the hydrazide residue of a drug or a compound of formula (III). (See, e.g., P. Hamann et al., Hinman, LM, et al., Cancer Res. 53, 3336-334, 1993; B. Laguzza et al., J. Med. Chem., 32; 548-555, 1959; P. Trai (I et al., Cancer Res., 57; 100-105, 1997). Containing triazole bond in the conjugate The linkage can be effected via click chemistry (Huisgen cycloaddition) to either the drug or a compound of formula (II This is achieved by reacting the 1-yne group of the compound I) with the other azide residue. (Lutz, JF. et al, 2008, Adv. Drug Del. Rev. 60, 958-970; Sletten , EM et al. 2011, Acc. Chem. Research 44, 666-676). The linkage containing the oxime bond in the cell-binding agent-drug conjugate is a linkage containing the oxime bond of the drug or the compound of formula (III) The ketone or aldehyde of the modified cell-binding agent and the hydroxy group of the drug or modified cell-binding agent of formula (III) This is achieved by reacting the hydroxyamine group with the cell-binding molecule of formula (I). To obtain the thioether bond in the -drug conjugate, the thiol-containing drug is added at pH 5.5–9. In an aqueous medium at 0.0, a compound of the formula ( III) can react with the modified cell-binding molecule conjugate. To obtain the conjugate, the thiol-containing drug is reacted with a compound of formula (III) having a pyridyldithio moiety. Disulfide exchange can be performed with the modified cell-binding molecule conjugate. Ether or thiol To obtain modified drugs with ether bonds, drugs with hydroxyl or thiol groups are The agent reacts with halogens, particularly carboxylic acids, in the presence of a mild base, e.g., pH 8.0-9.5. The modified bridge linker of formula (III) can be reacted with an acid alpha halide. To obtain crosslinks, agents containing hydroxyl groups are dehydrated in the presence of a dehydrating agent such as EDC or DCC. and then condensing with a cross-linked compound of formula (IV) having a carboxyl group, and then reacting with a compound of formula (III The drug-modified bridge linker of interest can be conjugated to a cell-binding molecule. To obtain a bridged conjugate, an amino group-containing drug is reacted with a cell-binding molecule of formula (III): On the linker, NHS, imidazole, carboxyl ester of nitrophenol; N- Hydroxysuccinimide (NHS); Phenol; Dinitrophenol; Pentafluoro Phenol; Tetrafluorophenol; Difluorophenol; Monofluorophenol Pentachlorophenol; Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol Lachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2 -ethyl-5-phenylisoxazolium-3'-sulfonate .

[0134] Synthetic conjugates can be prepared by standard biochemical methods, e.g., by precipitation with Sephadex G25 or Sephadex G25. Gel filtration using acryl S300 column, adsorption chromatography, ion exchange, or can be purified by dialysis, and in some cases, small molecules ( For example, when small molecule drugs (e.g., folic acid, melanocyte-stimulating hormone, EGF, etc.) are conjugated, Chromatography, such as HPLC, medium pressure column chromatography, or ion exchange It can be purified by chromatography.

[0135] a pair of free thiols on a cell-binding molecule, preferably an antibody, and a cytotoxic molecule of formula (II) To achieve higher yields of the bis-linked conjugate, a small proportion of organic co-solvent or This may require the addition of a phase transfer agent. First, a cross-linking reagent (linker) of formula (II) at a high concentration, e.g., 1 to 500 mM, in a water-miscible polar organic solvent, e.g., methanol , ethanol, propanol, etc., different alcohols, acetone, acetonitrile, tetrahydrofuran Hydrofuran (THF), 1,4-dioxane, dimethylformamide (DMF), Dissolve in dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO). On the other hand, it is possible to obtain a solution containing 1 to 3% of the hydroxybenzoate in an aqueous buffer solution having a pH of 4 to 9.5, preferably 6 to 8.5. A cell-binding molecule such as an antibody dissolved at 5 mg / ml is added to 0.5 to 20 equivalents of TCEP or DT After reduction, DT was purified by SEC chromatography. TCEP can also be optionally removed by SEC chromatography. Alternatively, it can be retained in the reaction mixture for the next step reaction without purification. Furthermore, in order to achieve cross-linking of cell-binding molecules simultaneously with TCEP reduction, In addition, reduction of an antibody or other cell binding agent with TCEP results in the formation of a cross-linker of formula (II). can be done.

[0136] The aqueous solution for the modification of cell-binding agents has a pH between 4 and 9, preferably between 6.0 and 7.5. It can be buffered and contain non-nucleophilic buffer salts useful in these pH ranges. Agents include phosphate, acetate, triethanolamine HCl, HEPES, and MOP. S buffers, and further, for example, dextrin, sucrose, salts (e.g., NaCl, KC The solution containing the reduced cell-binding molecule may contain additional components such as a compound of formula (I). After addition of the cross-linker I), the reaction mixture is heated to 4°C to 45°C, preferably 15°C to ambient temperature. The progress of the reaction can be monitored by a decrease in absorbance at 254 nm or by the increase in absorbance at 280 nm. by measuring the increase in absorption at or other suitable wavelength. After the reaction is complete, the modified cell-binding agent can be isolated by conventional methods, e.g., gel electrophoresis. Filtration chromatography, ion exchange chromatography, silica gel or alumina Adsorption chromatography or column chromatography, crystallization, preparative thin layer chromatography This can be done by chromatography, ion exchange chromatography, or HPLC. .

[0137] The degree of modification can be determined by the UV spectrum emitted by nitropyridinethiones, dinitropyridines, and Lysine dithione, pyridine thione, carboxyamidopyridine dithione, and dicarboxy The development can be evaluated by measuring the absorbance of the thiamidopyridinedithione group. For conjugates without a chromophore group, the modification or conjugation reaction can be monitored by LC-MS, preferably UPL. Monitored by C-QTOF mass spectrometry or capillary electrophoresis-spectrometry (CEMS) The bridge linkers described herein may be any of the following with appropriate substituents: These have a variety of functional groups that can react with drugs, preferably cytotoxic drugs, such as amino or Modified cell-binding molecules with hydroxy substituents are prepared using N-hydroxysuccinimide (NHS) ) modified cell-binding molecules having thiol substituents that can react with drugs having esters; The molecule can react with drugs that have maleimide or haloacetyl groups. Modified cell-binding molecules with hydroxyl substituents (ketones or aldehydes) can be used in combination with hydrazides or aldehydes. Those skilled in the art will recognize that the available alkyloxyamines on the linker can react with drugs having alkyloxyamines. The linker to be used can be readily determined based on the known reactivity of the available functional groups.

[0138] Cell-binding agents

[0139] The cell-binding molecule Cb constituting the conjugate and modified cell-binding molecule of the present invention can be used therapeutically. or other biologically modified residues of the cell population to be modified. It may be any molecule that is currently known or that will be discovered.

[0140] Cell binding agents include, but are not limited to, large molecular weight proteins, such as full-length antibodies (PPs). Reclonal or monoclonal), dimeric, multimeric, multispecific antibodies (e.g., bispecific single-chain antibodies; antibody fragments, e.g., Fab, Fab', F(ab')2, Fv [Parham, J. Immunol. 131, 2895-2902 (1983)], F Fragments obtained by ab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs, bispecific antibodies, trispecific antibodies, cancer cell antigens, viral antigens, microbial antigens, or The immunoglobulin is capable of recognizing and binding to a specific antigen or exhibiting a desired biological activity. epitope-binding fragments of any of the foregoing that immunospecifically bind to a protein produced in an immune system; Interferons (e.g., types I, II, III); 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 stimulator estrogen-releasing hormone), MSH (metastatic hormone), or androgen, estrogen steroid hormones such as steroids (e.g., steroid hormones) or melanocyte-stimulating hormone (MSH); growth factors and and colony-stimulating factors, e.g., epidermal growth factor (EFG), granulocyte-macrophage colony-stimulating factors stimulating factors (GM-CSF); transforming growth factors (TGF), e.g., TGF α, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II) G -CSF, M-CSF, and GM-CSF [Burgess, Immunology T Today, 5, 155-158 (1984)]; Vaccine Growth Factor (VGF); Fibroblast Growth Factor (FGF) Follicular growth factors (FGFs); small molecular weight proteins, polypeptides, peptides, and peptide phosphatase inhibitors steroids, such as bombesin, gastrin, and gastrin-releasing peptide; platelet-derived Growth factors; interleukins and cytokines, e.g., interleukin-2 (IL-2 ), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony -stimulating factor (GM-CSF); vitamins such as folic acid; apoproteins and glycoproteins, e.g. For example, transferrin [O'Keefe et al, J. Bio. Chem. 260 , 932-927 (1985)]; carbohydrate-binding proteins such as lectins or lipoproteins; vesicle nutrient transport molecules; and small molecule inhibitors, e.g., inhibition of prostate-specific membrane antigen (PSMA) drugs, small molecule tyrosine kinase inhibitors (TKIs), non-peptides, or other cell-binding molecules or materials, such as bioactive polymers (Dhar, et al., Proc. Natl. A cad.Sci.2008,105,17356-61), bioactive dendrimers (Le e,et al,Nat.Biotechnol.2005,23,1517-26;A lmutairi,et al;Proc.Natl.Acad.Sci.2009,1 06,685-90), nanoparticles (Liong, et al, ACS Nano, 200 8,19,1309-12;Medarova,et al,Nat.Med.2007 ,13,372-7;Javier,et al,Bioconjugate Chem .2008,19,1309-12), liposomes (Medinai,et al,Cu rr. Phar. Des. 2004, 10, 2981-9), viral capsid (Fle nniken,et al,Viruses Nanotechnol.2009,32 7,71-93).

[0141] Generally, if a suitable monoclonal antibody is available, it can be used to target the cell surface. Antibodies are preferred as binding molecules. Antibodies may be murine, human, humanized, chimeric, or derived from other species. So that's fine.

[0142] The antibodies used in the present invention can be produced using in vivo or in vitro production processes. The present invention also includes a method for preparing an anti-receptor peptide polyclonal antibody. is well known, for example, as shown in U.S. Pat. No. 4,493,795 (Nestor et al.). A typical method for preparing monoclonal antibodies is to generate antibodies from mice immunized with a specific antigen. This method involves fusing isolated mouse spleen cells with myeloma cells (Kohler ,G;Milstein,C.1975.Nature 256:495-497). Details For the correct procedure, see Antibodies-A Laboratory Manual. l, Harlow and Lane, eds., cold spring harbo Laboratory Press, New York (1988) The contents of this document are incorporated herein by reference. by immunizing a mouse, rat, hamster, or other mammal with the antigen of interest, Monoclonal antibodies can be obtained and used to target the antigen of interest, e.g., intact target cells. , antigens isolated from target cells, whole viruses, weakened whole viruses and viral proteins PEG6000 is used to fuse spleen cells with myeloma cells. The hybridomas obtained after the hybridization were screened using their sensitivity to HAT. Hybridomas producing monoclonal antibodies useful in the practice of the present invention are those that bind to specific targets. These antibodies are identified by their immunoreaction with target cell receptors or by their inhibition of receptor activity.

[0143] The monoclonal antibodies used in the present invention are those that secrete antibodies with appropriate antigen specificity. Initiate a culture of monoclonal hybridoma cells in a nutrient medium containing hybridoma cells. In the culture, the hybridoma cells release the antibody into the culture medium. It is necessary to maintain sufficient time and conditions for secretion. After collecting the antibody-containing culture supernatant, , well-known techniques, e.g., Protein A affinity chromatography; anion exchange chromatography; Chromatography, Cation Exchange Chromatography, Hydrophobic Interaction Chromatography and molecular sieve chromatography (especially affinity chromatography using antigen-crosslinking protein A) chromatography and molecular sieve chromatography; centrifugation, sedimentation, or other techniques The antibodies can be isolated by standard methods for purifying proteins.

[0144] Media useful for the preparation of these compositions are well known in the art and commercially available. and includes synthetic synthetic media. Exemplary synthetic media include Dulbecco's Minimum Essential Medium (DMEM) and MEM; Dulbecco et al., Virol. 8:396 (1959)), 4.5 g / L glucose, 0-20mM glutamine, 0-20% fetal bovine serum, ppm amounts of Some heavy metals (e.g., Cu, Mn, Fe, or Zn) or / and heavy metals added in salt form and antifoaming agents (e.g., polyoxyethylene-polyoxypropylene block copolymers) body).

[0145] In addition to cell fusion technology, we have also constructed cell lines for antibody production using the following methods: For example, direct transformation of B lymphocytes by oncogenic DNA can tion, or oncogenic viruses, such as Epstein-Barr virus (EBV, human Also known as herpes simplex virus 4 (HHV-4) or Kaposi's sarcoma-associated virus Transfection of the virus (KSHV) is described in detail in U.S. Patent No. 43417 61;4399121;4427783;4444887;4451570;44669 17;4472500;4491632;4493890). Monoclonal antibodies can be prepared by known methods using anti-receptor peptides or peptides containing terminal carboxyl groups. (For details, see Niman et al. Proc. Natl. Acad. Sci. USA,80:4949-4953(1983);Geysen et al. Proc.N atl. Acad. Sci. USA,82:178-182(1985); Lei et al. (See Chemistry 34(20):6675-6688(1995)). The anti-receptor polypeptide or polypeptide analog is an anti-receptor polypeptide of a monoclonal antibody. Used alone or conjugated to a cross-linked immunogenic carrier as an immunogen to prepare peptides. It can be used.

[0146] To produce monoclonal antibodies as binding molecules of the present invention, other well-known production methods can be used. Among them, the method of producing fully human antibodies has attracted particular attention. Display technology uses affinity selection to select antibodies specific to a known antigen from a fully human antibody library. Phage display technology itself is widely used in the literature to obtain fully human antibodies that bind to specific antigens. , vector construction, and library screening are described in detail. Dente et al. Gene. 148(1):7-13 (1994); Little et al. Biotechnol Adv.12(3):539-55(1994);Clacks on et al. Nature 352:264-628(1991); Huse et al. Scien See ce 246:1275-1281 (1989).

[0147] Monoclonal antibodies derived from non-human species (e.g., mice) using hybridoma technology For cloned antibodies, humanization is performed to avoid human anti-mouse antibodies when administered to humans. Among these, the well-known method for antibody humanization is the method of humanizing the complementarity-determining region (CDR) of the antibody. For more information, see U.S. Patent Nos. 5,859,205 and 6,859,205. , No. 797, 492; Liu et al., Immunol Rev. 222:9-27 (2008 );Almagro et al., Front Biosci.1;13:1619-33(200 8);Lazar et al., Mol Immunol.44(8):1986-98(2007 );Li et al. Proc.Natl.Acad.Sci.USA.103(10):355 7-62 (2006), the disclosure of which is incorporated by reference. The transgenic mouse, U, possesses most of the human immunoglobulin light and heavy chains. It can be prepared by immunizing herons, monkeys, and other mammals with an antigen. Examples of such mice include Xenomouse (Abgenix, Inc.), HuM ab-Mouse(Medarex / BMS), VelociMouse(Regene For details, see U.S. Patent Nos. 6,596,541, 6,207,418, No. 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,5 Nos. 45, 5,661,016, 5,545,806, 5,436,149 and 5, See US Pat. No. 569,825. In the course of human therapy, mouse antibody variable region genes and human antibody The immunogenicity of chimeric antibodies produced in the human body by integrating the human constant region genes is Much lower than mouse antibodies (Kipriyanov et al., Mol Biotech nol.26:39-60(2004);Houdebine,Curr Opin B Biotechnol. 13:625-9 (2002)). The disclosure of said document is incorporated by reference. Furthermore, site-directed mutagenesis of antibody variable regions can be used to improve the quality of antibody parental sequences. This can improve compatibility and specificity (Brannigan et al., Nat Rev M ol Cell Biol.3:964-70(2002);Adams et al., J.Imm Univ. Methods. 231:249-60 (1999)). By replacing the cytotoxicity of the cytotoxicity domain with the cytotoxicity of the immune effector cells, the cytotoxicity of the immune effector cells can be effectively promoted. The toxic effect can be enhanced.

[0148] Immunospecific antibodies against malignant cell antigens can be obtained commercially or by several conventional techniques. For example, they can be obtained by chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding immunospecific antibodies to Other similar databases, commercial sources, published literature, or routine cloning and It can be obtained by sequencing.

[0149] Besides antibodies, polypeptides or proteins can also be used as binding molecules to target cell surfaces. Interact with the corresponding receptor or epitope by binding, blocking, attacking, or other means These peptides or proteins bind to epitopes or their corresponding receptors. They do not have to belong to the immunoglobulin family, as long as they can bind effectively. The polypeptides are also isolated by a technique similar to that of phage display antibodies (Sza et al., rdenings,J Recept Signal Transduct Res.2 003;23(4):307-49). Peptides obtained from a random peptide library The polypeptide or protein fragments are similar in application to antibodies and antibody fragments. The molecule binds to the antigen by connecting to some macromolecule or medium via a binding molecule. These macromolecules include, but are not limited to, albumin, polymers, , liposomes, nanoparticles, or dendrimers.

[0150] The conjugates of the invention are used to treat cancer, autoimmune diseases, and / or infectious diseases. Antibodies for use in conjugating drugs include, but are not limited to, the following: 8 (anti-GD2 antibody), abagovomab (anti-CA-125 antibody), abciximab (anti-CD4 1 antibody (integrin α-IIb), adalimumab (anti-TNF-α antibody), adalimumab (anti-EpCAM antibody, CD326), afelimomab (anti-TNF-α); afutuzumab (anti-CD20 antibody), alacizumab pegol (anti-VEGFR2 antibody), body), ALD518 (anti-IL-6 antibody), alemtuzumab (also known as campath, mabca) Ampath, anti-CD52 antibody), altumomab (anti-CEA antibody), anatumomab (anti-tag- 72 antibody), anrukinzumab (IMA-638, anti-IL-13 antibody), apolizumab ( anti-HLA-DR antibody), arcitumomab (anti-CEA antibody), acelizumab (anti-L-ceramide Antibody to cytokinin (CD62L), Atlizumab (also known as tocilizumab, ac Temra, RoActemra, anti-IL-6 receptor antibody), Atorolimumab ( anti-rhesus factor antibody), bapineuzumab (anti-β-amyloid antibody), basiliximab ( Simulect, anti-CD25 (IL-2 receptor α chain) antibody), Bavituximab ) (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), bertilimumab (anti-CCL11 Eotaxin-1 antibody), besilesomab (also known as scintimun, anti-CEA-related antigen antibody) , bevacizumab (also known as Avastin, an anti-VEGF antibody), biciromab (also known as FibriScint , anti-fibrin II beta chain antibody), bivatuzumab (anti-CD44v6 antibody), blinatumomab (blinatumomab) (also known as BiTE, anti-CD19 antibody), brentuximab b) (cAC10, anti-CD30 TNFRSF8 antibody), Briakinumab (anti-IL-12, IL-23 antibody), canakinumab (also known as Ilaris, anti-IL-1 antibody) body), cantuzumab (also known as C242, anti-CanAg antibody), capromab, Catumaxomab (also known as removab, 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 Erbitux, IMC-C 225, anti-EGFR antibody), sitatuzumab (anti-EpCAM antibody), cixutumbam ( tumumab (anti-IGF-1 antibody), clenoliximab (anti-CD4 antibody), clivatuzumab (Clivatuzumab) (anti-MUC1 antibody), Conatumumab (anti-TRAIL- R2 antibody), CR6261 (anti-influenza A hemagglutinin antibody), dacetuzumab ( dacetuzumab) (anti-CD40 antibody), daclizumab (also known as Zenapax, anti-CD25C (IL -2 receptor alpha chain) antibody), daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase antibody), denosumab (also known as Prolia, anti-RANKL antibody) , detumomab (anti-B-lymphoma cell antibody), dorlimomab, dorxizumab ab), Ecromeximab (anti-GD3 ganglioside antibody), Eculizumab (also known as Soliris, anti-C5 antibody), edovacomab (anti-endotoxin antibody), edreco Romab (also known as Panorex, MAb17-A1, anti-EpCAM antibody), Efalizumab (also known as Name: Raptiva, anti-LFA-1 (CD11a) antibody), efungumab (also known as :Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody) , Elsilimomab (anti-IL-6 antibody), Enlimomab pegol (anti-ICA M-1 (CD54) antibody), Epitumomab (anti-episialin antibody), Ratuzumab (anti-CD22 antibody), Erlizumab (anti-ITGB2 (CD18 antibody) ) antibody), Ertumaxomab (also known as Rexomun, anti-HER2 / neu, C D3 antibody), etaracizumab (also known as Abegrin, anti-integrin αvβ3), exibibi Ilumab (anti-hepatitis B surface antigen antibody (HBs antibody)), Fanolesomab ( Other names: NeutroSpec, anti-CD15 antibody, faralimomab antibody (anti-interacting antibody) -Ferron receptor antibody), Farletuzumab (anti-folate receptor 1 antibody), Felvizumab (antibody against respiratory syncytial virus), Fezakinumab numab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor antibody) ), Fontolizumab (anti-IFN-γ antibody), Foravirumab ab) (anti-rabies virus glycoprotein antibody), fresolimumab (anti-TGF -β antibody), Galiximab (anti-CD80 antibody), Gantenerum ab) (anti-β-amyloid antibody), Gavilimomab (anti-CD147 (basigin) antibody body), gemtuzumab (anti-CD33 antibody), girentuximab (anti-decarboxylase antibody) Water enzyme 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 ( Other names: Indimacis-125, anti-CA-125 antibody), imciromab (other names: Myos cint, an anti-cardiac myosin antibody), infliximab (also known as Remicade, an 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 (TNF RSF8 antibody), keliximab (anti-CD4 antibody), labetuzumab (also known as :CEA-Cide, anti-CEA antibody), lebrikizumab (anti-IL-13 antibody), Lemalesomab (anti-NCA-90 (granulocyte antigen) antibody), lerdelimumab (Lerdelimumab) (anti-TGFβ-2 antibody), Lexatumumab (anti-TRAP IL-R2 antibody), ribivirumab (anti-hepatitis B surface antigen antibody), Linz Lintuzumab (anti-CD33 antibody), Lucatumumab (anti-CD40 antibody) antibody), Lumiliximab (anti-CD23 (IgE receptor) antibody), Patumumab (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), Milatsuzu Milatuzumab (anti-CD74 antibody), Minretumomab (anti-TAG -72 antibody), Mitumomab (also known as BEC-2, anti-ganglioside antibody- GD3), Morolimumab (anti-rhesus factor antibody), Motavizumab vizumab (also known as Numax, anti-RS virus antibody), muromonab-CD3 (also known as Name: Orthoclone OKT3, anti-CD3 antibody), Nacolomab (anti-C242 antibody), Naptumomab (anti-5T4 antibody), natalizumab (also known as Tysabri, anti-insulin Tegrin α4 antibody), Nebacumab (anti-endotoxin antibody), Necitumma Necitumumab (anti-EGFR antibody), Nerelimomab (anti-TNF-α antibody), body), nimotuzumab (also known as Theracim, Theraloc, anti-EGFR antibody), nofetumomab ( Nofetumomab), ocrelizumab (anti-CD20 antibody), odulimomab (also known as afolimoma) b, anti-LFA-1 (CD11a) antibody), ofatumumab (also known as Arzerra, anti-CD20 antibody Olaratumab (anti-PDGF-Rα antibody), Omalizumab uba) (also known as Xolair, anti-IgE Fc antibody), oportuzumab (anti EpCAM antibody), Oregovomab (also known as OvaRex, anti-CA-125 antibody) , Otelixizumab (anti-CD3 antibody), Pagibaximab ) (anti-LTA antibody), Palivizumab (also known as Synagis, Abbosynagis, anti-RS virus antibody) ), panitumumab (also known as Vectibix, ABX-EGF, anti-EGFR antibody), panobacumab Panobacumab (anti-Pseudomonas aeruginosa antibody), Pascolizumab (anti-IL-4 antibody) ), Pemtumomab (also known as Theragyn, anti-MUC1 antibody), Pertuzumab ( Other names: Omnitarg, 2C4, anti-HER2 / neu antibody, pexelizumab (anti-C5 antibody), pintumomab (anti-adenocarcinoma antigen antibody), priliximab (Pr iliximab (anti-CD4 antibody), pritumumab (anti-vimentin antibody), PR O140 (anti-CCR5 antibody), racotumomab (also known as: 1E10, anti-(N -Glycolylneuraminic acid (NeuGc, NGNA)-ganglioside (GM3) antibody) , Rafivirumab (anti-rabies virus glycoprotein antibody), Ramucirumab (R amucirumab) (anti-VEGFR2 antibody), ranibizumab (also known as Lucentis, anti-VEGF-A antibody), Raxibacumab (anti-anthrax toxin, protective antigen antibody), Rega Burma Regavirumab (anti-CMV glycoprotein B antibody), reslizumab (anti IL-5 antibody), rilotumumab (anti-HGF antibody), rituximab (also known as :MabThera, Rituxanmab, anti-CD20 antibody), Robatumumab (anti-IGF -1 receptor antibody), rontalizumab (anti-IFN-α antibody), rovelizumab Rovelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), Ruplizumab ( uplizumab) (also known as Antova, anti-CD154 (CD40L) antibody), Satumomab (anti-CD154 (CD40L) antibody), ab) (anti-TAG-72 antibody), Sevirumab (anti-CMV antibody), Sibrotuz Mab (anti-FAP antibody), Sifalimumab (anti-IFN-α antibody), Siltz Siltuximab (anti-IL-6 antibody), Siplizumab (anti-CD2 antibody), (Smart MI95 (anti-CD33 antibody), solanezumab (anti-β-amyloid antibody) Sonepcizumab (anti-sphingosine-1-phosphate antibody), Sontz Sontuzumab (anti-episialin antibody), Stamulumab (anti-myosin tatin antibody), sulesomab (also known as LeukoScan, anti-NCA-90 (granulocyte Antigen) antibody), Tacatuzumab (anti-alpha-fetoprotein antibody), Tadocizumab (anti-integrin αIIbβ3 antibody), talizumab (anti-I gE antibody), tanezumab (anti-NGF antibody), taplitumomab b) (anti-CD19 antibody), tefibazumab (also known as Aurexis, anti-clampicin) Anti-Tenatumomab (anti-Tenatumomab), anti ... SynC antibody), Teneliximab (anti-CD40 antibody), Teplizumab lizumab) (anti-CD3 antibody), TGN1412 (anti-CD28 antibody), ticilimumab (also known as :Tremelimumab, anti-CTLA-4 antibody), Tigatuzumab (anti-TRAI antibody), Tigatuzumab (anti-TRAI L-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-C TLA-4 antibody), Tucotuzumab celmoleukin (anti-EpC AM antibody), tuvirumab (anti-hepatitis B antibody), urtoxazumab ab) (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), Veltuzumab (anti CD20 antibody), Vepalimomab (anti-AOC3 (VAP-1)) antibody), Visilizumab (also known as Nuvion, an anti-CD3 antibody), Vitaxin (anti-angiogenic integrin a vb3 antibody), Volociximab (anti-integrin α5β1), Votumma Votumumab (also known as HumaSPECT, anti-tumor antigen CTAA16.88 antibody), Zaltumab HuMax-EGFr (anti-EGFR antibody), zanolimumab (anti-CD4 antibody), 4 antibody), Ziralimumab (anti-CD147 (basic immunoglobulin) antibody) , zolimomab (anti-CD5 antibody), etanercept (registered trademark "Enbrel"), Alefacept (registered trademark "Amevive"), abatacept (registered trademark "Oren") cia"), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron-regulating Protein 2) antibody], 14G2a (from Nat. Cancer Inst. for melanoma and solid tumors) anti-ganglioside GD2 antibody), J591 (from Weill Cornell Medical School for prostate cancer) Anti-PSMA antibodies for treating melanoma, 225.28S [anti-HMW-MA for melanoma A (high molecular weight melanoma-associated antigen) antibody, Sorin Radiofarmaci SRL (Milan, Italy). ], COL-1 (Anti-CEACAM3 antibody for colorectal cancer and gastric cancer from Nat. Cancer Institute) , CGM1), CYT-356 (registered trademark "Oncoltad", prostate cancer), HNK20 (Ora Vax Inc. for respiratory syncytial virus), ImmuRAIT (IMMUNOMEDICS for non-Hodgkin's for lymphoma), Lym-1 (anti-HLA-DR10 antibody, from Peregrine Pharm MAK-195F [anti-TNF-α from Abbott / Knoll for sepsis and toxic shock] (Tumor necrosis factor; TNFA, TNF-α; TNFSF2) antibody], MEDI-500 [another Name: T10B9, an anti-CD3 antibody for graft-versus-host disease from MedImmune Inc., TRαβ (T cell receptor α / β), RING SCAN [from Neoprobe Corp. for breast cancer, colon cancer, and colorectal cancer anti-TAG72 (tumor-associated glycoprotein 72) antibody for intestinal cancer)], Avicidin ( Anti-EPCAM (epithelial cell adhesion molecule) antibody, anti-TACSTD1 (tumor-associated calcium signaling Neural 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; from NeoRx Corp. for colon, ovarian, prostate, and non-Hodgkin's cancers CD326 for lymphoma; LYMPHOCIDE (IMMUNOMEDICS, NJ), Smart ID 10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTran splant, CA), anti-VEGF antibody (Genentech, CA); CEAcide (Immunome dics, NJ), IMC-1C11 (ImClone Systems, NJ), and cetuximab (ImC lone, NJ).

[0151] Other antibodies as cell binding molecules / ligands include, but are not limited to, the following antigens: Contains antibodies against: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian), 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 lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythema) tos), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD 38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, CD66e (cancer), CD70 (metastatic renal cell carcinoma and liquid tumors, multiple myeloma) CD74 (multiple myeloma), CD80 (lymphoma), C D98 (cancer), mucin (carcinoma), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer) , CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors) cancer), 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, Hemoglobin (hemoglobin, solid tumors), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecules, bladder, head and neck, colon cancer, NHL prostate cancer, and ovarian cancer), ERBB2 ( Epidermal growth factor receptor 2 (lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (Folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28G (cell surface glycosphingolipid) tumor, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 ( Lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (NHL), HL A-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor-1 receptor, solid tumors, blood cancers), IL-2 receptor (interleukin-2 receptor) Interleukin-2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin-6 receptor body, multiple myeloma, RA, Castleman's disease, IL6-dependent tumors), integrins (various αVβ3, α5β1, α6β4, αIIβ3, α5β5, αVβ5) for cancer of MA MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma) ), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (transmembrane 4-domain Main family A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or M UC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA12 5) (ovarian cancer), CEA (colon), gp100 (melanoma), MART1 (melanoma), MP G (melanoma), MS4A1 (transmembrane 4-domain family A member 1), small cell lung cancer, NHL), nucleolin, neurooncogene product (carcinoma), P21 (carcinoma), anti-(N-glucan Paratope of cholylneuraminic acid (breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase Tase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate), ROBO4, T AG72 (tumor-associated glycoprotein 72, leukemia (AML), gastric cancer, colorectal cancer, ovarian cancer) , T cell transmembrane protein (cancer), Tie (CD202b), TNFRSF10B (tumor Tumor necrosis factor receptor superfamily member 10B (cancer), TNFRSF13B (tumor Necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, R A and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TN F-related apoptosis ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCA M-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 immunot her. 54 (3), 187-207 (2005) Franke et al, cancer biother radiopharm. 2000, 15, 459-76).

[0152] The cell binding agent, more preferably an antibody, may bind to tumor cells, virus-infected cells, microbial-infected cells, Parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or mesenchymal stem cells It can be any agent capable of combating lanocytes. More specifically, A binding agent is any agent that can target any one of the following antigens or receptors: Agents / molecules can be: CD2, CD2R, CD3, CD3gd, CD3e, CD 4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD1 1a, CD11b, CD11c, CD12, CD12w, CD13, CD14, CD15 , CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17 , CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25 , CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33 , CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41 , CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R、CD45、CD45RA、CD45RB、CD45RO、CD46、CD4 7、CD47R、CD48、CD49a、CD49b、CD49c、CD49e、CD4 9f、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 、CD74、CD75、CD75s、CD76、CD77、CD78、CD79、CD7 9a、CD79b、CD80、CD81、CD82、CD83、CD84、CDw84、 CD85、CD86、CD87、CD88、CD89、CD90、CD91、CD92、 CDw92、CD93、CD94、CD95、CD96、CD97、CD98、CD99 、CD99R、CD100、CD101、CD102、CD103、CD104、CD1 05、CD106、CD107、CD107a、CD107b、CD108、CD109 、CD110、CD111、CD112、CD113、CDw113、CD114、CD 115、CD116、CD117、CD118、CD119、CDw119、CD120 a、CD120b、CD121a、CD121b、CDw121b、CD122、CD1 23、CDw123、CD124、CD125、CDw125、CD126、CD127 、CD128、CDw128、CD129、CD130、CD131、CDw131、C D132、CD133、CD134、CD135、CD136、CDw136、CD13 7、CDw137、CD138、CD139、CD140a、CD140b、CD141 、CD142、CD143、CD144、CD145、CDw145、CD146、CD 147、CD148、CD149、CD150、CD151、CD152、CD153、 CD154、CD155、CD156a、CD156b、CDw156c、CD157, CD158a、CD158b、CD159a、CD159b、CD159c、CD1 60、CD161、CD162、CD162R、CD163、CD164、CD165、 CD166、CD167、CD167a、CD168、CD169、CD170、CD1 71、CD172a、CD172b、CD172g、CD173、CD174、CD17 5、CD175s、CD176、CD177、CD178、CD179、CD180、C D181、CD182、CD183、CD184、CD185、CD186、CDw18 6、CD187、CD188、CD189、CD190、CD191、CD192、CD 193、CD194、CD195、CD196、CD197、CD198、CDw198 、CD199、CDw199、CD200、CD200a、CD200b、CD201、 CD202、CD202b、CD203、CD203c、CD204、CD205、CD 206、CD207、CD208、CD209、CD210、CDw210、CD212 、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、 CD220、CD221、CD222、CD223、CD224、CD225、CD22 6、CD227、CD228、CD229、CD230、CD231、CD232、CD 233, CD234, CD235a, CD235ab, CD235b, CD236, CD 236R, CD238, CD239, CD240, CD240CE, CD240D, CD 241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD25 7, CD258, CD261, CD262, CD263, CD265, CD266, CD 267, CD268, CD269, CD271, CD273, CD274, CD275, CD276(B7-H3), CD277, CD278, CD279, CD280, CD2 81, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD30 0a, CD300c, CD300e, CD301, CD302, CD303, CD304 , CD305, CD306, CD309, CD312, CD314, CD315, CD3 16, CD317, CD318, CD319, CD320, CD321, CD322, C D324, CDw325, CD326, CDw327, CDw328, CDw329, C D331, CD332, CD333, CD334, CD335, CD336, CD337 , CDw338, CD339, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activation 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 antibody Hara, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAF F (B-cell activating factor), 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), C CR4 (CC chemokine receptor type 4, CD194), CCR5, CD3E (epsilon) , CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (Claude fin-18), clumping factor A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte macrophage Dicolony stimulating factor (GM-CSF)), CSP4, CTLA4 (cytotoxic T lymphocytes) Related protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), CXC Chemokine receptor type 4, cADP-ribose hydrolase, Cyclin B1, CYP1 B1, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, D LL3 (Delta-like ligand 3), DLL4 (Delta-like ligand 4), DPP4 (Dipeptide Dilpeptidase 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, E GFRII, EGFRvIII, endoglin, endothelin B receptor, endotoxin , EpCAM (epithelial cell adhesion molecule), EphA2, episialin, ERBB2 (epithelial growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), Escherichia coli, E TV6-AML, FAP (fibroblast activation protein α), FCGR1, α-fetoprotein Protein, fibrin II, β chain, fibronectin ectodomain B, FOLR (folate receptor) folate receptor α, folate hydrolase, respiratory syncytial virus Fos-related antigen 1F protein Protein, Frizzled receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell Surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycol Gluneuraminic acid, GM3, GMCSF receptor α chain, growth differentiation factor 8, GP100, GPN MB (transmembrane protein NMB), GUCY2C (guanylate cyclase 2C, guanylate Guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase-C receptor Enterotoxin receptor (hSTAR), heat shock protein, hemocoagulation agglutinin, 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 / scatter factor), HHGFR, HIV-1, histone complex, HLA-D R (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic villus Gonadotropin, HNGF, human cell scatter factor receptor kinase, HPV E6 / E7, H sp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1 R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza IgE, IgE Fc region, IgE, 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 (IL-31RA), insulin-like growth factor 2), integrin (α4, α IIIb β3, αvβ3, α4β7, α5β1, α6β4, α7β7, αIIβ3, α5β5, αvβ5), interferon Gamma-inducing protein, ITAGA2, ITGB2, KIR2D, Kappa Ig, LCK , Le, legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-associated antigen 1), CD11 a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LTA, MA D-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAG EA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (macrophage Migration inhibitory factor or glycosylation inhibitor (GIF), MS4A1 (transmembrane 4 domain subunit phospholipase A member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface related (MUC1) or Polymorphic epithelial mucin (P EM), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein transmembrane type 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (transmembrane type 4) domain subfamily A), MYCN, myelin-associated glycoprotein, myostatin, N A17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22 ME), NGF, neuronal apoptosis-regulating proteinase 1, NOGO-A, Notch receptor Physiology, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX- 40, Oxidized low-density lipoprotein (OxLDL), OY-TES1, P21, and p53 unchanged Variant, P97, Page4, PAP, anti-(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed cell death protein) protein1), PDGF-Rα, (platelet-derived growth factor receptor α), PDGFR-β, PD L-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived growth factor receptor Body β, sodium phosphate cotransporter, PMEL17, polysialic acid, proteinase 3 (P R1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhP I)), Rhesus factor, RANKL, PhoC, Ra s mutant, RG55, ROBO4, respiratory syncytial virus, RON, ROR1, sarcoma metastasis break Point, SART3, sclerostin, SLAMF7 (SLAM family member 7) ), selectin P, SDC1 (syndecan 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX 2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG- 22 (tumor-associated glycoprotein 72), survivin, T-cell receptor, T-cell transmembrane protein Streptococcus aureus, 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 (trophoblast glycoprotein) , TRAIL-R1 (tumor necrosis-inducing ligand receptor 1), TRAILR2 ( Death receptor 5 (DR5), tumor-associated calcium signal transducer 2, MU Tumor-specific glycosylation of C1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF-2(C D309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or is a cell expressing any insulin growth factor receptor, or any epidermal growth factor receptor.

[0153] In another specific embodiment, the cell-binding ligand-drug conjugate of the present invention via the linker is used in the treatment of cancers, including but not limited to: Renal cortical carcinoma, anal cancer, bladder cancer, brain tumors (adult, brainstem glioma, children, cerebellar astrocytoma, brain Astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and visual Subfloor glioma), breast cancer, carcinoid tumor, gastrointestinal, carcinoma of unknown primary origin, cervical carcinoma, colon carcinoma, Endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family tumor (PNET), extracranial malignancy Germ cell tumors, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumors, extragonadal, pregnancy nutrition Membrane tumor, head and neck cancer, hypopharyngeal cancer, pancreatic islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal carcinoma, leukemia ( acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lips and oral cavity Cancer, liver cancer, lung cancer (non-small cell, small cell), lymphoma (AIDS-related, central nervous system, skin T-cell Cells, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, metastasis of unknown primary origin Metastatic squamous neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome , myeloproliferative syndrome, nasopharyngeal carcinoma, neuroblastoma, oral cancer, pharyngeal cancer, osteosarcoma, ovarian cancer (epithelial, Germ cell tumors, low malignant potential tumors), pancreatic cancer (exocrine gland, islet cell carcinoma), paranasal sinus and and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary cancer, plasmacytoma, prostate cancer, Chariosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Sézary disease syndrome, skin cancer, skin cancer (cutaneous T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft tissue cancer Osteosarcoma, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), childhood cancer These include common cancers, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0154] In another specific embodiment, the cell-binding-drug conjugate of the present invention is Therefore, it can be used for the treatment or prevention of autoimmune diseases. Autoimmune gastric achlorhydria, chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhage Leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, Ankylosing spondylitis, anti-GMB / TBM nephritis, antiphospholipid syndrome, antisynthetase syndrome , arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, auto Immune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune Lymphoproliferative syndrome, autoimmune peripheral nervous system disease, autoimmune pancreatitis, multiple autoimmune Endocrinopathy type I, II, III, autoimmune progesterone dermatitis, autoimmune thrombocytopenia Purpura purpura, autoimmune uveitis, Barlow's disease / Barlow's concentric sclerosis, Behcet's disease disease, Berger's disease, Bickerstaff encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, Chagas disease, chronic fatigue and immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy Neuropathy, chronic recurrent multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergies Granulomatous vasculitis, cicatricial pemphigoid, celiac disease, Cogan's syndrome, cold agglutinin disease, Body component C2 deficiency, cephalic arteritis, CREST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, cutaneous leukocytoclastic vasculitis, malignant atrophic papulopathy, painful steatosis, herpetiform skin dermatitis, dermatomyositis, type 1 diabetes, diffuse cutaneous scleroderma, myocardial infarction, discoid lupus erythematosus, eczema Eruptions, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum , idiopathic mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, fibrodysplasia Fibromyalgia, fibromyositis, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, renal bulbitis corpus nephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto Thyroiditis, hemolytic anemia, allergic purpura, herpes gestationis, hidradenitis suppurativa, Hughes' syndrome group (antiphospholipid syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic Pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy ( Berger's disease), inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymph node syndrome, lumbar Todd-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA Disease (LAD), Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-like hepatitis, lupus erythematosus , Blau syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed Syncytial connective tissue disease, scleroderma, Mucha-Jakob disease, Muckle-Wells syndrome, multiple myeloma , multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), Neuromuscular, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, thyroid inflammation, relapsing rheumatism, Panda syndrome (a childhood autoimmune neuropsychiatric disorder complicated by streptococcal infection) , tumor cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, parsonage Georgia syndrome, pars planus inflammation, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis , POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary liver cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, gangrenous purulence erythroderma, pure red cell aplastic anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Raynaud's disease Iter's syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis Idosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, spondyloarthropathy, sticky blood syndrome, Still's disease, stiff-man syndrome Skin, subacute bacterial endocarditis, Susac syndrome, acute febrile neutrophilic dermatosis, Sydenham chorea , sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse Myelitis vulcanis, ulcerative colitis (a type of idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spinal cord Spondyloarthropathy, vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, Westcott-A This includes Rudrich's syndrome.

[0155] In another specific embodiment, the bis-linkages of the present invention for the treatment or prevention of autoimmune diseases Linking molecules used for interbody conjugation include, but are not limited to, anti-elastic Antibody; Anti-epithelial cell antibody; Anti-basement membrane type IV collagen protein antibody; Anti-nuclear antibody Anti-double-stranded DNA antibody, anti-single-stranded DNA antibody, anti-cardiolipin antibody IgM, IgG; Anti-celiac antibodies; anti-phospholipid antibodies IgK, IgG; anti-SM antibodies; anti-mitochondrial antibodies ;Thyroid antibody; Microgranular antibody, T cell antibody; Thyroglobulin antibody, anti-scleroderma-70 antibody (A antiSCL-70; anti-Jo antibody (Anti-Jo), anti-U1RNP antibody (Anti -U1RNP);anti-La / SSB antibody;anti-SSA antibody;anti-SSB antibody;anti-parietal cell antibody;anti- Histone antibody; anti-RNP antibody; C-ANCA; P-ANCA; anti-centromere antibody; anti-F Antibody, anti-GBM antibody, anti-ganglioside antibody; anti-desmosomal glycoprotein 3 antibody Antibody (anti-Desmogein3); anti-p62 antibody; anti-sp100 antibody; anti-mitochondrial antibody Chondria (M2) antibody; rheumatoid factor antibody; anti-MCV antibody; anti-topoisomerase antibody; These include antineutrophil cytoplasmic (cANCA) antibodies.

[0156] In some preferred embodiments, the binding molecules used in the conjugates of the invention are By binding to receptors or receptor complexes expressed by disease-associated activated lymphocytes. The receptor or receptor complex can be, for example, a member of the immunoglobulin gene superfamily. Members of the CD11 receptor (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD 22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD 79, CD90, CD125, CD147, CD152 / CTLA-4, PD-1, or ICOS), TNF receptor superfamily (e.g., CD27, CD40, CD95 / Fas, CD134 / OX40, CD137 / 4-1BB, INF-R1, TNFR- 2, RANK, TACI, BCMA, osteoprotegerin, Apo2 / TRAIL-R 1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3), Tegrin, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins Examples of such proteins include complement regulatory proteins (type C, type S, or type I).

[0157] In another specific embodiment, the antibody is immunospecific for a viral or bacterial antigen. A useful conjugate is a humanized or human monoclonal antibody. The term "viral antigen" includes any viral peptide, polypeptide, or antigen capable of eliciting an immune response. glycoproteins (e.g., HIV gp120, HIV nef, RSV F glycoprotein, Influenza virus neuraminidase, influenza virus hemagglutinin, HTL Vtax, herpes simplex virus glycoproteins (e.g., gB, gC, gD, and gE), and hepatitis B surface antigen). The term "bacterial antigen" includes any microbial peptide, polypeptide, or antigen capable of eliciting an immune response. Protein, sugar, polysaccharide, or lipid molecules (e.g., bacteria, fungi, pathogenic protozoa, yeast, These include, but are not limited to, peptides (e.g., LPS and 5 / 8). Type I antibodies useful for treating viral or bacterial infections include palivizumab (used to treat RVS infections). Humanized anti-respiratory syncytial virus monoclonal antibody), PRO542 (HIV infection) CD4 fusion antibody used to treat hepatitis B virus), Ostavir (a hepatitis B virus treatment) antibody), PROTVIR (humanized IgG1 antibody used to treat cytomegalovirus antibodies), and anti-LPS antibodies.

[0158] The bis-linked cell-binding molecule-drug conjugates of the present invention can be used to treat infectious diseases. The infectious diseases include Acinetobacter infection, actinomycosis, African sleeping sickness ( African trypanosomiasis), AIDS (acquired immune deficiency syndrome), amebiasis, anaplasia Zuma, Anthrax, Bacillus anthracis, Bacillus tuberculosis infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Ast Rovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, bacteriologic Ides infection, Balantidiosis, Bailey's roundworm infection, BK virus infection, black sand hair , Blastocys hominis infection, Blastomyces, Bolivian hemorrhagic fever, Borrelia infection, Bo Turinism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus (Norovirus, Sapovirus), Campylobacter pylori infection Lactobacillus infection, Candida infection (candidiasis, thrush), cat scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), chancroid, chickenpox, plaque, plaque pneumonia Plasmodium infection, cholera, pigmented fungus, liver fluke disease, Clostridium difficile infection, Schizoidomycosis, Colorado tick fever, colds (acute viral nasopharyngitis, acute rhinitis), black flu Zfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, Cryptococcus, Cryptosporidium Umbilical cord disease, skin larval migration, Cyclospora infection, cysticercosis, cytomegalovirus infection, dengue Fever, diamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, hydatid disease, Urrichiasis, pinworms (pinworm infection), enterococcal infection, enterovirus infection, typhus Erythema infectiosum (fifth disease), acute exanthema in children, trematode fluke disease, thrombocytopenia, fatal familial insomnia , filariasis, food poisoning caused by Clostridium perfringens, non-parasitic amoebic infections, Fusobacterium infection, gas gangrene (clostridial myonecrosis), diotrichosis, gel Stomann-Straussler-Scheinker syndrome, Giardiasis, glanders, gnathostomiasis, Gonorrhea, granuloma groin (donovanosis), group A streptococcus infection, group B streptococcus infection, influenza Hand, foot and mouth disease (HFMD), Hantavirus pulmonary syndrome, Helicobacter pylori Lori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D Hepatitis, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human Balkan Virus infection Dye, human ehrlichiosis Evans, human granulocytic anaplasmosis, human metapneumovirus human monocytic aerosol infection, human papillomavirus infection, human parainfluenza Virus infection, microtaeniasis, influenza, isosporosis, Kawasaki disease, mononucleosis , Kim's disease, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease leishmaniasis, leprosy, leptospirosis, listeriosis, leprosy Lyme disease (Lyme borrelia), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, ria, Marburg hemorrhagic fever, measles, melioidosis (Whitmore's disease), meningitis, meningococcal disease , metagonism, microsporidiosis, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis (ophthalmia neonatorum), Zfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, onchocerciasis (blindness) filariasis), paracoccidioidomycosis (South American blastomycosis), paragonimiasis, pasture disease Head lice, head lice, body lice, pubic lice (Pubic lice, Crabb ice), Pelvic inflammatory disease, Whooping cough ugh), plague, pneumococcal infection, Pneumocystis carinii pneumonia, pneumonia, polio, Prevotella infection, PA ME, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial Viral infections, rhinovirus infections, rickettsial infections, rickettsia, Rift Valley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), Organ syndrome), scabies, schistosomiasis, sepsis, diarrhea (dysentery), shingles (Herpes zoster) ster), smallpox, sporotrichum, staphylococcal food poisoning, staphylococcal infection, nematodes, syphilis , Taeniasis, Tetanus (trismus), Barber's itch, Ringworm of the Hands , pityriasis nigricans, tinea pedis, tinea unguium, tinea versicolor, toxocariasis (ocular larva migrans), Toxocara visceral larva migrans, toxoplasmosis, trichinosis, trichomoniasis, cryptobiosis (Whipworm infection), pulmonary tuberculosis, tularemia, urea-degrading Mycoplasma infection, Venezuelan equine Encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobacterium balanitis, Mycobacterium pseudotuberculosis Infectious diseases, including but not limited to yersiniosis, yellow fever, and zygomycosis.

[0159] Cell-binding molecules, preferably antibodies against the pathogenic strains described in this patent, The sexual strains include Acinetobacter baumannii, Actiomyces israelii, and Actinobacter Ises odontolyticus gelenceria, Propionibacterium propionicus, Trypanosoma brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anap Plasma, Bacillus anthracis, Methylschaemolyticum (Arcanobacterium hae molyticum), Junin virus, Ascaris, Aspergillus, Astroviridae, Babesia spp., Bacillus cereus spp., Multiplex bacteria, Bacteroides spp., Colon P. Ciliate nematode, Roundworm nematode, BK virus, Piedra hortae iahortae), Blastocystis hominis, dermatitis blasticulosis, Macupo virus , Borrelia spp., Clostridium botulinum, Sabia, Brucella spp., Burkholderia cepacia, and and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae Milli, Campylobacter, usually Candida albicans and other Candida species, Baltic Bartonella henselae, group A streptococci, and Staphylococcus aureus, Trypanosoma cruzi, Haemophilus ducreyi, varicella-zoster virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Fonseca pediatric Losoi, Clonorchiasis, Clostridium difficile, Coccidioides immitis, Coccidioides posadaci, Colorado tick fever virus, rhinovirus, coronavirus Creutzfeldt-Jakob disease prion, Crimean-Congo hemorrhagic fever virus, Crip Tocox neoformans, Cryptosporidium sp., Cat hookworm, Co-parasites, Cyclos Pola, Taenia solium, Cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - Flavivirus, Dentaramoebae, Corynebacterium disponible Phtheria, Diphyllobothrix, Dracunculusmedinensis, Boravirus, Echinococcus, Ehrlichia, Pinworms, Enterococcus, Enterococcus Virus genera, typhus rickettsiae, parvovirus B19, human herpesvirus 6 type, human herpesvirus type 7, fatty liver fluke, liver fluke and liver fluke giant, FFI prion, filar Lithoidea, Clostridium perfringens, Fusobacterium, Clostridium perfringens, other Clostridia genus, Geotrichum candidum, GSS prion, Giardia lamblia lamblia), Burkholderia mallei, Gnathostoma nematode, Gnathostoma striata, Neisseria gonorrhoeae, Granulomatosis bacillus , Streptococcus pyogenes, Streptococcus agalactiae, Haemophilus influenzae, enteric viruses most Coxsackie A viruses, enterovirus type 71, and 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, Simple Herpes virus type 1, herpes simplex virus type 2, Histoplasma capsulatum, Duodenal hookworm, American hookworm, Haemophilus influenzae, Boca human virus, Ehrlichia eupnea Ehrlichia ewingii, Anaplasma phagocytophilum , human metapneumovirus, Ehrlichia chaffeensis, human papillomavirus Human parainfluenza virus, dwarf tapeworm, tapeworm, Epstein-Barr virus Rus, Orthomyxoviridae, Isospora belli , Kingella kingae, Klebsiella pneumoniae, Klebsiella otis Klebsiellarhinoscl eromotis), Couloury pneumonia, Lassa fever virus, Legionella pneumophila Lactobacillus casei, Legionella pneumophila, Leishmania, Mycobacterium leprae and Mycobacterium Lepromatosis (Mycobacterium lepromatosis), Lepto Spira spp., Listeria monocytogenes, Borreliosis and other Borrelia species, Wuchereria bancrofti and Malaria - Heartworms, Lymphocytic choriomeningitis virus (LCMV), Plasmodium sodiumgenus), Marburg virus, measles virus, Burkholderia pseudomallei (Burkh olderia pseudomallei), meningococcus, fluke yokogawai, microsporidia, infection Molluscum contagiosum virus (MCV), mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, various bacteria (actinomycetoma) and fungi (mycetoma), parasitic flies Larvae of Diptera, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia Steroides and other Nocardia species, Onchocerca volvulus, Blastomyces brasiliensis, Paragonimus and and other Paragonimus species, Pasteurella species, head lice, body lice, Phthylus puvis ( Phthiruspubis), Bordetella pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis cysticerci poliovirus, Prevotella, Naegleria ameba, JC virus, psittacosis Lamidia, Coxiella burnetii, rabies virus, bead chain E. coli and rat Bite fever spirochete, respiratory respiratory syncytial virus, Rhinosporidium severii, rhino Rickettsia spp., Rickettsia mites, Rift Valley fever virus, Rocky Mountain spotted fever Kettsia, rotavirus, rubella virus, Salmonella, atypical pneumonia coronavirus, scabies Mite, Schistosoma, Shigella, Varicella-zoster virus, Variola major or minor, Sporotrich Schenckii, Staphylococcus spp., Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Syphilis spirochete Cetacea, Tapeworm, Clostridium tetani, Ringworm, Trichophyton tonsurans, Ringworm, Epidermophyte Trichophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophyton, Trichophyton rubrum, Holtea wellne Ringworm, Malassezia, Roundworm, Toxocara canis, Toxocara catis, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis S. cerevisiae, Trichuris, Mycobacterium tuberculosis, Tula hot Francis bacteria, Ureaplasma urealyticum , Venezuelan equine encephalitis virus, Vibrio cholerae, Guanarito virus, West Nile virus, White Trichosporonium, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mucorales (mucormycosis) and Insects Insect mesh mold (Entomophthora), Pseudomonas aeruginosa, Campylobacter fetus (Vibrio), Aeromonas, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae, Shigella entella, Shigella solanacearum Typhimurium, Treponema pertenue, Treponema carateneum, Fen Borrelia burgdorferi, Borrelia burgdorferi, Leptospira hemorrhagic jaundice, New Mocystis carinii, Brucella abortus, Brucella abortus, Malta fever, Mycoplasma spp., typhus Rickettsia, rickettsia tsutsugamushi, Chlamydia spp., pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); protozoa (dysentery Amoeba, Trichomonas vaginalis, Trichomonas mansoni, Trypanosoma gambiens, Rhodesia Trypanosoma, Leishmania donovani, Leishmania tropicalis, Leishmania brasiliensis Pneumocystis carinii pneumonia, Plasmodium vivax, Plasmodium falciparum, malignant malaria or helminths (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium and hookworms) These include, but are not limited to:

[0160] Other antibodies as cell-binding ligands for use in the present invention for the treatment of viral diseases Examples of antibodies include, but are not limited to, antibodies against pathogenic virus antigens, and the pathogenic virus Examples of viruses include, but are not limited to, viruses from the family Poxyirida e), Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae , Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, Influenza influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial Viruses, rubella, arboviruses, rhabdoviruses, arenaviridae, non-A / non-B hepatitis inflammation viruses, rhinoviruses, coronaviruses, rotaviruses, tumor viruses [e.g. HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma-associated herpes virus ( Kaposi's sarcoma), EB virus (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma tumor), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatitis C virus) caused by viruses such as HTLV-I (adult T-cell leukemia / lymphoma); Immune diseases: [e.g., human immunodeficiency virus (AIDS)], CNS viruses: [ For example, JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (liver vein leukoencephalopathy), Lymphocytic choriomeningitis), arboviral encephalitis, Orthomyxoviridae (presumed) (Encephalitis lethargica), RV (Rabies), Vesicular stomatitis, Herpes virus meningitis, Ramsay Hunt syndrome type II; poliovirus (acute poliomyelitis, post-polio syndrome), HTLV- I (tropical spastic paralysis)]; cytomegalovirus (CMV) retinitis, HSV (herpes simplex virus) Cardiovascular diseases (e.g., CBV (pericarditis, myocarditis)); respiratory / acute nasopharyngeal Headache / viral pneumonia: [EB virus (EBV infection / infectious mononucleosis), cytomegalovirus (HBV) Rovirus, SARS coronavirus (Severe Acute Respiratory Syndrome), Orthomyxovirus Influenza virus A / B / C (influenza / avian influenza) ), Paramyxovirus: Human parainfluenza virus (parainfluenza), R SV (human respiratory syncytial virus), hMPV]; digestive system viruses [MuV (mumps) esophagitis), cytomegalovirus (CMV esophagitis); adenovirus (adenovirus infection) Infection); Rotavirus, Norovirus, Astrovirus, Coronavirus, HBV (type B) Hepatitis virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus) )]; urogenital viruses [e.g., BK virus, MuV (mumps)] It can be enjoyed.

[0161] According to a further object, the present invention provides a conjugate of the present invention and a pharmaceutically acceptable carrier, A pharmaceutical composition for treating cancer, an infectious disease, or an autoimmune disease, together with a diluent or excipient. The present invention also relates to compositions for treating cancer, infectious diseases, and autoimmune diseases. in vitro, in vivo or ex vivo An example of an in vitro therapy is to develop a desired mutant that does not express the target antigen. To kill all cells except the mutant, or to kill mutants expressing undesired antigens. Examples of ex vivo therapy include cell culture treatments to induce cell proliferation and proliferation in the host. Hematopoietic stem cells (HSCs) are treated prior to transplantation to kill diseased or malignant cells. This includes returning the cells to the patient, for example prior to autologous transplantation in the treatment of cancer and autoimmune diseases. to remove cancer cells or lymphocyte cells from the bone marrow or to prevent graft-versus-host disease To achieve this, T cells and other lymphocytes are removed from allogeneic bone marrow or tissue prior to transplantation. Clinical ex vivo treatment for the treatment of a patient or other individual can be carried out by: After obtaining bone marrow cells from the body, the present invention is Incubate the cells in serum-containing medium containing the conjugate at 37°C for 30 minutes to approximately 48 hours. The conditions and incubation time (=dose) can be easily determined by an experienced clinician. The bone marrow cells are then washed with serum-containing medium and returned to the body by known methods such as intravenous injection. During bone marrow cell procurement and reinfusion therapy, the patient may not be receiving other treatments (e.g., ablative chemotherapy or systemic chemotherapy). If the bone marrow cells have been irradiated, the processed bone marrow cells are frozen in liquid nitrogen using standard medical equipment. It is then frozen and stored.

[0162] Drugs / cytotoxic drugs for conjugation

[0163] Drugs that can be conjugated to cell-binding molecules in the present invention include small molecules, including cytotoxic agents. A small molecule drug can be linked to a cell-binding molecule directly or after modification. The "drug" is a compound having a molecular weight of, for example, 100 to 2500, more suitably 200 to 2000. Organic, inorganic, or organometallic compounds are widely used. For a better definition of small molecule drugs, see W Nos. 005058367A2 and 4,956,303, as well as other references. These drugs include known drugs. and potential drugs.

[0164] Known drugs include, but are not limited to:

[0165] (1) Chemotherapeutic agents: a) an alkylating agent selected from the group consisting of: nitrogen mustard; chlorambucil; chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifos Famide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobromide Nititol, melphalan, mitolactol, pipobroman, nobembrine, fenesterol thiazol-10, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-10 65 (including adozelesin, carzelesin, bizelesin, or their synthetic analogs); cuocarmycin (its synthetic analogues KW-2189, CBI-TMI, or CBI dimers); benzodiazepine dimers (e.g., pyrrolobenzodiazepines (PBDs) , tomaymycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or dimers of oxazolidinobenzodiazepines; nitrosourea compounds (carmustine , including lomustine, chlorozotocin, fotemustine, nimustine, and ranimustine); Alkyl sulfonates (busulfan, treosulfan, improsulfan, and pirosulfan) including posulfan); triazenes or dacarbazine; platinum-containing compounds (carboplatin , cisplatin, oxaliplatin); benzodopa, carboquone, metholedopa, or Aziridines such as Ledopa; ethyleneimines, as well as altretamine, triethyleneimine, amine, triethylene phosphoramide, triethylene thiophosphoramine, and trimethylol Methylamelamines, including methylamelamines.

[0166] b) Plant alkaloids: Vinca alkaloids (vincristine, vinblastine, vincristine ... taxoids (including paclitaxel, docetaxel, maytansinoids (DM1, DM2, DM3, DM4, maytansine, and ansamitocin) and their analogues, cryptophycins (especially Cryptophycin 1 and Cryptophycin 8); Epothilones, Eleuterobins, Di Scodermolide, bryostatins, dolostatins, auristatins, tuburi sarcodictin; spongiostatin; pancratistatin; cephalostatin; Ng et al.

[0167] c) DNA topoisomerase inhibitors: epipodophyllins (9-aminocamptothecin , camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate, Irinotecan, mitoxantrone, novantrone, retinoic acid (or retinols), Teniposide, topotecan, 9-nitrocamptothecin (RFS 2000); Mitomycin Mitomycins (mitomycin C) and their analogues.

[0168] d) Antimetabolites: {[Antifolates: (methotrexate, trimetrexate, denopteris containing folic acid analogs, pteropterin, aminopterin (4-aminopteroic acid), or folic acid analogs DHFR inhibitors; IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurin) ribonucleotide reductase inhibitors (including hydroxyurea, ribavirin, and EICAR); a, including deferoxamine)]; [pyrimidine analogs: uracil analogs: (ancitabine azacitidine, 6-azauridine, capecitabine (Xeloda), carmofur, Rabin, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil , floxuridine, raltitrexed (Tomudex); cytosine analogs: (including cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathi oprin, fludarabine, mercaptopurine, thiamiprine, and thioguanine)]; folic acid supplements, floric acid}, etc.

[0169] e) Hormonal therapy agents: {Receptor antagonists: [Antiestrogens: (megestrol, raloxacin, LHRH agonists: (including goserelin, leuprorelin acetate) Antiandrogens: (including bicalutamide, flutamide, calcitriol, propionamide) Dromostanolone onate, epithiostanol, goserelin, leuprolide, mepitiostanol androgen inhibitors, including stenosine, nilutamide, testolactone, trilostane, and other Retinoids / Deltoids: [Vitamin D3 Analogs: (CB1093, EB1089 , KH1060, cholecalciferol, ergocalciferol); photodynamic Therapeutic Agents: (Verteporfin, Phthalocyanine, Photosensitizer Pc4, Demethoxy-Hypocrethrin Cytokines: (interferon alpha, interferon gamma, tumor catabolism tumor necrosis factor (TNF), including TNF domain-containing human proteins)]}, etc.

[0170] f) Kinase inhibitors: BIBW2992 (anti-EGFR / Erb2), imatinib, GEF ritinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib tinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEG FR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-4 06), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, Ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab tuximab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispinesib, etc.

[0171] g) Poly(ADP-ribose) polymerase (PARP) inhibitors: Olaparib, Nirapa Rib, iniparib, talazoparib, veliparib, CEP9722 (Cephalon), E7016 (Eisai), BGB-290 (BeiGene), or 3-aminobenz Amides etc.

[0172] h) Antibiotics: Enediyne antibiotics (calicheamicins, calicheamicin γ1, δ1, α1 and β1, J. Med. Chem., 39(11), 2103-2117(1996), Angew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A and deoxydynemicin Micin; Esperamicin, Kedarcidin, C-1027, Maduropeptin, and Neoca Ruzinostatin chromophore and related enediyne antibiotic chromophores. a, etc.), aclacinomycins, actinomycin, anthramycin, azaserine, Rheomycins, cactinomycin, carabicin abicin), carminomycin, carzinophilin; chromomycins, dactinoma Isin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2 -pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin , idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogalamycin cin, olivomycins, peplomycin, potfilomycin, puromycin, chlomycin elamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, Ubenimex, zinostatin, zorubicin, etc.

[0173] i) Others: Polyketides (acetogenins), especially bullatacin and bullatacinone; gem Citabine, epoxomicins (carfilzomib, etc.), bortezomib, thalidomide, Nalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, S TA-9090, Stimuvax, Allovectin-7, Zygeba, Provenge, Elvoy, isoprenylation inhibitors and lovastatin, dopaminergic Toxins and 1-methyl-4-phenylpyridine ions, cell cycle inhibitors (staurosporine, etc. ), actinomycins (actinomycin D, dactinomycin, etc.), bleomycin (bleomycin A2, bleomycin B2, peplomycin, etc.), anthracyclines Drugs (daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin cin, pirarubicin, zorubicin, etc.), mitoxantrone, MDR inhibitors or verapamil , Ca 2+ ATP inhibitors or thapsigargin, histone deacetylase inhibitors (borino Stat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD 0103, etc.), belinostat, PCI-24781, entinostat, SB939, Resminostat, gibinostat, AR-42, CUDC-101, sulforaphane , trichostatin A, etc.); thapsigargin, celecoxib, glitazones, epigallocatechin Quingallate, disulfiram, salinosporamide A, antiadrenal drugs (aminoglutethimide, mitotane, trilostane); aceglatone; aldophosphamide Aminolevulinic acid; Amsacrine; Arabinoside, Bestravsil; Bisantrene ; edatrexate; defofamine, demecolcine, diazicon, elfornithine ( DFMO), elfomycin; elliptinium acetate, etocluzid; gallium nitrate; Tosine; hydroxyurea; ibandronate, lentinan; lonidamine; mitoguazone; Mitoxatrone; Mopidamol; Nitraerin; Pentostatin; Fenamet; Pilar Bicine; Podophyllic acid; 2-ethylhydrazide; Procarbazine; PSK® ;Razoxane;Rhizoxin;Schizophyllan;Spirogermanium;Tenuazonic acid;Tri Azicon; 2,2',2''-trichlorotriethylamine; Trichothecenes (T2 Toki cin, verrucarin A, roridin A, and anguidine, etc.); urethane, siRNA, an chemotherapeutic drugs; nucleolytic enzymes, etc.

[0174] (2) Anti-autoimmune disease drugs include, but are not limited to, cyclosporine, cyclosporine Phosphorus A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, Roquine, cyclophosphamide, corticosteroids (amcinonide, betamethasone, budesonide) Sonide, hydrocortisone, flunisolide, fluticasone propionate, fluo Cortodanazol, dexamethasone, triamcinolone acetonide, dipropionate bec lometasone), DHAE, etanercept, hydroxychloroquine, ibuprofen, infliximab, meloxicam, methotrexate, mycophenolate mofetil, prednisolone These include donisone, sirolimus, and tacrolimus.

[0175] (3) Anti-infective agents include, but are not limited to: a) aminoglycosides Amikacin, astromycin, gentamicins (netimicin, sisomicin, iromycin) Sepamicin), hygromycin B, kanamycins (amikacin, arbekacin, bekacin, Kanamycin, dibekacin, tobramycin), neomycins (framycetin, paromycin) momycin, ribostamycin), netilmicin, spectinomycin, streptomycin Isin, tobramycin, verdamycin; b) amphenicols: azidamphenicol c) Ansama Isin: geldanamycin, herbimycin; d) Carbapenem: biapenem, doripenem Penem, ertapenem, imipenem / cilastatin, meropenem, panipenem; e) cef Cefamides: carbacephems (loracarbef), cephacetrile, cefaclor, cephradine , cefadroxil, cephalonium, cephaloridine, cephalothin or cephalosporin Phosphate, Cephalexin, Cephaloglycin, Cefamandole, Cephapirin, Cephato Lysine, Cefazaflur, Cefazedone, Cefazolin, Cefbuperazone, Cefcapene, Cefazolin Fudaloxime, cefepime, cefminox, cefoxitin, cefprozil, cephalosporin Ceftezol, cefuroxime, cefixime, cefdinir, cefditoren, cef Epim, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone , ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin, cef Pimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome, Cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephems (flomoxef, Latamoxef; f) Glycopeptides: Bleomycin, Vancomycin (Oritavancin) , telavancin), teicoplanin (dalbavancin), ramoplanin; g) glycyrrhizin Icrine: tigecycline; h) β-lactamase inhibitors: penams (sulbactam, Zobactam), clavam (clavulanic acid); i) lincosamides: clindamycin, j) Lipopeptides: Daptomycin, A54145, calcium dependent Antibiotics (CDA); k) Macrolides: Azithromycin, Cethromycin, Clarithromycin Thromycin, dirithromycin, erythromycin, flurithromycin, josamycin Synthon, ketolides (telithromycin, cethromycin), midecamycin, miokamycin rifamycin, oleandomycin, rifamycins (rifampicin, rifampin, rifamycin abutin, rifapentine), rokitamycin, roxithromycin, spectinomycin Spiramycin, tacrolimus (FK506), troleandomycin, telithromycin isinin; l) monobactams: aztreonam, tigemonam; m) oxazolidinones : Linezolid; n) Penicillins: Amoxicillin, Ampicillin, Pivampicillin, Hexyl Citrate Tacillin, Bacampicillin, Methampicillin, Talampicillin, Azidocillin, Azlocillin Phosphorus, Benzylpenicillin, Benzathine Benzylpenicillin, Benzathine Phenoxymethicone Procaine benzylpenicillin, carbenicillin (potassium Indacillin), cloxacillin, dicloxacillin, epicillin, flucloxacillin, Mecillinam (pivmecillinam), mezlocillin, methicillin, nafcillin, oxacillin , penamecillin, penicillin, phenethicillin, phenoxymethylpenicillin, piperacillin phospholipid, propicillin, sulbenicillin, temocillin, ticarcillin;o) polypeptides : Bacitracin, colistin, polymyxin B; p) Quinolones: Alatrofloxacin , balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, di Floxacin, Enoxacin, Enrofloxacin, Floxacin, Garenoxacin, Gachi Floxacin, gemifloxacin, grepafloxacin, canotrovafloxacin, levofloxacin Floxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin floxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, Trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, te Mafloxacin, tosufloxacin, trovafloxacin;q) Streptogramins: Pristinamycin, quinupristin / dalfopristin; r) sulfonamides: Enid, Prontosil, Sulfacetamide, Sulfamethizole, Sulfanilamide, Sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulf Amethoxazole (cotrimoxazole); s) Steroidal antibacterial agents: Selected from fusidic acid t) Tetracyclines: doxycycline, chlortetracycline, chlor Mocycline, demeclocycline, lymecycline, meclocycline, methacycline minocycline, oxytetracycline, penimepicycline, rolitetracycline cyclosporine, tetracycline, glycylcycline (including tigecycline); u) other cyclosporine Antibiotics of the following types: annonacin, arsphenamine, bacitra, Synthon), DADAL / AR inhibitor (cycloserine), dictyostatin, discodel Molide, eleutherobin, epothilone, ethambutol, etoposide, faropenem, Fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirozin Mycolactone, NAM synthesis inhibitor (fosfomycin), nitrofurantoin, Ritaxel, platensimycin, pyrazinamide, quinupristin / dalfopristin, Rifampin (rifampin), tazobactamcinidazole, uvaricin;

[0176] (4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, vicrivir Lock, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab); b) I Integrase inhibitors: raltegravir, elvitegravir, globoidin A; c) maturation inhibitors: Bevirimat, Vivicon; d) neuraminidase inhibitors: oseltamivir, Zanamivir, Peramivir; e) Nucleosides and nucleotides: Abacavir, Acyclovir adefovir, amdoxovir, apricitabine, brivudine, cidofovir, Vudine, dexelvucitabine, didanosine (DDI), elvucitabine, emtricitabine FTC, entecavir, famciclovir, fluorouracil (5-FU), 3' -Fluoro-substituted 2',3'-deoxynucleoside analogs (3'-fluoro-2',3' -dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine including the group consisting of FLG), fomivirsen, ganciclovir, idoxuridine, Lamivudine (3TC), L-nucleosides (β-L-thymidine and β-L-2'-deoxycysteine) including the group consisting of cicitidine), penciclovir, rasvir, ribavirin, stampidine , stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir fluridin, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), Zidovudine (AZT); f) Non-nucleosides: amantadine, ateviridin, capraviridin Diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol , emivirine, efavirenz, foscarnet (phosphoryl formate), imiquimod, Tarferon α, Loviride, Rhodenosine, Methisazone, Nevirapine, NOV-205, Pe Interferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimo g) Protease inhibitors: amprenavir, atazavir Naviru, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, Nelfinavir, Pleconaril, Ritonavir, Saquinavir, Telaprevir (VX-950 ), Tipranavir; h) Other antiviral drugs: Abzyme, Arbidol, Calanolide A, ceragenin, cyanovirin-N, diallylpyrimidine, epigallocatechin gallate ( EGCG), foscarnet, Griffithsin, taribavirin (viramidine), hydroxyl Ciurea, KP-1461, Miltefosine, Pleconaril, Portmanteau inhibitors, Bavirin, seliciclib;

[0177] (5) The drugs used in the conjugation via the bis-linker of the present invention may also include radioisotopes. Examples of radioisotopes (radionuclides) 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, 1 25 I, 131 I, 133 Xe, 177 Lu, 211 At, or 213 Bi is one example Radiolabeled antibodies are very useful in receptor-targeted imaging experiments, or for example For example, the invention of antibody-drug conjugates (Wu et al. (2005) Nature Biotechnology 23(9):1137-114 6) Cell-binding molecules, such as antibodies, that can be used directly for specific targeted therapy. As previously mentioned, the radioisotopes are conjugated, chelated, or otherwise complexed with the present bridges. This labeling technique is described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, N .Y., Pubs. (1991). Chelating agents capable of forming complex metal complexes include D OTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, Tex. ) is included.

[0178] (6) Pharmaceutically acceptable salts, acids, derivatives, hydrates, or hydrates of any of the above drugs. or a salt; or a crystal structure; or an optical isomer, racemate, diastereomer, or enantiomeric form Mah.

[0179] In another embodiment, the drug / cytotoxic molecule of formula (I) and / or (II) is a cell-bound The conjugates can be used to detect, monitor, or study the interaction of the conjugate molecule with a target cell. The chromophore molecule can be a molecule that can emit UV light, fluorescent light, IR light, near IR light, A chromophore is a compound that has the ability to absorb certain types of light, such as light and visual light; Classes of chromatophores, erythrophores, iridophores, leucophores, melanophores, cerebrophores, and fluorophores or The phosphors include subclasses of fluorescent chemicals that re-emit light with light; Class or subclass of visual light transmitting molecules; Class or subclass of light emitting molecules; Luminescence A class or subclass of molecules; a class or subclass of luciferin compounds.

[0180] Chromophore molecules include, but are not limited to, xanthene derivatives (fluorescein, rhodopsin, amine, Oregon Green, Eosin, and Texas Red); cyanine derivatives (cyanine , indocarbocyanines, oxacarbocyanines, thiacarbocyanines, and merocyanines squalene derivatives and ring-substituted squaraines (Seta, SeTau, and Squa) including re dyes); naphthalene derivatives (dansyl and prodan derivatives); coumarin derivatives oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazoles; anthracene derivatives (DRAQ5, DRAQ7, and CyT Anthraquinones, including RAK Orange; pyrene derivatives (e.g., Cascade Blue) ;Oxazine derivatives (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170, etc.). Acridine derivatives (proflavine, acridine orange, acridine yellow) Arylmethine derivatives (auramine, crystal violet, malachite glycol) Tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin) and other non-tannin compounds The phosphor can be selected from a group consisting of phosphorus, phosphate, phosphate-containing organic phosphors, and phosphate-containing organic phosphors.

[0181] Alternatively, the chromophore molecule is selected from any analogues and derivatives of the following fluorophore compounds: CF dye (Biotium), DRAQ and CyTRAK probes (Bio-Status ), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLig ht Fluor (Thermo Scientific, Pierce), Atto and Trance (Sigma A ldrich), FluoProbes (Interchim), Abberior dye (Abberior), DY and MegaStokes dyes (Dyomics), SulfoCy dyes (Cyandye), Hi Lyte Fluor (AnaSpec), Seta, SeTau and Square dyes (SETA BioMedicals), Quasar and Cal Flour dyes (Biosearch Technologies) , SureLight dyes (APC, RPEPerCP, phycobilisomes) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech).

[0182] Examples of widely used fluorophore compounds that can react or conjugate with the conjugates of the present invention include: , allophycocyanin (APC), aminocoumarin, APC-Cy7 conjugate, BODIP Y-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5 .5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, Lissamine rhodamine B, Lucifer Yellow, Methoxycoumarin, NBD, Pacific Blue, P Acific Orange, PE-Cy5 conjugate, PE-Cy7 conjugate, PerCP, R-Phycoerythrin (PE), Red 613, Seta-555-azide, Seta- 555-DBCO, Seta-555-NHS, Seta-580-NHS, Seta- 680-NHS, Seta-780-NHS, Seta-APC-780, Seta-P erCP-680, Seta-R-PE-670, SeTau380-NHS, SeTa u405-maleimide, SeTau405-NHS, SeTau425-NHS, SeT Examples include au647-NHS, Texas Red, TRITC, TruRed, and X-rhodamine. It can be obtained.

[0183] Fluorophores that can be linked to the conjugates of the invention for the study of nucleic acids or proteins The compound is selected from the following compounds or derivatives thereof: 7-AAD (7-aminoadenine dihydrochloride) Cutinomycin D, CG selective), acridine orange, chromomycin A3, CyTR AK Orange (Biostatus, red excitation dark), DAPI, DRAQ5, DRAQ7, Ethidi ammonium bromide, Hoechst 33258, Hoechst 33342, LDS751, Mithramycin Synthon, propidium iodide (PI), SYTOX Blue, SYTOX Green, SYTO X Orange, Thiazole Orange, TO-PRO: Cyanine Monomer, TOTO-1, TO -PRO-1, TOTO-3, TO-PRO-3, YOseta-1, YOYO-1. Fluorescent dyes that can be linked to the conjugates of the invention for cell studies include the following compounds: Selected from its derivatives: DCFH (2'7'dichlorodihydro-fluorescein, acid Dihydrorhodamine 123 (oxidized form), DHR (dihydrorhodamine 123, oxidized form, light catalyzes oxidation), Fluo -3 (AM ester, pH > 6), Fluo-4 (AM ester, pH 7.2), Ind o-1 (AM ester, low / high calcium (Ca 2+ )), and SNARF (pH 6 / 9 ) Preferred conjugates that can be linked to the conjugates of the present invention for protein / antibody studies are: The fluorescent dye is selected from the following compounds or their derivatives: allophycocyanin (APC) ), AmCyan1 (tetramer, Clontech), AsRed2 (tetramer, Clontech), Aza mi Green (monomer, MBL), azurite, B-phycoerythrin (BPE), celeriac Ann, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP", Clontech) ch), EBFP, EBFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen), EYFP (weak dimer, Clontech), GFP (S65A mutant ), GFP (S65C mutant), GFP (S65L mutant), GFP (S65T mutant) , GFP (Y66F mutant), GFP (Y66H mutant), GFP (Y66W mutant), GFP uv , HcRed1, J-Red, Katyusha, Kusabira Orange e (monomer, MBL), mCFP, mCherry, mCitrine, Midrish i Cyan (dimer, MBL), mKate (TagFP635, monomer, Evrogen), m Keima-Red (monomer, MBL), mKO, mOrange, mPlum, mRas pberry, mRFP1 (monomer, Tsien Lab), mStrawberry, mTFP1, mTurquoise2, P3 (phycobilisome complex), Peridi nin Chlorophyll (PerCP), R-Phycoerythrin (RPE), T -Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrog en), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTo mato (tandem dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboGFP (dimer, Evrogen), T urboRFP (dimer, Evrogen), TurboYFP635 (dimer, Evrogen), Nuss, native GFP, YPet, Zs Green 1 (tetramer, Clontech), Zs Yellow 1 (tetramer, Clontech).

[0184] An example of the structure of an antibody-chromophore molecule conjugate via a bridge linker is Ac01 shown below: Ac02, Ac03, Ac04, Ac05, Ac06, and Ac07:

[0185] [ka]

[0186] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. R 12 and R 12 ' is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) n COOH, O(CH2C H2O) p CH2CH2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH 2CH2O) p CH2CH2NH2, O(CH2CH2O)p CH2CH2COOH,N H(CH2CH2O) p CH2CH2COOH, O(CH2CH2O) p CH2CH2N HSO3H, NH(CH2CH2O) p CH2CH2NHSO3H, R1-NHSO3H , NH-R1-NHSO3H, O(CH2CH2O) p CH2CH2NHPO3H2,N H(CH2CH2O) p CH2CH2NHPO3H2, R1-NHPO3H2, R1-O PO3H2, O(CH2CH2O) p CH2CH2OPO3H2, NH(CH2CH2O ) p CH2CH2NHPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H 2, NH-Ar-COOH, NH-Ar-NH2, where p is 0 to 5000. Aa is an amino acid; R1, m1, n, L1, and L2 have the same definitions as in formula (I). be.

[0187] In another embodiment, the agents of Formula (I) and Formula (II) are capable of inhibiting cell proliferation when administered to a mammal. It may also be a polyalkylene glycol, which is used to extend the half-life of the binding molecule. Alkylene glycols include poly(ethylene glycol) (PEG), poly(propylene glycol) glycol), and copolymers of ethylene oxide and propylene oxide. Particularly preferred is PEG, and even more particularly preferred is is a monofunctional activated hydroxyPEG (e.g., hydroxyPEG-monocarboxylic acid reaction) Hydroxy PEG-monoaldehyde, hydroxy PEG-monoamine, hydroxy ...aldehyde, hydroxy PEG-monoaldehyde, hydroxy PEG-monoaldehyde, HydroxyPEG-monohydrazide, HydroxyPEG-monocarbazate, HydroxyPE G-Monoiodoacetamide, HydroxyPEG-monomaleimide, HydroxyPEG- Mono-orthopyridyl disulfide, HydroxyPEG-mono-orthopyridyl disulfide , HydroxyPEG-monoxime, HydroxyPEG-monophenylcarbonate, Hydroxyl PEG-monophenylglyoxal, Hydroxyl PEG-monothiazolidine PEG-2-thione, hydroxyl PEG-monothioester, hydroxyl PEG-monothio ol, hydroxyl PEG-monotriazine, and hydroxyl PEG-monovinyl The hydroxy PEGs are activated at a single terminus, including hydroxy PEGs containing methyl methyl hydroxybenzoates.

[0188] In such embodiments, the polyalkylene glycol has a viscosity of from about 10 Daltons to about 20 0 kDa, preferably about 88 Da to about 40 kDa; and more preferably, the two branches each have a molecular weight of about 88 Da to about 40 kDa; and In one particular embodiment, the polyalkylene glycol is Poly(ethylene) glycol, about 10 kDa; about 20 kDa, or about 40 kDa In certain embodiments, the PEG has a molecular weight of 10 kDa (linear or branched), PEG 20kDa (linear or branched), or PEG 40kDa (linear or branched). Numerous U.S. patents, such as U.S. Patents 5,428,128; 5,621,039; 5,622,986; 5,643,575; 5,728,56 0; 5,730,990; 5,738,846; 5,811,076; 5,824,701; 5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974; 5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,0 11,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095 Preparation of linear or branched "non-antigenic" PEG polymers and their derivatives or conjugates The structure of the antibody-polyalkylene glycol conjugate via a bridge linker is The following Pg01, Pg02, and Pg03 are mentioned:

[0189] [ka]

[0190] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. p is 1 to 5000; R1, L1, and L2 have the same definitions as in formula (I). Preferably, R1 and R3 are independently H, OH, OCH3, CH3, or OC2H5. do.

[0191] In yet another embodiment, the present invention provides a method for conjugating a cell-binding molecule via a bridge linker. Preferred cytotoxic agents are tubulysins, maytansinoids, taxanoids (taxanoids), sans), CC-1065 analogs, daunorubicin and doxorubicin compounds, Amatoki Synthins (including amanitins), indolecarboxamides, benzodiazepine dimers (e.g. For example, pyrrolobenzodiazepines (PBDs), tomaymycin, anthramycin, indoli benzodiazepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines Dimers of zepines), calicheamicin and enediyne antibiotics, actinomycin , azaserine, bleomycin, epirubicin, eribulin, tamoxifen, ida Rubicin, dolastatins, auristatins (e.g., monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2- AQ, 6-AQ, EB (AEB), and EFP (AEFP) and their analogues), cuocarmycins, geldanamycins or other HS P90 inhibitors, centanamycin, methotrexates, thiotepa, vindesines, vin Cristines, hemiasterins, nazumamides, microgyres Microginins, radiosumins, sto Streptonigtins, SN38 or other camptothecins ( Camptothecin analogues or metabolites, alterobactins bactins, microsclerodermicins s), theonellamides, esperamicins ramicins), PNU-159682; and their analogs and derivatives, pharmaceutical or a photoacceptable salt, acid, derivative, hydrate or hydrated salt thereof; or a crystal structure thereof; or a photoacceptable salt, acid, derivative, hydrate or hydrated salt ... crystal structure thereof; They are optical isomers, racemates, diastereomers, or enantiomers.

[0192] Tubulysins, which are preferred compounds for the conjugates of the present invention, are well known in the art. , can be extracted from natural products according to known methods or synthesized by known methods ( For example: Balasubramanian, R.; et al. J. Med. Chem., 2009, 52, 238-240. Wipf, P.; et al. Org. Lett., 2004, 6, 4057-4060. Pando, O.; et al. J. Am. Chem. Soc., 201 1, 133, 7692-7695. Reddy, JA; et al. Mol. Pharmaceutics, 2009, 6, 1518-1525. Raghavan, B.; et al. J. Med. Chem., 2008, 51, 1530-1533. Patterson, AW; et al J. Org. Chem., 2008, 73, 4362-4369. Pando, O.; et al. Org. Lett., 2009, 11 (2 4), pp 5567-5569. Wipf, P.; et al. Org. Lett., 2007, 9 (8), 1605-1607. tad, GK; Org. Lett., 2004, 6, pp 3249-3252. Hillary M. Peltier, HM; et al. J. Am. Chem. Soc., 2006, 128, 16018-16019. Chandrasekhar, S.; et al. J. Org. Ch em., 2009, 74, 9531-9534. Liu, Y.; et al. Mol. Pharmaceutics, 2012, 9, 168-175. Friestad, GK; et al. Org. Lett., 2009, 11, 1095-1098. Kubicek, K.; et al., An gew Chem Int Ed Engl, 2010. 49: p. 4809-12. Chai, Y.; et al., Chem Biol, 2010, 1 7: 296-309. Ullrich, A.; et al., Angew Chem Int Ed Engl, 2009, 48, 4422-5. Sani, M.; et al. Angew Chem Int Ed Engl, 2007, 46, 3526-9. Domling, A.; et al., Ang ew Chem Int Ed Engl, 2006. 45, 7235-9. Patent applications: Zanda, M. ; et al, C the. Pat. Appl. CA 2710693 (2011). Chai, Y.; et al. Time. Pat. Appl. 2174947 (2 010), WO 2010034724. Leamon, J.; et al, WO 2010033733, WO 2009002993. Ellman, J .; et al, WO 2009134279; WO 2009012958, US appl. 20110263650, 20110021568, Mats chiner, G.; et al, WO 2009095447.Vlahov, I.; et al, WO 2009055562, WO 2008112873 . Low, P.; et al, WO 2009026177. Richter, W., WO 2008138561. Kjems, J.; et al, W O 2008125116. Davis, M.; et al, WO 2008076333. Diener, J.; et al, US Pat. Appl . 20070041901, WO 2006096754. Matschiner, G.; et al, WO 2006056464. Vaghefi, F.; et al, WO 2006033913. Doemling, A., Ger. Offen. DE 102004030227; WO 200400532 7; WO 2004005326; WO2004005269. Stanton, M.; et al, US Pat. Appl. Publ. 200402 49130. Hoefle, G.; et al, Ger. Offen. DE 10254439; DE 10241152; DE 10008089. Le ung, D.; et al, WO 2002077036. Reichenbach, H.; et al, Ger. Offen. DE 19638870; Wolfgang, R.; US 20120129779, Chen, H., US appl. 20110027274.). together with cell-binding molecules Preferred structures of tubulysins for use in the treatment of cancer are described in PCT / IB2012 / 053554 is described in.

[0193] An example of the structure of an antibody-tubulysin analog conjugate via a bis-linker is shown below. T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, and T11:

[0194] [ka] JPEG2025138629000060.jpg248168

[0195] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; R 12 are OH, NH2, and NHR1 , NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH- (Aa) n COOH, O(CH2CH2O) p CH2CH2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2CH2NH2, NR1R1', NHOH, NHOR1, O(CH2CH2O) p CH2CH2COOH, NH(CH2C H2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O(C H2CH2O) p CH2CH2NHSO3H, NH(CH2CH2O) p CH2CH2N HSO3H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O) p CH2CH2NHPO3H2, NH(CH2CH2O) p CH2CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O) p CH2C H2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH 2CH2NH) p CH2CH2NH2, NH(CH2CH2S) p CH2CH2NH2, NH(CH2CH2NH) p CH2CH2OH, NH(CH2CH2S) p CH2CH2 OH, NH-R1-NH2, or NH(CH2CH2O) p CH2CH2NHPO3H2 wherein Aa is 1 to 8 amino acids; n and m1 are independently 1 to 20. p is 1 to 5000; preferably, R1, R1', R2, R3, and R4 are independently H, C1-C8 straight or branched alkyl, amide, or amine; C2-C8 aryl alkyl, alkenyl, alkynyl, heteroaryl, heteroalkyl, alkylcycloalkoxy amine, ester, ether, heterocycloalkyl, or acyloxylamine; or 1 to A peptide containing eight amino acids, or a peptide of the formula (OCH2CH2) p Or (OCH2C H(CH3) p where p is an integer from 1 to about 5000. Two R: R1R2, R2R3, R1R3, or R3R4 are alkyl, aryl , heteroaryl, heteroalkyl, or alkylcycloalkyl group forming a 3- to 8-membered ring X3 can be H, CH3, CH2CH3, C3H7, or X1'R1'. where X1' is NH, N(CH3), NHNH, O, or S, and R1' is H or C1-C8 linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxyamine; R3' is H or a C1-C6 straight group. Z3 is H, COOR1, NH2, NHR1, OR1, CO NHR1, NHCOR1, OCOR1, OP(O)(OM1)(OM2), OCH2OP (O)(OM1)(OM2), OSO3M1, R1, or O-glycoside (glucoside, Galactosides, mannosides, glucuronosides / glucuronides, allosides, fructosides), NH-glycoside, S-glycoside, or CH2-glycoside; M1 and M2 are are each independently H, Na, K, Ca, Mg, NH4, and NR1R2R3; L1 and L2 has the same definition as in formula (I).

[0196] The calicheamicins and related enzymes preferred in the cell-binding molecule-drug conjugates of the present application The lendiine antibiotics are described in Nicolaou, KC et al, Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-5888), US Pa tent Nos. 4,970,198; 5,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,71 2,374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,5 62; 6,124,310; 8,153,768. Antibody-calicheamicin analog covalent bonds via this bridge linker. Examples of the structure of the character include C01 and C02 below.

[0197] [ka]

[0198] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. p is 1 to 5000; R1, L1, and L2 have the same definitions as in formula (I).

[0199] Maytansin compounds, including maytansinol and its analogues, are preferably used in the present invention. Nos. 4,256,746, 4,361,650, 4 ,307,016, 4,294,757, 4,294,757, 4,371,533, 4,424,219, 4,331,598, 4,450,254, 4,36 4,866, 4,313,946, 4,315,929 4,362,663, 4,322,348, 4,371,533, 4,424,219, 5,208,02 0, 5,416,064, 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821, 7,276,497, 7,301,019, 7,303,749, 7,368,565, 7,411,063, 7,851,432, and 8,163,888. An example of the structure of a bond-mediated antibody-maytanosinoid conjugate is My01, These include My02, My03, My04, My05, and My06.

[0200] [ka] JPEG2025138629000065.jpg169163

[0201] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. p is 1 to 5000; R1, L1, and L2 have the same definitions as in formula (I).

[0202] Preferred cytotoxic natural products for conjugation include paclitaxel (Taxol). Taxanes and their semi-synthetic derivatives, docetaxel (Taxotere), and their derivatives The derivatives are described in the following literature: K. C. Nicolaou et al., J. Am. Chem. Soc. 117, 2409-2420, (1995); Ojima et al, J. Med. Chem. 39:3889-3896 (1996); 40:267-278 ( 1997); 45, 5620-5623 (2002); Ojima et al., Proc. Natl. Acad. Sci., 96:4256-4261 (1999; Kim et al., Bull. Korean Chem. Soc., 20, 1389-1390 (1999); Miller, et al. J. Med. Chem., 47, 4802-4805(2004); U.S. Patent Nos. 5,475,011 5,728,849, 5,811,452 ; 6,340,701; 6,372,738; 6,391,913, 6.436,931; 6,589,979; 6,596,757; 6,706,708; 7 ,008,942; 7,186,851; 7,217,819; 7,276,499; 7,598,290; and 7,667,054.

[0203] As an example of the structure of the antibody-taxane conjugate via the linker of the present invention, the following Tx0 1, Tx02, and Tx03.

[0204] [ka]

[0205] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. R1, L1, and L2 have the same definitions as in formula (I).

[0206] CC-1065 analogs and Duocarmycin analogs are also Also preferred for use in the conjugates containing bis-bridges of the present invention. Examples of duocarmycin analogues and their synthesis are described in the following documents: For example, Warpehoski, et al, J. Med. Chem. 31:590-603 (1988), D. Boger et al., J. Org. Chem; 66; 6654-6661, 2001; U.S. Patents: 4169888, 4391904, 4671958, 4816567 , 4912227, 4923990, 4952394, 4975278, 4978757, 4994578, 5037993, 5070092, 508446 8, 5101038, 5117006, 5137877, 5138059, 5147786, 5187186, 5223409, 5225539, 5288 514, 5324483, 5332740, 5332837, 5334528, 5403484, 5427908, 5475092, 5495009, 553 0101, 5545806, 5547667, 5569825, 5571698, 5573922, 5580717, 5585089, 5585499, 55 87161, 5595499, 5606017, 5622929, 5625126, 5629430, 5633425, 5641780, 5660829, 5 661016, 5686237, 5693762, 5703080, 5712374, 5714586, 5739116, 5739350, 5770429, 5773001, 5773435, 5786377 5786486, 5789650, 5814318, 5846545, 5874299, 5877296, 5877397, 5885793, 5939598, 5962216, 5969108, 5985908, 6060608, 6066742, 6075181, 6103236, 6114598, 6130237, 6132722, 6143901, 6150584, 6162963, 6172197, 618037 0, 6194612, 6214345, 6262271, 6281354, 6310209, 6329497, 6342480, 6486326, 6512 101, 6521404, 6534660, 6544731, 6548530, 6555313, 6555693, 6566336, 6,586,618, 6593081, 6630579, 6,756,397, 6759509, 6762179, 6884869, 6897034, 6946455, 7,049, 316, 7087600, 7091186, 7115573, 7129261, 7214663, 7223837, 7304032, 7329507, 7,3 29,760, 7,388,026, 7,655,660, 7,655,661, 7,906,545, and 8,012,978. As an example of the structure of an antibody-CC-1065 analog conjugate via C02, CC03, and CC04.

[0207] [ka]

[0208] where mAb is an antibody; Z3 is H, PO(OM1)(OM2), SO3M1, C H2PO(OM1)(OM2), CH3N(CH2CH2)2NC(O)-, O(CH2 CH2)2NC(O)—, R1, or a glycoside; [ka] is either a single or double bond or is absent; X1, X5, Y1, and Y 2 are independently O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1 mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 ∼20; R1, L1, and L2 have the same definitions as in formula (I).

[0209] Daunorubicin / doxorubicin analogs are also preferred for conjugation with the bis-linkage of this patent. Its preferred structure and synthesis method are exemplified below: Hurwitz, E., et al., Cancer Res. 35, 1175-81(1975). Yang, HM, and Reisfeld, RA, Proc. Natl. Acad. Sci. 85, 1189-93(1988); Pietersz, CA, E., et al., E., et al.," Cancer Res. 48, 926-311(1988); Trouet, et al., 79, 626-29(1982); Z. Brich et al., J. C ontrolled Release, 19, 245-58(1992); Chen et al., Syn. Comm., 33, 2377-90, 2003 ; King et al., Bioconj. Chem., 10, 279-88, 1999; King et al., J. Med. Chem., 45, 4336-43, 2002; Kratz et al., J Med Chem. 45, 5523-33, 2002; Kratz et al., Biol Pharm Bull. Jan. 21, 56-61, 1998; Lau et al., Bioorg. Med. Chem. 3, 1305-12, 199 5; Scott et al., Bioorg. Med. Chem. Lett. 6, 1491-6, 1996; Watanabe et al., Tok ai J. Experimental Clin. Med. 15, 327-34, 1990; Zhou et al., J. Am. Chem. Soc. 1 26, 15656-7, 2004; WO 01 / 38318; U.S. Patents 5,106,951; 5,122,368; 5,146,064; 5,177,0 16; 5,208,323; 5,824,805; 6,146,658; 6,214,345; 7,569,358; 7,803,903; 8,084,586; 8 ,053,205. An example of the structure of an antibody-CC-1065 analog conjugate via a linker of this patent is shown below. Then, Da01, Da02, Da03, Da04, Da05, Da06, and Da07 below. , and Da08.

[0210] [ka] JPEG2025138629000072.jpg145145

[0211] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently and is O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; R 12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, NH-R1-COOH, NH-(Aa) n COOH, O(CH2CH2O) p CH2CH 2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2 CH2NH2, NR1R1’, NHOH, NHOR1, O(CH2CH2O) p CH2C H2COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-COOH , NH-Ar-NH2, O(CH2CH2O) p CH2CH2NHSO3H, NH(CH 2CH2O) p CH2CH2NH-SO3H, R1-NHSO3H, NH-R1-NHS O3H, O(CH2CH2O) p CH2CH2NHPO3H2, NH(CH2CH2O) p CH2-CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO3H2 , O(CH2CH2O) p CH2CH2OPO3H2, OR1-NHPO3H2, NH- R1-NHPO3H2, NH(CH2CH2NH) p CH2CH2NH2, NH(CH2 CH2S) p CH2CH2NH2, NH(CH2CH2NH) p CH2CH2OH, NH (CH2CH2S) pCH2CH2OH, NH-R1-NH2, or NH(CH2CH2 O) p CH2CH2NHPO3H2, where Aa is 1 to 8 amino acids; p is 1 to 5000; mAb is an antibody, preferably a monoclonal antibody; n and m 1 is independently 1 to 20; R1, L1, and L2 have the same definitions as in formula (I).

[0212] Auristatins and dolastatins ins) are preferred for conjugation via the bis-linkers of the present patent. Auristatins (e.g., Auristatin E (AE), auristatin EB (AEB), auristatin EFP (A EFP), monomethyl auristatin E (MMAE), monomethyl auristatin (MM AF), auristatin F phenylenediamine (AFP), and MMAE phenylalanine Indole-1-4-one (a variant of Indole-1-4-one) is a synthetic analogue of the dolastatin family and is described in the following literature: t. J. Oncol. 15:367-72 (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S. Patent Applications 11134826, 20060074008, 2006022925. U.S. Patent No. 4414 205, 4753894, 4764368, 4816444, 4879278, 4943628, 4978744, 5122368, 5165923, 516 9774,5286637, 5410024, 5521284, 5530097, 5554725, 5585089, 5599902, 5629197, 563 5483, 5654399, 5663149, 5665860, 5708146, 5714586, 5741892, 5767236, 5767237, 57 80588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065, 6048720, 6054297, 6 054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 6342219, 6342221, 6407213, 6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097840, 7098305, 7098308, 7498298, 7375078, 7462352, 7553816, 7659241, 7662387, 7745394 , 7754681, 7829531, 7837980, 7837995, 7902338, 7964566, 7964567, 7851437, 799413 5. An example of the structure of the antibody-auristatin conjugate via the linker of this patent is shown below. Au01, Au02, Au03, Au04, Au05, Au06, Au07, Au08 , Au09, Au10, Au11, Au12, and Au13.

[0213] [ka] JPEG2025138629000075.jpg117169

[0214] During the ceremony, [ka] is optionally either a single or double bond or absent; X1 and Y1 are independently O, NH, NHNH, NR5, S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R1), N(R1)C(O)N(R1), CH, C(O)NHNHC(O), and C( O)NR1; R 12 is OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-CO OH, NH-R1-COOH, NH-(Aa) n COOH, O(CH2CH2O) p CH 2CH2OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2CH2NH2, NR1R1’, NHOH, NHOR1, O(CH2CH2O) p C H2CH2COOH, NH(CH2CH2O) p CH2CH2COOH, NH-Ar-C OOH, NH-Ar-NH2, O(CH2CH2O) p CH2CH2NHSO3H, NH (CH2CH2O) p CH2CH2NH-SO3H, R1-NHSO3H, NH-R1- NHSO3H, O(CH2CH2O) p CH2CH2NHPO3H2, NH(CH2CH 2O) p CH2-CH2NHPO3H2, OR1, R1-NHPO3H2, R1-OPO 3H2, O(CH2CH2O) p CH2CH2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, NH(CH2CH2NH) p CH2CH2NH2, NH( CH2CH2S) p CH2CH2NH2, NH(CH2CH2NH) p CH2CH2OH , NH(CH2CH2S) p CH2CH2OH, NH-R1-NH2, or NH(CH2 CH2O) pCH2CH2NHPO3H2, where Aa is 1 to 8 amino acids Yes; p is 1-5000; mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20; p is 1-5000; Preferably, R1, R2, R3, and R4 are independently H; C1-C8 straight or branched chain; Alkyl, aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, es ter, ether, amide, amine, heterocycloalkyl, or acyloxylamine; or a peptide containing 1 to 8 amino acids, or a peptide of the formula (OCH2CH2) p Or (O CH2CH(CH3)) p where p is from 1 to about 5000. is an integer. Two R: R1R2, R2R3, R1R3, or R3R4 are alkyl, aryl, hydroxyl, or hydroxyl. Forming a 3- to 8-membered ring of heteroaryl, heteroalkyl, or alkylcycloalkyl group can; X3 is H, CH3, or X1'R1', where X1' is NH, N(CH3) , NHNH, O, or S, and R1' is H or C1-C8 linear or branched alkyl; Aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxya Min is; R3' is H or C1-C6 straight or branched alkyl; Z3' is H, COOR1, NH2, NHR1, OR1, CONHR1, NHCOR1 , OCOR1, OP(O)(OM1)(OM2), OCH2OP(O)(OM1)(OM 2), OSO3M1, R1, or O-glycosides (glucosides, galactosides, mannosides , glucuronosides / glucuronides, arosides, fructosides), NH-glycosides, S- M1 and M2 are each independently H, N, or a, K, Ca, Mg, NH4, NR1R2R3; L1 and L2 have the same definitions as in formula (I).

[0215] Benzodiazepine dimers (e.g., pyrrolobenzoxazoles) are preferred as cytotoxic agents of the present invention. Diazepines (PBD) or (tomaymycin), indolinobenzodiazepines, imidazoline dimers of oxazolidinobenzodiazepines or oxazolidinobenzodiazepines) are known in the prior art. Exemplary in: U.S. Patent Nos. 8,163,736; 8,153,627; 8,034,808; 7,834,005; 7,741 ,319; 7,704,924; 7,691,848; 7,678,787; 7,612,062; 7,608,615; 7,557,099; 7,528,12 8; 7,528,126; 7,511,032; 7,429,658; 7,407,951; 7,326,700; 7,312,210; 7,265,105; 7,202,239; 7,189,710; 7,173,026; 7,109,193; 7,067,511; 7,064,120; 7,056,913; 7,0 49,311; 7,022,699; 7,015,215; 6,979,684; 6,951,853; 6,884,799; 6,800,622; 6,747, 144; 6,660,856; 6,608,192; 6,562,806; 6,977,254; 6,951,853; 6,909,006; 6,344,451 ; 5,880,122; 4,935,362; 4,764,616; 4,761,412; 4,723,007; 4,723,003; 4,683,230; 4 ,663,453; 4,508,647; 4,464,467; 4,427,587; 4,000,304; U.S. Patent Application Nos. 20100203007, 2 0100316656, 20030195196. Conjugation of antibody-benzodiazepine dimers via bridge linkers Examples of body structures are: PB01, PB02, PB03, PB04, PB05, and PB0 6, PB07, PB08, PB09, PB10, PB11, PB12, PB13, PB1 4, PB15, PB16, PB17, PB18, PB19, PB20, PB21, and One example is the PB22.

[0216] [ka] JPEG2025138629000078.jpg246167JPEG2025138629000079.jpg228167JPEG2025138629000080.jpg85169

[0217] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; Ab is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. L1, L2, Z1, and Z2 have the same definitions as in formula (I). R1, R2, R3, R 1', R2', and R3' are independently H; F; Cl; ═O; ═S; OH; SH; C1- C8 linear or branched alkyl, aryl, alkenyl, heteroaryl, heteroalkyl , alkylcycloalkyl, ester (COOR5 or -OC(O)R5), ether ( OR5), amide (CONR5), carbamate (OCONR5), amine (NHR5, NR5R5'), heterocycloalkyl, or acyloxylamine (-C(O)NHO H, -ONHC(O)R5); or a peptide containing 1 to 8 natural or unnatural amino acids or formula (OCH2CH2) p or (OCH2CH(CH3)) p Polyethylene oxy unit, where p is an integer from 1 to about 5000. Two R: R1R2, R2 R3, R1R3, R1'R2', R2'R3', or R1'R3' are independently alkyl , aryl, heteroaryl, heteroalkyl, or alkylcycloalkyl group of 3 to 8 X and Y are independently N, CH, or CR; where R5 is H, OH, NH2, NH(CH3), NHNH2, COOH, SH, or OZ 3, SZ3, or C1-C8 linear or branched alkyl, aryl, heteroaryl, hetero Z3 is H, OP(O )(OM1)(OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, or O-glycosides (glucosides, galactosides, mannosides, glucuronosides / glucuronides) glycosides, alosides, fructosides), NH-glycosides, S-glycosides, or CH2-glycosides M1 and M2 are each independently H, Na, K, Ca, Mg, NH4, It is NR1R2R3.

[0218] Several poisonous mushroom genera, most notably Amanita phalloides (Amanita phalloides) At least 10 toxins found in Mushroom Species (Mushroom spp.) and several other mushroom species A subgroup of compounds, the amatoxins, are also preferred for conjugation in this patent. tin, β-amanitin, γ-amanitin, ε-amanitin, amanulin, amanurinic acid, Ten amatoxins, named amaninamide, amanine, and proamanulin, It is a rigid bicyclic peptide, synthesized as a 35 amino acid proprotein, from which The last eight amino acids are cleaved by prolyl oligopeptidase (Litten, W. 1975 Science ntific American232 (3): 90-101;HE Hallen, et al 2007 Proc. Nat. Aca. Sci. US A 104, 19097-101; . K. Baumann, et al, 1993 Biochemistry 32 (15): 4043-50; Karl son-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, PA et al. 19 78 Arch. Microbio. 118 (3): 317-9). Amatoxins inhibit RNA polymerase II ( Potent and selective inhibitor of Pol II, halting gene transcription and protein biosynthesis (Brodner, OG and Wieland, T. 1976 Biochemistry, 15( 16): 3480-4; Fiume, L., Curr Probl Clin Biochem, 1977, 7: 23-8; Karlson-Stiber C, Persson H. 2003, Toxicon 42(4): 339-49; Chafin, DR, Guo, H. & Price, D. H . 1995 J. Biol. Chem. 270 (32): 19114-19; Wieland (1983) Int. J. Pept. Protein R es. 22(3): 257-76.) Amatoxins were isolated from collected Amanita phalloides (Aman ita phalloides) from mushrooms (Yocum, RR 1978 Biochemistry 17(18): 3786-9; Zhang, P. et al, 2005, FEMS Microbiol. Lett. 252(2), 223-8) or basidio Fermentation using bacteria (Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89(1): 73-6), or from fermentation using A. fissa (Guo, XW, et al., 2006 Wei Sheng W u Xue Bao 46(3): 373-8), or Galerina fasciculata or G Produced from the culture of A. alerina helvoliceps (WO / 1990 / 009799, JP11137291) However, the yields from these isolations and fermentations were very low (culture Over the past 30 years, several amatoxins and their analogues have been identified. Chemical or synthetic production has been reported (W. E. Savige, A. Fontana, Chem. Commun. . 1976, 600-1; Zanotti, G., et al, Int J Pept Protein Res, 1981. 18(2): 162-8; W ieland, T., et al, Eur. J. Biochem. 1981, 117, 161-4; PA Bartlett, et al, Te trahedron Lett. 1982, 23, 619-22; Zanotti, G., et al., Biochim Biophys Acta, 198 6. 870(3): 454-62; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 323–9; Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 450-9; Zanot ti, G., et al., Int J Pept Protein Res, 1988. 32(1): 9-20; G. Zanotti, T. et al , They. J. Peptide Protein Res. 1989, 34, 222-8; Zanotti, G., et al., Int J Pept Protein Res, 1990. 35(3): 263-70; Mullersman, JE and JF Preston, 3rd, Int J Pept Protein Res, 1991. 37(6): 544-51; Mullersman, JE, et al, Int J Pept Pro tein Res, 1991. 38(5): 409-16; Zanotti, G., et al, Int J Pept Protein Res, 1992. 40(6): 551-8; Schmitt, W. et al, J. Am. Chem. Soc. 1996, 118, 4380-7; Anderson , MO, et al, J. Org. Chem., 2005, 70(12): 4578-84; JP May, et al, J. Org. Chem. 2005, 70, 8424-30; F. Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; JP May, DM Perrin, Chem. Eur. J. 2008, 14, 3404-9; JP May, et al, Chem. Eur. J. 2008, 14, 3410-17; Q. Wang, et al, Eur. J. Org. Chem. 2002, 834-9; May, JP and DM Perrin, Biopolymers, 2007. 88(5): 714-24; May, J. P ., et al., Chemistry, 2008. 14(11): 3410-7; S. De Lamo Marin, et al, Eur. J. Org Chem. 2010, 3985-9; Pousse, G., et al., Org Lett, 2010. 12(16): 3582-5; Luo, H ., et al., Chem Biol, 2014. 21(12): 1610-7; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5), most of these preparations were by partial synthesis. Due to their extremely potent and unique cytotoxic mechanism, amatoxins are the most commonly used conjugates. (Fiume, L., Lancet, 1969. 2 (7625): 853-4; Barbanti-Broda no, G. and L. Fiume, Nat New Biol, 1973. 243(130): 281-3; Bonetti, E., M. et al, Arch Toxicol, 1976. 35(1): p. 69-73; Davis, M. T., Preston, J. F. Science 1981 , 213, 1385-1388; Preston, J.F., et al, Arch Biochem Biophys, 1981. 209(1): 63-7 1; H. Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, L.S., et al., Pr oc Natl Acad Sci U S A, 1981. 78(5): 3034-8; Faulstich, H. and L. Fiume, Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al, Toxicon, 1987. 25(9): 981-7; Khalacheva, K., et al, Eksp Med Morfol, 1990. 29(3): 26-30; U. Bermbach, H. Faul stich, Biochemistry 1990, 29, 6839-45; Mullersman, J. E. and J. F. Preston, Int . J. Peptide Protein Res. 1991, 37, 544-51; Mullersman, J.E. and J.F. Preston, B iochem Cell Biol, 1991. 69(7): 418-27; J. Anderl, H. Echner, H. Faulstich, Beils tein J. Org. Chem. 2012, 8, 2072-84; Moldenhauer, G., et al, J. Natl. Cancer Ins t. 2012, 104, 622-34; A. Moshnikova, et al; Biochemistry 2013, 52, 1171-8; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5; Zhou, B., et al., Biosens Bioele ctron, 2015. 68: 189-96; WO2014 / 043403, US20150218220, EP 1661584). For some time, we have been working on amatoxin conjugates. Examples of the structure of the syn conjugate are preferably the following Am01, Am02, Am03, and Am04: An example is m04.

[0219] [ka]

[0220] During the ceremony, [ka] can optionally be a single or double bond, or optionally be absent; X and Y are independently O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1. mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 2 0; R7, R8, and R9 are independently H, OH, OR1, NH2, NHR1, C 1-C6 alkyl or absent; Y2 is O, O2, NR1, NH or absent; R 10are CH2, O, NH, NR1, NHC(O), NHC( O)NH, NHC(O)O, OC(O)O, C(O), OC(O), OC(O)(NR1 ), (NR1)C(O)(NR1), C(O)R1, or absent; R 11 are OH, NH2, NHR1, NHNH2, NHNHCOOH, O-R1-COOH, N H-R1-COOH, NH-(Aa) n COOH, O(CH2CH2O) p CH2CH2 OH, O(CH2CH2O) p CH2CH2NH2, NH(CH2CH2O) p CH2C H2NH2, NR1R1', O(CH2CH2O) p CH2CH2COOH, NH(CH 2CH2O) p CH2CH2COOH, NH-Ar-COOH, NH-Ar-NH2, O (CH2CH2O) p CH2CH2NHSO3H, NH(CH2CH2O) p CH2CH 2NHSO3H, R1-NHSO3H, NH-R1-NHSO3H, O(CH2CH2O ) p CH2CH2NHPO3H2, NH(CH2CH2O) p CH2CH2NHPO3H 2, OR1, R1-NHPO3H2, R1-OPO3H2, O(CH2CH2O) p CH 2CH2OPO3H2, OR1-NHPO3H2, NH-R1-NHPO3H2, or N H(CH2CH2O) p CH2CH2NHPO3H2, where Aa is 1 to 8 alkyl groups. amino acid; n and m1 are independently 1 to 20; p is 1 to 5000; R1, L1 and L2 have the same definition as in formula (I); L1, L2, R1, Z1, and Z2 are The definition is the same as in formula (I).

[0221] In yet another embodiment, the immunotoxin is conjugated to a cell-binding molecule via a conjugate of the present application. The immunotoxin used herein is generally a diphtheria toxin (DT), a cholera toxin (CTH), or the like. Trichosanthin (CT), trichosanthin (TCS), dianthin, Pseudomonas exotoxin A (ET A'), bacterial or plant proteins such as erythrotoxin, diphtheria toxin, AB toxin, and type III exotoxin It is a macromolecular drug that is a cytotoxic protein derived from proteins. It could be a highly toxic bacterial pore-forming protoxin that requires proteolytic processing for its release. Examples of such protoxins include proaerolysin and its genetically modified forms, and topsalysin. Topsalysin is a prostate A modified recombinant protein designed to be selectively activated by an enzyme, It causes localized cell death and tissue destruction without damaging the underlying tissues and nerves.

[0222] In yet another embodiment, the cell-binding ligand or cell receptor agonist is a bis-linked These conjugated cell-binding ligands can be conjugated to cell-binding molecules via A targeting compound or receptor, particularly an antibody-receptor conjugate, is used to deliver the conjugate to malignant cells. act as inductors / directors, but also modulate or costimulate the desired immune response or to alter signal transduction pathways.

[0223] In immunotherapy, cell-binding ligands or receptor agonists bind to TCR (T cell receptor ) T cells, or CAR (chimeric antigen receptor) T cells, or B cell receptor (BCR), Preferably, the antibody is conjugated to an antibody of a natural killer (NK) cell or a cytotoxic cell. Such antibodies are preferably anti-CD3, CD4, CD8, CD16 (FcγRIII), CD27, CD40, CD40L, CD45RA, CD45RO, CD56, CD57, CD57 bright , TNFβ, Fas ligand, MHC class I molecules (HAL-A, B, C), or NKR-P1. The cell-binding ligand or receptor is not limited to However, folate derivatives (folate receptors, proteins overexpressed in ovarian cancer and other malignancies) (Low, PS et al 2008, Acc. Chem. Res. 41, 120-129); glutamateuria A derivative of thiazolinone (binding to prostate-specific membrane antigen, a surface marker of prostate cancer cells) (Hillier, S. M.et al., 2009, Cancer Res. 69, 6932-6940); somatostatin (growth hormone inhibitor) Mon (GHIH), somatotropin-releasing inhibitory factor (SRIF), or somatotropin-releasing inhibitory hormones) and their derivatives, such as octreotide ( doxorubicin) and lanreotide (somatulin) (especially for neuroendocrine tumors, GH-producing pituitary gland adenoma, paraganglioma, dysfunctional pituitary adenoma, pheochromocytoma) (Ginj, M., et al, 2006, Pro c. Natl. Acad. Sci. USA 103, 16436-16441); in general, somatostatin and its receptors The receptor subtypes (sst1, sst2, sst3, sst4, and sst5) are Many types of tumors, including secretory tumors, especially GH-secreting pituitary adenomas (Reubi JC, Landolt, A. M. 1984 J. Clin. Endocrinol Metab 59: 1148-51; Reubi J. C., Landolt A. M. 1987 J Clin Endocrinol Metab 65: 65-73; Moyse E, et al, J Clin Endocrinol Metab 61: 98-103) and gastrointestinal and pancreatic tumors (Reubi J. C., et al, 1987 J Clin Endocrinol Metab 65: 11 27-34; 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 J. C., et al, 1992 J Cl in Endocrinol Metab 74: 1082-9), neuroblastoma (Prevost G, 1996 Neuroendocrinolo gy 63:188-197; Moertel, C. L, et al 1994 Am J Clin Path 102:752-756), medullary thyroid carcinoma (Reubi, J. C, et al 1991 Lab Invest 64:567-573), small cell lung carcinoma (Sagman U, et al, 1990 Cancer 66:2129-2133), and non-neuroendocrine tumors including brain tumors such as meningioma, medulloblastoma, and glioma (Reubi J. C., et al 1986 J Clin Endocrinol Metab 63: 433-8; Reubi J . C., et al 1987 Cancer Res 47: 5758-64; Fruhwald, M. C, et al 1999 Pediatr Res . C., et al 1987 Cancer Res 47: 5758-64; Fruhwald, M. C, et al 1999 Pediatr Res 45: 697-708), breast cancer (Reubi J. C., et al 1990 Int J Cancer 46: 416-20; Srkalov ic G, et al 1990 J Clin Endocrinol Metab 70: 661-669), lymphoma (Reubi J. C., e t al 1992, Int J Cancer50: 895-900), renal cell carcinoma (Reubi J. C., et al 1992, Cance r Res 52: 6074-6078), mesenchymal tumors (Reubi J. C., et al 1996 Cancer Res 56: 1922-3 1), prostate (Reubi J. C., et al 1995, J. Clin. Endocrinol Metab 80: 2806-14; et al 1989, Prostate 14:191-208; Halmos G, et al J. Clin. Endocrinol Metab 85: 2564 -71), ovary (Halmos, G, et al, 2000 J Clin Endocrinol Metab 85: 3509-12; Reubi J. C., et al 1991 Am J Pathol 138:1267-72), stomach (Reubi J. C., et al 1999, Int J Cancer 81: 376-86; Miller, G. V, 1992 Br J Cancer 66: 391-95), hepatocellular (Kourou malis E, et al 1998 Gut 42: 442-7; Reubi J. C., et al 1999 Gut 45: 66-774), and pharyngeal cancer (Loh K. S, et al, 2002 Virchows Arch 441: 444-8); specific aromatic sulfonamides specific for carbonic anhydrase IX (a marker for hypoxia and renal cell carcinoma) ​Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-777); Pituitary adenylate cyclase-activating peptide (PACAP) for transglandular tumors 1) for cancers of the lung, stomach, colon, rectum, breast, prostate, pancreatic duct, liver, bladder, and epithelial carcinomas Vasoactive intestinal peptide (VIP) and its receptor subtypes (VPAC1, VCA P2); α-melanocyte-stimulating hormone (α-MSH) receptors for various tumors; small cell lung Cholecystokinin for cancer, medullary thyroid carcinoma, astrocytoma, insulinoma, and ovarian cancer (CCK) / gastrin receptors and their receptor subtypes (CCK1 (formerly CCK -A) and CCK2; renal cell carcinoma, breast cancer, lung cancer, gastric cancer, and prostate cancer, as well as neuroblastoma ( and bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-) for neuroblastoma (neuroblastoma). Trp-Ala-Val-Gly-H is-Leu-Met-NH2) / gastrin-releasing peptide (GRP) and its receptor Body subtypes (BB1, GRP receptor subtype (BB2), BB3, and BB4) (Oh lsson, B., et al, 1999, Scand. J. Gastroenterology 34 (12): 1224-9; Weber, H. C ., 2009, Cur. Opin. Endocri. Diab. Obesity 16(1): 66-71, Gonzalez N, et al, 2008 , Cur. Opin. Endocri. Diab. Obesity 15(1), 58-64); small cell lung cancer, neuroblastoma, pancreatic Neurotensin (NTR1, NTR2, NTR3) for pancreatic and colon cancer; glial Substance P and its receptor subtypes in tumors (e.g., NK1 receptor for glial tumors, Hennig IM, et al 1995 Int. J. Cancer 61, 786-792); Neuropeptides for breast cancer Y thiol and its receptor subtypes (Y1-Y6); RGD (Arg-Gly-Asp), NGR (Asn -Gly-Arg), a dimeric and multimeric cyclic RG that recognizes receptors (integrins) on tumor surfaces D peptide (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, Antica Ncer Research, 19(2A), 959-968; Thumshirn, G, et al, 2003 Chem. Eur. J. 9, 2717- 2725), and TAASGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor) and F3 peptide (cell surface-expressed nucleolin receptor) 31 amino acid peptide that binds to the IL-1 receptor (Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139 -5143, Temminga, K., 2005, Drug Resistance Updates, 8, 381-402; P. Laakkonen an d K. Vuorinen, 2010 Integrative Biol, 2(7-8), 326-337; MA Burg, 1999 Cancer R es., 59(12), 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11) , 7444-9); cell-penetrating peptides (CPPs) (Nakase I, et al. l, 2012, J. Control Release. 159(2),181-188); peptide hormones, e.g., testosterone As well as steroid production, it also regulates the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Luteinizing hormone-releasing hormone (LHRH) agonists and antagonists that act by targeting agonists, as well as gonadotropin-releasing hormone (GnRH) agonists, such as Buserene Phosphorus (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), gonadorelin (Pyr-His- Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtB u)-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 (Py r-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, deslorelin, ava Relics (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu- isopropylLys-Pro-DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl) Lysyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4- chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(calcium phosphate) bamoyl)-Leu-isopropylLys-Pro-D-Ala-NH2), and ganirelix (Ac-D-2Nal-D-4-ch loroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N9,N10-diethyl)-homoArg-Leu-(N9,N10-diethyl) Ethyl)-homoArg-Pro-D-Ala-NH2)(Thundimadathil, J., J. Amino Acids, 2012, 96734 7, doi:10.1155 / 2012 / 967347; Boccon-Gibod, L.; et al, 2011, Therapeutic Advances in Urology 3 (3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696; Schally A. V; Nagy, A. 1999 Eur J Endocrinol 141:1-14; Koppan M, et al 1999 Pros tate 38:151-158); and small molecules such as imiquimod, guanidine, and adenosine Lipopolysaccharide (LPS), nucleic acids (CpG DNA, poly I;C) and lipopeptides Pam3CSK4 (Kasturi, SP, et al, 2011, Nature 470, 543-547; Lan e, T., 2001, JR Soc. Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmu 1, 227-228; Dudek, AZ, et al, 2007, Clin. Cancer Res. 13, 7119-7125) Toll-like receptors (TLRs), which recognize large and complex biological macromolecules such as C type lectins, and Nod-like receptors (NLRs) (Fukata, M., et al., 2009, Semin. Immu nol. 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, 2 000, Life Sci. 68, 241-258) and other pattern recognition receptors (PRRs). A 32-amino acid neuropeptide that is broadly involved in regulating calcium levels through its effects on 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); various cells, especially osteoclasts, It is expressed on the surface of endothelial cells and tumor cells and generally plays an important role in angiogenesis. Integrin receptors and their receptor subtypes (e.g., αVβ1, αVβ3, αV β5, αVβ6, α6β4, α7β1, αLβ2, αIIbβ3) (Ruoslahti, E. et a l, 1994 Cell 77, 477-8; Albelda, SM et al, 1990 Cancer Res., 50, 6757-64). A short peptide and GRGDSPK that showed high binding affinity to integrin receptors, and Cyclic RGD pentapeptides such as cyclo(RGDfV)(L1) and their derivatives [cyclo( -N(Me)R-GDfV), cyclo(R-Sar-DfV), cyclo-(RG-N(M e)D-fV), cyclo(RGD-N(Me)fV), cyclo(RGDf-N(Me) V-)(Cilengitide)](Dechantsreiter, MA et al, 1999 J. Med. Ch em. 42, 3033-40, Goodman, SL, et al, 2002 J. Med. Chem. 45, 1045-51).

[0224] Cell-binding ligands or cell receptor agonists bind to Ig-based and non-Ig-based proteins. The Ig-based scaffold can be, but is not limited to, Nanobodies (VHH (camelid Ig) derivatives) (Muyldermans S., 20 13 Annu Rev Biochem. 82, 775-797); domain antibodies (dAb, VH or VL domains) derivatives) (Holt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); bispecific T cell induction (BiTE, bispecific dimer) (Baeuerle, P. A, et al, 2009, Curr. Opin. M ol. Ther. 11, 22-30); dual affinity retargeting (DART, bispecific dimer) (Moore P A. P, et al. 2011, Blood 117(17), 4542-4551; tetravalent tandem antibody (TandAb , bispecific dimer) (Cochlovius, B, et al. 2000, Cancer Res. 60(16):4336-4341) Non-Ig scaffolds can be selected from, but are not limited to, anticalins ( Lipocalin derivatives) (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 (FN3 (Finally bronectin)) (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 Re s. 17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 23(4):211-219); Developed ankyrin repeat proteins (DARPins) (ankyrin repeat (AR) ) Protein derivatives) (Boersma, YL, et al, 2011 Curr Opin Biotechnol. 22(6): 8 49-857), such as DARPinC9, DARPinEc4, and DARPinE69_L Z3_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. 286(48),41273-41285; Avimer (Domain A / Low Density Low-density lipoprotein (LDL) receptor) (Boersma Y. L, 2011 J. Biol. Chem. 286(48): 4 1273-41285; Silverman J, et al, 2005 Nat. Biotechnol., 23(12):1556-1561). You can choose.

[0225] The present invention relates to a method for the preparation of a bis-linked antibody and a cell-binding ligand, a cell receptor agonist, or Examples of drug conjugate structures are shown below: LB01 (folic acid conjugate), LB02 (PMSA conjugate), Ligand conjugate), LB03 (PMSA ligand conjugate), LB04 (PMSA ligand conjugate), LB05 (somatostatin conjugate), LB06 (somatostatin conjugate), L B07 (octreotide, somatostatin analogue conjugate), LB08 (lanreotide, somatostatin analogue conjugate), Somatostatin analogue conjugate), LB09 (vapreotide (Sanvar), somatostatin LB10 (CAIX ligand conjugate), LB11 (CAIX ligand conjugate) conjugate), LB12 (gastrin-releasing peptide receptor (GRPr), MBA conjugate), L B13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate) , LB15 (GnRH antagonist, abarelix conjugate), LB16 (cobalamin , VB12 analogue conjugate), LB17 (cobalamin, VB12 analogue conjugate), LB18 (α v β3 integrin receptor, cyclic RGD pentapeptide conjugate), LB19 (VEG Hetero-bivalent peptide-ligand conjugate for F receptor), LB20 (neuromedin B conjugate) conjugate), LB21 (bombesin conjugate for G protein-coupled receptors), LB22 (T TLR2 conjugate for oll-like receptor), LB23 (for androgen receptor), LB24(α v Cilengitide / cyclo(-RGDfV)- for integrin receptors ) conjugate), LB23 (fludrocortisone conjugate), LB25 (rifabutin analogue conjugate) LB26 (rifabutin analogue conjugate), LB27 (rifabutin analogue conjugate) ), LB28 (fludrocortisone conjugate), LB29 (dexamethasone conjugate), LB 30 (fluticasone propionate conjugate), LB31 (beclomethasone dipropionate conjugate) ), LB32 (triamcinolone acetonide conjugate), LB333 (prednisone conjugate ), LB34 (prednisolone conjugate), LB35 (methylprednisolone conjugate), L B36 (betamethasone conjugate), LB37 (irinotecan analogue conjugate), LB38 ( LB39 (bortezomib analogue conjugate), LB40 (calf LB41 (carfilzomib analogue conjugate), LB42 (lufilzomib analogue conjugate) LB43 (triptorelin analogue conjugate), LB44 (clinda mycin conjugate), LB45 (liraglutide analogue conjugate), LB46 (semaglutide analogue conjugate) LB47 (retapamulin analogue conjugate), LB48 (indibulin analogue conjugate) analogue conjugate), LB49 (vinblastine analogue conjugate), LB50 (lixisenatide analogue conjugate) LB51 (osimertinib analogue conjugate), LB52 (nucleoside analog conjugate), LB53 (erlotinib analog conjugate), and LB54 (lapatinib analog conjugate) (Rim conjugate).

[0226] [ka] JPEG2025138629000085.jpg251170JPEG2025138629000086.jpg254169JPEG202 5138629000087.jpg255162JPEG2025138629000088.jpg228159JPEG20251386290 00089.jpg233148JPEG2025138629000090.jpg252170JPEG2025138629000091.j pg234170JPEG2025138629000092.jpg233148JPEG2025138629000093.jpg239159

[0227] During the ceremony, [ka] is optionally either a single or double bond, or absent; X1 and Y1 are independently Then, O, NH, NHNH, NR5, 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), CH, C(O)NHNHC(O), and C(O)NR1; mAb is an antibody, preferably a monoclonal antibody; n and m1 are independently 1 to 20. L1, L2, R1, R1', R2, Z1, and Z2 have the same definitions as in formula (I). . X3 is CH2, O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(NR), R, NHR, NR, or C(O)R, or absent; X4 is H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1 ), R1, NHR1, NR1, C(O)R1, or C(O)O; X5 is H, CH3 , F, or Cl; M1 and M2 are each independently H, Na, K, Ca, Mg, NH4, NR1R2R3; R6 is 5'-deoxyadenosyl, Me, OH, or C It's N.

[0228] In yet another embodiment, one, two, or more of DNA, RNA, mRNA, small molecule interferon, Interfering RNA (siRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) is preferably conjugated to a cell-binding molecule via the bis-linker of the present application. NAs (siRNA, miRNA, piRNA) and long non-coding antisense RNAs A is known to be involved in epigenetic changes within cells (Goodchil d, J (2011), Methods in molecular biology (Clifton, NJ). 764: 1-15). DNA, RNA, mRNA, siRNA, miRNA, or piRNA in It may be single-stranded or double-stranded, having from 1 to 1 million nucleotide units, Some of the oxidases are phosphodiesterases, exemplified by fomivirsen. thioate-linked oligonucleotides or mipomersen (Mipomers en), rather than the phosphodiester bonds of natural RNA and DNA. The sugar moiety in the center of the molecule is deoxyribose, and the two nucleotides with 2'-O-methoxyethyl modified ribose at one end, or peptides thiophosphoramide (PNA), morpholino, phosphorothioate, thiophosphoramidate, or 2'-O-Methoxyethyl (MOE), 2'-O-Methyl, 2'-Fluoro, Locked Nucleus oligonucleotides made with ribose sugar bicyclic nucleic acids (LNA) or ribose sugar bicyclic nucleic acids (BNA) oxide, or a nucleus modified to remove the 2'-3' carbon bond in the sugar ring structure It may also be in a non-natural (synthetic) form, such as an acid (Whitehead, KA; et al (2011), Annual Report eview of Chemical and Biomolecular Engineering 2: 77-96; Bennett, CF; Swayze, EE (2010), Annu. Rev. Pharmacol. Toxicol. 50: 259-29). Preferably, oligonucleotides The length of the nucleotides ranges from about 8 to over 100 nucleotides. Examples of conjugate structures are shown below. Shown below:

[0229] [ka]

[0230] In the formula, mAb, m1, n, X1, L1, L2, Z1, Z2, [ka] is the same as in formula (I) or as defined above; [ka] is a single strand of DNA, RNA, mRNA, siRNA, miRNA, or piRNA It is double-stranded; Y is preferably O, S, NH, or CH2.

[0231] In yet another embodiment, one, two, or more different functional molecules or agents and an IgG antibody. The conjugates with preferably have the following structures: ST1, ST2, ST3, ST4, ST5, or As shown in ST6, the disulfide bonds between the light chain and the heavy chain, the upper disulfide bonds between the two heavy chains, and a pair of thiols (by reduction of disulfide bonds) on the lower side between the two heavy chains, which is specific is conjugated to:

[0232] [ka] JPEG2025138629000099.jpg132166

[0233] In the formula, Z1, Z2, X, Y, L1, L2, [ka] , m1, and the cytotoxic molecule have the same definition as X1 in formula (I) above.

[0234] Additionally, cytotoxic molecules containing the same or different bis-linkers can be sequentially conjugated to cell-binding molecules. Alternatively, different cytotoxic molecules containing the same or different bis-linkers may be used to bind to the cell-binding moiety. When added stepwise to a conjugation reaction mixture containing ATP, the ATP is transferred to different conjugation sites on the cell-binding molecule. The cytotoxic molecule and m1 in may be different.

[0235] Formulation and application

[0236] The conjugates of the present application may be formulated in liquid form or lyophilized and then reconstituted into a liquid formulation. Suitable for conjugating active ingredients to patients without high levels of antibody aggregation. Liquid formulations containing the compound at a concentration of 0.1 g / L to 300 g / L may contain polyols (e.g., sugars), pH 4.5-7.5 buffer, surfactant (e.g., polysorbate 20 or 80), oxidizing agents (e.g., ascorbic acid and / or methionine), tonicity agents (e.g., mannitol chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes; biodegradable polymers such as polyesters; preservatives (e.g., The composition may contain one or more of: glycerol, ...

[0237] Suitable buffering agents for use in the formulation include, but are not limited to, citric acid, ascorbic acid, organic acid salts such as salts of carboxylic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; thris, tromethamine (tris(hydroxymethyl)-aminomethane) hydrochloride, or phosphate Buffer solutions include amino acid components. The amino acid components include, but are not limited to, arginine, glycine, glycylglycine, and histidine. Arginine buffers include arginine acetate, arginine hydrochloride, and arginine acetate. Examples include arginine phosphate, arginine sulfate, and arginine acid succinate. In the example, an arginine buffer is arginine acetate. An example of a histidine buffer is histidine acetate. Histidine Hydrochloride-Arginine Hydrochloride, Histidine Acetate-Arginine Acetate, Histidine Phosphate - Arginine phosphate, Histidine sulfate - Arginine sulfate, Histidine succinate Buffer formulations include arginine succinate, arginine succinate, etc. Buffer formulations range from pH 4.5 to pH 7.5 , preferably about 4.5 to about 6.5, more preferably about 5.0 to about 6.2. In this embodiment, the concentration of the organic acid salt in the buffer is from about 10 mM to about 500 mM.

[0238] A "polyol" that may optionally be included in the formulation is a substance that has multiple hydroxyl groups. Polyols act as stabilizers and / or tonicity enhancers in both liquid and lyophilized formulations. Polyols can be used as barrier agents against both physical and chemical degradation pathways. Preferentially excluded cosolvents can protect biopharmaceuticals at the protein interface. Increase the effective surface tension of the solvent in the solution, thereby promoting the most energetically favorable conformation. Polyols include sugars (reducing and non-reducing sugars) "Reducing sugars" include sugars that can reduce metal ions or proteins. containing a hemiacetal group capable of covalently reacting with lysine and other amino groups in proteins "Non-reducing sugars" are those that do not have these properties of reducing sugars. , for example, fructose, mannose, maltose, lactose, arabinose, xylose Non-reducing sugars include ribose, rhamnose, galactose, and glucose. Sugars include sucrose, trehalose, sorbose, melezitose, and raffinose. Cole contains mannitol, xylitol, erythritol, maltitol, and lactitol. , erythritol, threitol, sorbitol, and glycerol. Acids include L-gluconate and its metal salts. Preferably, non-reducing sugars: 0.01% in the formulation. Sucrose or trehalose at a concentration of about 10% to 15% is selected. The solution stability of lehalose makes it preferable to sucrose.

[0239] Surfactants optionally included in the formulation include polysorbates (polysorbate 20, polysorbate 21, polysorbate 22, polysorbate 23, polysorbate 24, polysorbate 25, polysorbate 26, polysorbate 27, polysorbate 28, polysorbate 29 ... Polysorbate 40, Polysorbate 65, Polysorbate 80, Polysorbate 81, Polysorbate rubet 85, etc.); poloxamers (e.g., poloxamer 188, poly(ethylene oxy) do)-poly(propylene oxide), poloxamer 407 or polyethylene polypropylene glycol, etc.), Triton; sodium dodecyl sulfate (SDS), sodium lauryl sulfate sodium octylglycoside; lauryl-, myristyl-, linoleyl-, or Stearyl sulfobetaine; lauryl, myristyl, linoleyl, or stearyl Linolyl, myristyl, or cetyl betaine; Lauramidopro Pill-, Cocamidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, Pall isostearamidopropyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl) myristamidopropyl-, palmidopropyl-, or isostearamidopropyl -dimethylamine; sodium methyl cocoyl taurate or methyl oleyl taurine Sodium; Dodecyl Betaine, Dodecyl Dimethylamine Oxide, Cocamidopropyl Betaine Tine, and cocoamphoglycinate; "MONAQUAT™" (trademark) series ( For example, isostearyl ethylimidonium ethosulfate); polyethylene glycol Coal, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.) and the like. Preferred surfactants are poly Oxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80 (Tween 20, 40, 60, or 80). In certain embodiments, the concentration of the surfactant ranges from 0.0001% to about 1.0%. The concentration is from about 0.01% to about 0.1%. In one embodiment, the surfactant concentration is about 0.0 It is 2%.

[0240] "Preservatives" that are optionally included in the formulation are compounds that reduce bacterial activity in the formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexadecyldimethylbenzylammonium chloride, Benzalkonium chloride (the alkyl group is a long-chain compound) Benzethonium chloride, a mixture of dimethylammonium chloride, and benzethonium chloride. Types of preservatives include phenol, butyl and benzyl alcohol, methyl or propyl phenol Alkylparabens such as lavendone, catechol, resorcinol, cyclohexanol, 3- Preservatives include aromatic alcohols such as pentanol and m-cresol. In one embodiment, the anti-oxidant in the present specification is less than 5%. The preservative is benzyl alcohol.

[0241] Suitable free amino acids for optional use in the formulation include, but are not limited to, arginine, Leucine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine Glutamic acid, or aspartic acid. Basic amino acids include arginine, Lysine and / or histidine are preferred. When the composition contains histidine, it acts as a buffer. The histidine buffer may act both as a buffer and as a free amino acid. Typically, these contain a histidine buffer and a non-histidine free amino acid, including lysine. Amino acids may be present in their D and / or L forms, although the L form is typical. The amino acid may be present as any suitable salt, such as a hydrochloride salt, such as arginine-HCl. The concentration of the amino acid ranges from 0.0001% to about 15.0%. Preferably, the concentration is 0.01 %~5%.

[0242] The formulation optionally contains methyl as an antioxidant at a concentration of about 0.01 mg / ml to 5 mg / ml. onic acid or ascorbic acid; the formulation optionally contains 0.01 mM to 2 mM A chelating agent such as EDTA, EGTA, etc. may be included in the solution at a concentration of 0.1%.

[0243] The final formulation may be prepared by adding modifiers (e.g., HCl, H2SO4, acetic acid, H3PO4, citric acid) Acids such as NaOH, KOH, NH3OH, ethanolamine, diethanolamine, or triethanolamine, sodium phosphate, potassium phosphate, trisodium citrate The pH can be adjusted to a desired value with a base such as tromethamine, and the formulation The solution must be controlled to be "isotonic," meaning that it has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Tonicity can be measured, for example, using vapor pressure or ice osmolality.

[0244] Other additives that may be useful in either the liquid or lyophilized formulations of the present application include, for example, cose, cellobiose, maltotriose, melibiose, octulose, ribose, Xylitol, arginine, histidine, glycine, alanine, methionine, glutamine Acid, Lysine, Imidazole, Glycyrglycine, Mannosylglyceride, Triton X-1 00, Pluronic F-127, cellulose, cyclodextrin, dextran (1 0, 40, and / or 70 kD), polydextrose, maltodextrin, Ficoll , gelatin, hydroxypropyl methacrylate, sodium phosphate, potassium phosphate, ZnCl2 , zinc, zinc oxide, sodium citrate, trisodium citrate, tromethamine, copper, Ibronectin, heparin, human serum albumin, protamine, glycerin, glycerol EDTA, metacresol, benzyl alcohol, phenol, polyhydroalcohol Polyhydric alcohols or polyalcohols, primary or secondary hydric These include hydrogenated forms of carbohydrates in which the hydroxyl group has been reduced to a carbonyl group.

[0245] Other possible additives that may be utilized in the aqueous pharmaceutical compositions of the present application include, for example, flavoring agents. , antibacterial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, cholesterol steroids such as steroids, serum albumin (human serum albumin) and other protein excipients, recombinant Contains human albumin, gelatin, casein, and salt-forming counter ions such as sodium. These and other suitable pharmaceutical excipients and / or additives for the formulations of the present invention are, for example, e Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., America n Pharmaceuticals Association (2003); Remington: the Science and Practice of Pha As shown in Medicine, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005) Such techniques are well known in the art.

[0246] In a further embodiment, the present invention provides a method for preparing a formulation comprising the steps of: (a) a lyophilization step to powderize the formulation containing the conjugate, excipients, and buffer system; and (b) Lyophilizing step (a) in a reconstitution medium such that the reconstituted formulation is stable. Reconstitution of the mixture. The formulation of step (a) is selected from the group consisting of bulking agents, salts, surfactants, and preservatives. The reconstitution solvent may further comprise a stabilizer and one or more excipients selected from the following: Some dilute organic acids or water, i.e., sterile water, bacteriostatic water for injection (BWFI), may be used. The reconstitution solvent is water, i.e., sterile water, water for injection (BWFI), or about 10 to about 250 ml M amounts of acetic acid, propionic acid, succinic acid, sodium chloride, magnesium chloride, sodium chloride from the group consisting of an acidic solution of sodium, an acidic solution of magnesium chloride, and an acidic solution of arginine. You can choose from:

[0247] Liquid pharmaceutical formulations of the present conjugates should exhibit various predefined properties. One of the main concerns for pharmaceuticals is the soluble and insoluble nature of proteins / antibodies during manufacturing and storage. It is stable because it has a tendency to form aggregates. Various chemical reactions occur in the sugars (e.g., sugar topping, high fructose corn syrup), resulting in a decrease in product levels and / or physiological activity. Preferably, the conjugate in the liquid or lyophilized formulation is The conjugate in the liquid or lyophilized formulation should exhibit a shelf life of at least 18 months at 25°C. Preferably, the liquid should exhibit a shelf life of at least 24 months at 25°C. The formulation should exhibit a shelf life of approximately 24-36 months at 2-8°C. The lyophilized formulation should exhibit a shelf life of approximately 24-36 months at 2-8°C. It should preferably exhibit a shelf life of approximately 60 months at 8°C. Both the liquid and lyophilized formulations The product should exhibit a shelf life of at least 2 years at -20°C or -70°C.

[0248] In certain embodiments, the formulation is frozen (e.g., at -20°C or -70°C) and subsequently The formulation is stable to thawing, eg, 1, 2, or 3 cycles of freezing and subsequent thawing. Stability is assessed by drug / antibody (protein) ratio and assessment of aggregate formation (e.g., UV and size using exclusion chromatography or by turbidity measurements and / or visual inspection); Cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or by assessing charge heterogeneity using capillary zone electrophoresis; Terminal or carboxy-terminal sequence analysis; mass spectrometry or matrix-assisted laser desorption ionization / Time-of-flight mass spectrometry (MALDI / TOF MS) or HPLC-MS / MS; SDS-PAGE analysis comparing the purified and intact antibodies; peptide maps (e.g., transcripts); lyptic or LYS-C) analysis; evaluation of antibody biological activity or antigen-binding function; etc. It can be assessed qualitatively and / or quantitatively in a variety of different ways. Aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (hinge region fragmentation, etc.) , succinimide formation, cysteine ​​unpairing, N-terminal extension, C-terminal processing, differential glycosylation It may also include the following:

[0249] A stable conjugate also means that in a pharmaceutical formulation, the pharmaceutical formulation remains stable over a given period of time, e.g., 12 months. As manufactured, antigen binding assays and / or in vitro cytotoxicity assays, etc. Within about 20%, preferably within about 10% of the biological activity determined by the assay (assay error). The compound must "retain biological activity" within the range of 0.1 to 1.5%.

[0250] A pharmaceutical container or vessel is a container or vessel for any of the conjugates of the present application. These containers are used to hold pharmaceutical preparations. syringe, or a prefilled auto-injector syringe.

[0251] For in vivo clinical applications, the bis-linked conjugates of the present invention may be administered in solution or injected. It is provided in the form of a lyophilized solid that can be redissolved in sterile water for injection. Examples of administration methods for the conjugate are as follows: The conjugate may be administered daily, weekly, every two weeks, or every three weeks. It is administered as an intravenous bolus every 4 weeks or once a month for 8 to 54 weeks. Dissolve the dose in 50 to 1000 mL of physiological saline, add human serum albumin to the physiological saline, and (e.g., 0.5 to 1 ml of concentrated human serum albumin solution can be added to 100 ml The drug dosage is approximately 50 μg to 20 mg / kg body weight per week, administered intravenously (each time). The injection dose ranges from 10 μg to 200 mg / kg. After 4 to 54 weeks of treatment, The patient may receive a second course of treatment. The exact treatment method, including dosage and duration, can be determined by an experienced surgeon. do.

[0252] Treating diseases by selectively killing cell populations using in vivo or ex vivo methods Examples include any type of cancer, autoimmune diseases, transplant rejection, and infectious diseases (including (including viruses, bacteria or parasites).

[0253] Several factors affect the amount of conjugated drug required for a desired biological effect. The characteristics of the compound, the efficacy and bioavailability of the conjugate drug, the type of disease, the patient's race, the patient's The administration schedule and dosage are determined by taking these factors into account. The route is determined.

[0254] Generally, the bis-linked conjugates of the present invention are useful at concentrations of 0.1 to 10% w / v. The conjugate may be dissolved in a physiological buffer solution to form a formulation for parenteral administration. Typical dose ranges are 1 μg / kg body weight to 0.1 g / kg body weight per day, given weekly for 2 weeks. weekly, three-weekly, or monthly. The preferred dose range is 0.01 mg / kg per day. ~20 mg / kg body weight weekly, every two weeks, every three weeks, or monthly, or equivalent to the human dose The preferred drug dosage depends, for example, on the type and degree of progression of the disease or disorder, the individual the patient's overall health, the relative biological activity of the selected drug, the compound's dosage form, and administration Mode (intravenous, intramuscular, or other), pharmacokinetics of the drug for the selected mode of administration characteristics, as well as the rate of administration (single injection or continuous infusion) and schedule of administration (administration over a period of time). The appropriate dependence on variables such as the frequency of administration.

[0255] The conjugates of the present invention via the linker may also be administered in unit dosage amounts, where "unit dosage" refers to "Quantity" means a single dose administered to one patient and is to be used in simple and convenient packaging. The active conjugates can be physically and / or therapeutically effective as active conjugates themselves or as pharmaceutically acceptable compositions as described below. It maintains a stable dosage and chemically stable unit dose. The dosage range is 0.01 to 100 mg / kg body weight. General guidance is: The unit dosage ranges from 1 to 3000 mg per day, week, biweekly, or month. The dosage is preferably 1 mg to 500 mg administered 1 to 4 times a week, and more preferably Preferably, 1 mg to 100 mg is administered once a week. The pharmaceutical composition may be prepared by adding one or more pharmaceutically acceptable excipients to the pharmaceutical composition. The unit dose of the drug may be packaged as a tablet, simple capsule, or soft capsule for oral administration. for intranasal administration as a powder, nasal drops, or aerosol; or for dermal administration For administration, the agent may be administered as an ointment, cream, lotion, gel, or spray or skin patch. It can be administered as follows.

[0256] In yet another embodiment, a therapeutically effective amount of a conjugate of formula (II) or any of the compounds described throughout this patent is administered. Pharmaceutical compositions containing any of the conjugates may be used in chemotherapy, radiation therapy, immunotherapy, autoimmune disease, and the like. and other conjugates such as anti-cancer agents, anti-infective agents, or other conjugates for the synergistically effective treatment or prevention of cancer. The synergistic agent may be administered simultaneously with the therapeutic agent. Preferably, the drug is selected from the following: abatacept (Orencia), abiraterone acetate (Zytig) a®), Abraxane, acetaminophen / hydrocodone, adalimumab, Afatinib dimaleate (Gilotrif®), alectinib (Alecensa), lemtuzumab (Campath®), alitretionin (Panretin®), Ado-trastuzumab (Kadcyla™), amphetamine mixed salts (amphetamine Adderall XR), anastrozole (Arimidex (registered trademark) Registered trademark), aripiprazole, atazanavir, atezolizumab (MPDL3280A), Vastatin, axitinib (Inlyta®), avelumab, belinostat (Be Leodaq™), bevacizumab (Avastin®), cabazitaxel (Jevtana (Registered Trademark), cabozatinib (Cometriq™), bexarotene (Targretin blinatumomab (Blincyto™), bortezomib (Velcade® ), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), Trademark), budesonide, budesonide / formoterol, buprenorphine, capecitabine , carfilzomib (Kyprolis®), celecoxib, ceritinib (LDK378 / Zyk adia), cetuximab (Erbitux®), cyclosporine, cinacalcet, Rizotinib (Xalkori®), Cosentyx, CTL0 19. Dabigatran, dabrafenib (Tafinlar®), daratumab (Darzalex ), darbepoetin alfa, darunavir, imatinib mesylate (Gleevec®), da Satinib (Sprycel®), denileukin diftitox (Ontak®) ), denosumab (Xgeva®), Depakote, dexlansoprazole Lu, dexmethylphenidate, dexamethasone, Dignitana DigniCa Cooling System, dinutuximab (Unituxin™), doxycycline Clin, duloxetine, duvelisib, elotuzumab, emtricitabine / rilpibili emtricitabine / tenofovir / efavirenz , enoxaparin, enzalutamide (Xtandi®), epoetin alfa, erlotinib Nib (Tarceva®), esomeprazole, eszopiclone, etanercept, Everolimus (Afinitor®), exemestane (Aromasin®), Verolimus (Afinitor®), ezetimibe, ezetimibe / simvastatin, phenytoin Nofibrate, filgrastim, fingolimod, fluticasone propionate, flu Ticazone / salmeterol, fulvestrant (Faslodex®), Gadiva (Ga zyva), gefitinib (Iressa®), glatiramer, goserelin acetate (Zo ladex), icotinib, imatinib (Gleevec), ibritumomab tiuxetan (Zevali n®), ibrutinib (Imbruvica™), idelalisib (Zydelig®), Trademark), infliximab, iniparib, insulin aspart, insulin detemir insulin glargine, insulin lispro, interferon beta-1a, interferon eron beta 1b, lapatinib (Tykerb®), ipilimumab (Yervoy®) )), ipratropium bromide / salbutamol, Ixazomi (Ninlaro®), Kanuma, lanreotide acetate (Somatuline® Depot), Lenalid lenaliomide (Revlimid®), lenvatinib mesylate (Lenvima®), (trademark), letrozole (Femara®), levothyroxine, lidocaine Cain, linezolid, liraglutide, lisdexamfetamine, LN-144 (Lion Bio tech), MEDI4736 (AstraZeneca, Celgene), memantine, methylphenidate , metoprolol, mekinist, modafinil, mometasone, nilotinib (Tasigna (Registered Trademark), niraparib, nivolumab (Opdivo®), ofatumumab (Arzer®), a®), obinutuzumab (Gazyva™), olaparib (Lynparza™) ), olmesartan, olmesartan / hydrochlorothiazide, omalizumab, omega-3 fat acid ethyl ester, oseltamivir, oxycodone, palbociclib (Ibrance® palivizumab, panitumumab (Vectibix®), panobinostat (Fa rydak®), pazopanib (Votrient®), pembrolizumab (Keytr uda®), pemetrexed (Alimta), pertuzumab (Perjeta™), pulmonary Streptococcus pneumoniae conjugate vaccine, pomalidomide (Pomalyst®), pregabalin, Pros caVax, propranolol, quetiapine, rabeprazole, radium-223 chloride (Xofigo®), raloxifene, raltegravir, ramucirumab (Cyramza®), Registered Trademark), ranibizumab, regorafenib (Stivarga®), rituximab (Rituxan®), rivaroxaban, romidepsin (Istodax®), Savastatin, ruxolitinib phosphate (Jakafi™), salbutamol, sevelamer , sildenafil, siltuximab (Sylvant™), sitagliptin, sitagliptin metformin, solifenacin, solanezumab, sorafenib (Nexavar (registered trademark) (trademark), sunitinib (Sutent®), tadalafil, tamoxifen, tafin Tafinlar, talazoparib, telaprevir, temsirolimus (Torisel) Registered Trademark), tenofovir / emtricitabine, testosterone gel, thalidomide (Im munoprin, Talidex), tiotropium bromide, toremifene (Fareston®), Trametinib (Mekinist®), trastuzumab, tretinoin (Vesanoid Registered trademark), ustekinumab, valsartan, veliparib, vandetanib (Caprelsa Registered Trademark), vemurafenib (Zelboraf®), venetoclax, vorinos tat (Zolinza®), divaflibercept (Zaltrap®), and Zolinza® Starbucks, as well as their analogs, derivatives, and pharmaceutically acceptable salts, A carrier, diluent or excipient, or a combination thereof.

[0257] The drugs / cytotoxic agents used in the conjugation via the bridge linkers of this patent are The compound may be any analogue and / or derivative of the drug / molecule. Those skilled in the art will recognize that the resulting compound still retains the specificity and / or activity of the starting compound. It is readily apparent that each of the drugs / cytotoxic agents described herein can be modified to Those skilled in the art will also appreciate that many of these compounds are also compounds that are suitable for use with the drugs / cell injury compounds described herein. It will be understood that the drug / cytotoxic agent of the present invention may be used in place of the cytotoxic agent. Agents include analogs and derivatives of the compounds described herein.

[0258] All references cited in the specification and examples that follow are expressly incorporated by reference in their entirety. It can be enjoyed. [Example]

[0259] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention. The cell lines described in the following examples are not intended to be representative unless otherwise specified. American Type Culture Collection (ATCC), German Collection of Microbial Cell Cultures Based on the conditions specified by the Shanghai Institute of Cell Culture, Chinese Academy of Sciences (DSMZ) Unless otherwise stated, all cell culture reagents were purchased from Invitrogen. All anhydrous solvents were obtained commercially and stored in nitrogen-filled sealed bottles. All other reagents and solvents were purchased according to the highest standards and used without further purification. Preparative HPLC separations were performed on a Varain PreStar HPLC. R spectra were detected on a Varian Mercury 400 MHz instrument. The chemical shift (Δ) is in ppm, and the bond constant is 0.00 relative to tetramethylsilane. The unit of (J) is Hz. Mass spectrometry data was obtained from Waters Acquity UPL Waters Xevo QTO equipped with a C separator and Acquity TUV detector F mass spectrometry.

[0260] Example 1: Di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-) Synthesis of oxoethyl)hydrazine-1,2-dicarboxylate [ka]

[0261] Di-tert-butyl hydrazine-1,2-dicarboxylate (8.01g, 34 A solution of NaH (60% in oil, 6.76 g, 68.4 mmol) in DMF (150 ml) After stirring at room temperature for 30 minutes, tert-butyl bromoacetate (1.8 mmol) was added. The mixture was stirred overnight and methanol (3 ml ), concentrated, and diluted with EtOAc (100 ml) and water (100 ml). The aqueous layer was extracted with EtOAc (2 x 50 ml). Drying with O4, filtration, evaporation, and SiO2 column chromatography (EtOAc / Hexane The title compound was obtained as a colorless oil (12.98 g). , yield 82%). MS ESI m / z calcd for C 22 H 41 N2O8[M+H] + 461.28, found 461.40.

[0262] Example 2: Synthesis of 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0263] Di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-)oxoethanoate (ethyl)hydrazine-1,2-dicarboxylate (6.51 g, 14.14 mmol) To the 1,4-dioxane solution (40 ml) was added HCl (12 M, 10 ml). The mixture was stirred for 30 minutes, diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and Coevaporation with dioxane (20 ml) and toluene (40 ml) to dryness gave the title compound. A crude product of the compound (2.15 g, 103% yield, approximately 93% purity) was obtained. Used in the next step without further purification. MS ESI m / z calculated for C4H9N2O4 [M+H] + 149.05, found 149.40.

[0264] Example 3: 2,2'-(1,2-bis((benzyloxy)carbonyl)hydrazine-1 Synthesis of (2-diyl)diacetic acid [ka]

[0265] A mixture of THF (200 ml) and NaH2PO4 (0.1 M, 250 ml, pH 8.0) Benzyl carbonochloride was added to 2,2'-(hydrazine-1,2-diyl)diacetic acid in the mixture. Dart (benzyl carbonochroridate) (5.01g, 29.4 7 mmol) was added in four portions over 2 hours. The mixture was stirred for an additional 6 hours, concentrated, and Purification was performed on an iO2 column eluted with H2O / CH3CN (1:9) containing 1% formic acid. The title compound was obtained (2.26 g, yield 73%, purity approximately 95%). MS ESI m / z calcd for C 20 H 21 N2O8[M+H] + 417.12, found 417.40.

[0266] Example 4: Dibenzyl 1,2-bis(2-chloro-2-oxoethyl)hydrazine-1 Synthesis of ,2-dicarboxylate [ka]

[0267] 2,2'-(1,2-bis((benzyloxy)carbonyl)hydrazine-1,2-di In a solution of 350 mg (0.841 mmol) of methyltrimethylsilyldiacetic acid in 30 ml of dichloroethane , (COCl)2 (905 mg, 7.13 mmol) was added, followed by 0.030 ml of D After stirring at room temperature for 2 hours, the mixture was diluted with toluene, concentrated, and dichloromethane was added. The title compound was obtained by coevaporation with hexane (2 x 20 ml) and toluene (2 x 15 ml). The product was obtained (365 mg, 96% yield) and used in the next step without further purification. ESI m / z calculation for C 20 H 19 Cl2N2O6[M+H] + 453.05, found 453.50.

[0268] Example 5: Di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-ol) Synthesis of 1,2-dihydroxyethylhydrazine-1,2-dicarboxylate [ka]

[0269] of NaH (0.259 g, 6.48 mmol, 3.0 equiv.) in anhydrous DMF (2 ml). The suspension was treated with di-tert-butyl hydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol, 1.0 equiv) in anhydrous DMF (8 ml) was added to 1 The mixture was stirred at room temperature for 10 minutes and then cooled to 0°C. -butyl 2-bromoacetate (1.4 ml, 8.61 mmol, 4.0 eq) was added dropwise. The resulting mixture was allowed to warm to room temperature and stirred overnight. Saturated ammonium chloride solution (10 0 ml) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 50 ml). The combined organic solution was washed with water and brine, dried over anhydrous Na2SO4, and purified by SiO2 column Concentration and purification by chromatography (10:1 hexane / EtOAc) afforded the title compound was obtained as a colorless oil (0.94 g, 99.6% yield). ESI MS m / z [M+Na] + 483.4.

[0270] Example 6: Synthesis of compound 2,2'-(hydrazine-1,2-diyl)diacetic acid [ka]

[0271] At 0°C, di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-on hydroxyethyl)hydrazine-1,2-dicarboxylate (0.94 g, 2.04 mmol To a solution of ) in DCM (4 ml) was added TFA (4 ml). The resulting mixture was stirred for 30 min. The mixture was stirred, then allowed to warm to room temperature and stirred overnight. The mixture was concentrated, diluted with DCM and concentrated. This procedure was repeated three times to obtain a white solid. ) and collected by filtration (0.232 g, 76.8% yield). ESI MS m / z [M+ H] + 149.2.

[0272] Example 7: 2,2'-(1,2-bis(2-chloroacetyl)hydrazine-1,2-diyl) Synthesis of hydroxydiacetic acid [ka]

[0273] At 0°C, 2,2'-(hydrazine-1,2-diyl)diacetic acid (0.232 g, 1.57 2-Chloroacetyl chloride was added to a solution of 1.0 mmol (1.0 equiv.) of 2-chloroacetyl chloride in anhydrous THF (10 ml). (0.38 ml, 4.70 mmol, 3.0 equiv) was added over 10 minutes. The mixture was warmed to room temperature, stirred overnight, and concentrated. The residue was co-evaporated with THF three times to give a white solid (0 .472g, theoretical yield). ESI MS m / z [M+H] + 301.1.

[0274] Example 8: tert-Butyl 2,8-dioxo-1,5-oxazocane-5-carboxylate Synthesis of silates [ka]

[0275] At 4°C, 3,3'-azanediyldipropanoic acid (10.00 g, 62.08 mmol) In a 1.0 M NaOH solution (300 ml), di-tert-butyl dicarbonate (2 A solution of 2.10 g (101.3 mmol) of HCl in THF (200 ml) was added over 1 hour. After the addition, the mixture was stirred and maintained at 4°C for 2 hours. The mixture was diluted with 0.2M H3PO4. Carefully acidify to H₄₄, concentrate under vacuum, extract with CH₂Cl₂, and dry with Na₂SO₄. The mixture was evaporated and purified by flash SiO2 chromatography using AcOH / MeOH / CH2C Purified by elution with I2 (0.01:1:5) and 3,3'-((tert-butoxycarbonyl) (13.62 g, 84% yield) 1,3-dihydroxyazanediyldipropanoic acid was obtained. ESI MS m / z C 11 H 19 NO6[M+H] + , calcd. 262.27, found 262.40.

[0276] At 0°C, 3,3'-((tert-butoxycarbonyl)azanediyl)dipropanoic acid (8.0 g, 30.6 mmol) in CH2Cl2 (500 ml) The mixture was stirred at 0° C. for 2 hours and then at room temperature. Stir for 1 h, filter through a short SiO2 column, and dilute with EtOAc / CH2Cl2 (1:6) The filtrate was concentrated and triturated with EtOAc / hexane to give the title compound. The product was obtained (5.64 g, 74% yield). ESI MS m / z C 11 H 17 NO5[M+H] + , calcd. 244.11, Found 244.30.

[0277] Example 9: Synthesis of tert-butyl 3-(benzyloxy)amino)propanoate [ka]

[0278] O-benzylhydroxylamine hydrochloride (10.0 g, 62.7 mmol) in THF The solution (100 ml) was mixed with Et3N (15 ml) and tert-butyl acrylate (12.1 g, 94.5 mmol) was added. The mixture was refluxed overnight, concentrated, and diluted with EtOAc / Hexane. The product was purified on a SiO2 column eluted with ethanol (1:4) to give the title compound 3 (13.0 8g, 83% yield. 1 H NMR (CDCl3) 7.49-7.25 (m, 5H), 4.75 (s, 2H), 3.20 (t, J=6 .4Hz, 2H), 2.54 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI MS m / z + C 14 H 21 NNaO3(M+Na), calcd 274.15, found 274.20.

[0279] Example 10: Synthesis of tert-butyl 3-(hydroxyamino)propanoate [ka]

[0280] In a hydrogenation bottle, tert-butyl 3-(benzyloxy)amino)propanoate (13.0 g, 51.76 mmol) in methanol (100 ml) The reaction system was placed under vacuum and hydrogen was added at 2 atmospheres. After being placed under gas, the reaction mixture was stirred at room temperature overnight. The crude reaction was poured onto a short pad of Celite. The column was filtered, rinsed with ethanol, concentrated, and eluted with MeOH / DCM (1:10 to 1:5). The resulting mixture was purified by SiO2 column to give the title compound (7.25 g, 87% yield). 1 H NMR (CDCl3) 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4Hz, 2H), 1.49 (s, 9H); ESI M S m / z + C7H 15 NNaO3(M+Na), calcd. 184.10, found 184.30.

[0281] Example 11: Synthesis of tert-butyl 3-((tosyloxy)amino)propanoate [ka]

[0282] tert-Butyl 2-methylpropional in a mixture of DCM (50 ml) and pyridine (20 ml) at 4°C. 3-(hydroxyamino)propanoate (5.10 g, 31.65 mmol) Syl chloride (12.05 g, 63.42 g) was added. The mixture was stirred at room temperature overnight and concentrated. The product was concentrated and purified on SiO2 by eluting with EtOAc / DCM (1:10 to 1:6). The title compound was obtained (8.58 g, 86% yield). 1 H NMR (CDCl3) 7.81 (s, 2H), 7.46 (s, 2H), 3.22 (t, J=6.4Hz, 2H), 2.55 (t, J=6.4 Hz, 2H), 2.41 (s, 3H), 1.49 (s, 9H); ESI MS m / z + C 14 H 21 NNaO5S (M+Na) calcd 338.1 1, found 338.30.

[0283] Example 12: Di-tert-butyl 3,3'-(hydrazine-1,2-diyl)dipropionate Synthesis of Noate [ka]

[0284] tert-Butyl 3-aminopropanoate (3.05 g, 21.01 mmol) HF solution (80 ml) was added to tert-butyl 3-((tosyloxy)amino)propanoate. The mixture was stirred at room temperature for 1 hour, and then The mixture was stirred at 45° C. for 5 hours. The mixture was concentrated and purified by SiO column with CHOH / DCM / Et3N (1:12:0.01 to 1:8:0.01) to purify the title compound. (2.89 g, 62% yield). ESI MS m / z + calcd for C 14 H 28 N2NaO4(M+Na) 311.2 0, found 311.40.

[0285] Example 13: Di-tert-butyl 3,3'-(1,2-bis(3-(2,5-dioxo)methyl) -2,5-dihydro-1H-pyrrol-1-yl)propanoyl)hydrazine-1,2-di Synthesis of yl)dipropanoate [ka]

[0286] A solution of 3-maleido-propionic acid (1.00 g, 5.91 mmol) in DCM (50 ml l), oxalyl dichloride (2.70 g, 21.25 mmol) and DMF (50 μL) The mixture was stirred at room temperature for 2 hours, evaporated and co-evaporated with DCM / toluene. Crude 3-maleidopropionic acid chloride was obtained. DC of tetrahydrofuran-1,2-diyl)dipropanoate (0.51 g, 1.76 mmol) To the mixture of M (35 ml) was added crude 3-maleidopropionic acid chloride. Stir overnight, evaporate, concentrate, and run on a SiO2 column in EtOAc / DCM (1:15 to 1: 8) to give the title compound (738 mg, 71% yield). / z+ C 28 H 38 N4NaO 10 (M+Na),calcd 613.26, found 613.40.

[0287] Example 14: 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)propanoyl)hydrazine-1,2-diyl)dipropion Acid synthesis [ka]

[0288] Compound 14 (700 mg, 1.18 mmol) in dioxane (4 ml) was dissolved in concentrated HCl. (1 ml) was added. The mixture was stirred for 30 min and then diluted with EtOH (10 ml) and toluene ( Dilute with 10 ml of EtOAc, evaporate, and co-evaporate with 10 ml of EtOAc and 10 ml of toluene. Evaporation gave the crude title product (560 mg) which was used in the next step without further purification. I MS m / z- C 20 H 21 N4O 10 (MH), calcd 477.13, found 477.20.

[0289] Example 15: Bis(2,5-dioxopyrrolidin-1-yl)-3,3'-(1,2-biphenyl)- bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid Synthesis of (1,2-diyl)hydrazine-1,2-diyldipropanoate [ka]

[0290] 3,3'-(1,2-bis(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone) Coarsening of (( ... The compound (~560 mg, ~1.17 mmol) in DMA solution (8 ml) was added to NHS (400 mg, 3.47 mmol) and EDC (1.01 g, 5.26 mmol) were added. The mixture was stirred overnight, evaporated, concentrated and purified by SiO2 column with EtOAc / DCM The title compound was purified by elution with 1:12 to 1:7 dichloromethane to give 520 mg (obtained in two steps). rate 65%). ESI MS m / z + C 28 H 28 N6NaO 14 (M+Na), calcd. 695.17, found 695.40.

[0291] Example 16: tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy) Synthesis of (ethoxy)propanoate [ka]

[0292] In anhydrous THF (350 ml), 80 mg (0.0025 mol) of sodium metal and 1 50.1 g (1.00 mol) of triethylene glycol was added with stirring. After the rubber was completely dissolved, tert-butyl acrylate (24 ml, 0.33 mol) was added. The solution was stirred at room temperature for 20 hours and neutralized with 8 ml of 1.0 M HCl. The residue was suspended in brine (250 ml) and extracted with ethyl acetate (3 x 125 ml). The combined organic layers were washed with brine (100 ml) and then with water (100 ml). The resulting colorless oil was dried under vacuum and evaporated. 69.78 g of the title product was obtained (76% yield). 1 H NMR: 1.41 (s, 9H), 2.49 (t, 2H, J=6.4Hz), 3.59-3.72 (m, 14H). ESI MS m / z-C 13 H 25 O6(MH), calcd. 277.17, found 2 77.20.

[0293] Example 17: tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)methyl Synthesis of (ethoxy)propanoate [ka]

[0294] tert-Butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) A solution of propanoate (10.0 g, 35.95 mmol) in acetonitrile (50.0 m l) was treated with pyridine (20.0 ml). Toluene chloride was added to 50 ml of acetonitrile. A solution of (7.12 g, 37.3 mmol) was added dropwise via a funnel over 30 min. TLC analysis after 5 hours revealed the reaction to be complete. The gin was filtered and the solvent was removed. The residue was purified by hexane with neat ethyl acetate on silica gel. The mixture was purified by elution with 20% ethyl acetate in hexane to give 11.2 g of the title compound (76% yield). rate). 1 H NMR: 1.40 (s, 9H), 2.40 (s, 3H), 2.45 (t, 2H, J=6.4Hz), 3.52-3.68 (m, 1 4H), 4.11 (t, 2H, J=4.8Hz), 7.30 (d, 2H, J=8.0Hz), 7.75 (d, 2H, J=8Hz), ESI MS m / z+ C 20 H 33 O8S (M+H), calcd. 433.18, found 433.30.

[0295] Example 18: tert-Butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy Synthesis of hydroxy)propanoate [ka]

[0296] In 50 ml of DMF, tert-butyl 3-(2-(2-(2-tosyloxy)ethoxy)ethoxy (4.0 g, 9.25 mmol) and azide Sodium (0.737 g, 11.3 mmol) was added with stirring. The reaction was heated to 80°C. After 4 hours, TLC analysis revealed the reaction was complete. The mixture was cooled to room temperature and quenched with water (25 ml). The aqueous layer was separated and diluted with ethyl acetate (3×35 The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. The crude azide product (2.24 g, 98% yield, approximately 9% purity by HPLC) was removed under reduced pressure. 3%) was used in the next step without further purification. 1 H NMR(CDCl3): 1.40 (s, 9H), 2.45 (t , 2H, J=6.4Hz), 3.33 (t, 2H, J=5.2Hz), 3.53-3.66 (m, 12H), ESI MS m / z+ C 13 H 26 N3O 8(M+H), calcd. 304.18, found 304.20.

[0297] Example 19: 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propion Acid synthesis [ka]

[0298] tert-Butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionate To a solution of panoate (2.20 g, 7.25 mmol) in 1,4-dioxane (40 ml) HCl (12M, 10 ml) was added. The mixture was stirred for 40 minutes and dioxane (20 Diluted with dioxane (20 ml) and toluene (40 ml), evaporated, and Coevaporation with ethanol (40 ml) to dryness gave the crude title compound (1.88 g, yield The yield was 105%, purity by HPLC was approximately 92%, and it was used in the next step without further purification. ESI m / z calculation for C9H 18 N3O5[M+H] + 248.12, found 248.40.

[0299] Example 20: 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester tert-butyl 13-amino-bis(4,7,10-trioxadodecanoate) Synthesis of sterol [ka]

[0300] Crude azide material 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propionate Dissolve phosphoric acid (5.0 g, 14.84 mmol) in ethanol (80 ml) and mg of 10% Pd / C was added. The reaction system was evacuated under vacuum and the hydrogenation reaction was carried out with vigorous stirring. The reaction was then placed under a 2 atm hydrogen atmosphere via a furnace. The reaction was then stirred at room temperature overnight and T LC showed the disappearance of the starting material. The crude reaction was purified by ethanol through a short pad of Celite. The solvent was removed and the residue was washed with methanol (5% to 15%) and 1% methylene chloride. The amine was purified on silica gel using a mixture of 10% triethylamine as eluent. 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ester (1.8 3g, 44% yield, ESI MS m / z+ C 13 H 27NO5(M+H), calcd. 278.19, found 278.30) and 13-amino-bis(4,7,10-trioxadodecanoic acid tert-butyl ester) ) (2.58g, 32% yield, ESI MS m / z+ C 26 H 52 NO 10 (M+H), calcd. 538.35, found 538.40).

[0301] Example 21: 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propion Synthesis of Acid HCl Salts [ka]

[0302] 13-amino-4,7,10-trioxadodecanoic acid tert-butyl ether in 30 ml of dioxane t-butyl ester (0.80 g, 2.89 mmol) in 10 ml of HCl (36%) After 0.5 hours, TLC analysis revealed that the reaction was complete. The reaction mixture was evaporated and co-evaporated with EtOH and EtOH / toluene to give the title product. The HCl salt of (>90% purity, 0.640 g, 86% yield) was formed without further purification. ESI MS m / z+ C9H 20 NO5(M+H), calcd. 222.12, found 222.20.

[0303] Example 22: 13-amino-bis(4,7,10-trioxadodecanoic acid HCl salt [ka]

[0304] 13-aminobis(4,7,10-trioxadodecanoic acid tert-butyl ester) (1.00 g, 1.85 mmol) in dioxane (30 ml) with 36% HCl (10 ml) was added with stirring. After 0.5 hours, TLC analysis showed the reaction was complete. After clarification, the reaction mixture was evaporated and co-evaporated with EtOH and EtOH / toluene. The title product was obtained as the HCl salt without further purification (90% pure, 0.71 g, yield rate 91%). ESI MS m / z+ C 18 H 36 NO 10 (M+H), calcd 426.22, found 426.20.

[0305] Example 23: tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy) Synthesis of (ethoxy)propanoate [ka]

[0306] 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 ml, 410.75 mmol, 3.0 eq) in anhydrous THF (200 ml) The mixture was stirred until the Na disappeared, and then tert-butyl acetate (0.1 g) was added. Acrylate (20.0 ml, 137.79 mmol, 1.0 eq) was added dropwise. The mixture was stirred overnight and then quenched with HCl solution (20.0 ml, 1N) at 0°C. The THF was removed by rotary evaporation, and brine (300 ml) was added. The resulting mixture was extracted with EtOAc (3 x 100 ml). The organic layer was washed with brine (3 x 300 ml). l), dried over anhydrous Na2SO4, filtered and concentrated to give a colorless oil (30.2 0g, 79.0% yield) and used without further purification. MS ESI m / z calcd for C13 H2 7O6[M+H] + 278.1729, found 278.1730.

[0307] Example 24: tert-Butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)methyl Synthesis of (ethoxy)propanoate [ka]

[0308] At 0°C, tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) (tox)propanoate (30.20 g, 108.5 mmol, 1.0 eq) and TsC l (41.37 g, 217.0 mmol, 2.0 eq) in anhydrous DCM (220 ml) To the mixture was added TEA (30.0 ml, 217.0 mmol, 2.0 eq). The mixture was stirred at room temperature overnight, washed with water (3 x 300 ml) and brine (300 ml), and then diluted with anhydrous Na2S Dry over O4, filter, concentrate, and eluate by SiO2 column chromatography (3:1 hexahydrate). Purification with hexane / EtOAc gave a colorless oil (39.4 g, 84.0% yield). MS ESI m / z calcd for C 20 H 33 O8S [M+H] + 433.1818, found 433.2838.

[0309] Example 25: tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy Synthesis of (thoxy)propanoate [ka]

[0310] tert-Butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy (39.4g, 91.1mmol, 1.0eq) dissolved in anhydrous DMF Into the solution (100 ml), add NaN3 (100 ml) (20.67 g, 316.6 mmol, 3 0.5 eq) was added. The mixture was stirred at room temperature overnight. Water (500 ml) was added and Et The combined organic layer was extracted with water (3 x 900 ml) and brine ( 900 ml), dried over anhydrous Na2SO4, filtered, concentrated, and then passed through a SiO2 column. Purification by chromatography (5:1 hexanes / EtOAc) gave a light yellow oil (23.8g, 85.53%). MS ESI m / z calcd for C 13 H 25 O3N5Na [M+Na] + 326.2, f ound 326.2.

[0311] Example 26: tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy Synthesis of (thoxy)propanoate [ka]

[0312] Raney Ni (7.5 g, suspended in water) was dissolved in water (3 times) and isopropyl alcohol (3 times). Wash and tert-butyl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy Mix with a solution of propanoate (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H2 balloon at room temperature for 16 hours and then filtered through a pad of Celite. The pad was washed with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography. The product was purified by filtration (5-25% MeOH / DCM) to give a light yellow oil (2.60 g, yield 57%). MS ESI m / z calculated for C 13 H 28 NO5[M+H] + 279.19, found 279.19.

[0313] Example 27: Synthesis of 2-(2-(dibenzylmino)ethoxy)ethanol [ka]

[0314] 2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq ) and K2CO3 (83.00 g, 600 mmol, 3.0 eq) in acetonitrile (350 ml) was added BnBr (57.0 ml, 480 mmol, 4 eq). The mixture was refluxed overnight. Water (1 L) was added and extracted with EtOAc (3 x 300 ml). The combined organic layers were washed with brine (1000 ml), dried over anhydrous Na2SO4, and concentrated. and filtered by SiO2 column chromatography (4:1 hexane / EtOAc). A colorless oil was obtained (50.97 g, 89.2% yield). MS ESI m / z calculated for C 18 H 23 NO2 Na [M+Na] + 309.1729, found 309.1967.

[0315] Example 28: tert-Butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy) Synthesis of propanoate [ka]

[0316] 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mm ol, 1.0eq), tert-butyl acrylate (72.0ml, 495.9mmol l, 3.0 eq), and n-Bu4NI (6.10 g, 16.53 mmol, 0.1 eq ) in DCM (560 ml) and add sodium hydroxide solution (300 ml, 50%) The mixture was stirred overnight. The organic layer was separated and the aqueous layer was washed with EtOAc (3 x 100 ml). The organic layer was washed with water (3 x 300 ml) and brine (300 ml) and extracted with anhydrous Na The extract was dried over SO4, filtered, concentrated, and purified by SiO2 column chromatography (7:1 hexahydrate). Purification by hexane / EtOAc gave a colorless oil (61.08 g, 89.4% yield) MS ESI m / z calculated for C 25 H 36 NO4[M+H] + 414.2566, found 414.2384.

[0317] Example 29: tert-Butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate Synthesis of acetone [ka]

[0318] tert-Butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanol Noate (20.00 g, 48.36 mmol, 1.0 eq) in THF (30 ml) and To a solution of MeOH (60 ml) was added Pd / C (2.00 g, 10%). Shake overnight at 1 atm H2, filter through Celite (filter aid), and Concentration gave a colorless oil (10.58 g, 93.8% yield). MS ESI m / z calculated for C 11 H2 4NO4[M+H] +234.1627, found 234.1810.

[0319] Example 30: tert-Butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanol Synthesis of Noate [ka]

[0320] 2,2'-oxydiethanol (19.7 ml, 206.7 mmol, 3.0 eq) Sodium (0.1 g) was added to anhydrous THF solution (100 ml). The mixture was stirred until 100°C, and then tert-butyl acrylate (10.0 ml, 68.9 ml) was added. mol, 1.0 eq) was added dropwise. The mixture was stirred overnight and brine (200 ml) was added. The organic layer was washed with brine (3 x 300 ml). Wash, dry over anhydrous Na2SO4, filter, concentrate, and then eluate by SiO2 column chromatography. Purification with hexanes / EtOAc (1:1) gave a colorless oil (8.10 g, yield rate 49.4%). MS ESI m / z calcd for C 11 H 23 O5 [M+H] + 235.1467, found 235.1667.

[0321] Example 31: tert-butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy) Synthesis of propanoate [ka]

[0322] At 0°C, tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate ate (6.24 g, 26.63 mmol, 1.0 eq) and TsCl (10.15 g, A solution of 53.27 mmol, 2.0 eq) in anhydrous DCM (50 ml) was added to pyridine (4.3 ml, 53.27 mmol, 2.0 eq) was added. The mixture was stirred at room temperature overnight, and then The combined organic layer was washed with water (100 ml) and the aqueous layer was extracted with DCM (3 x 50 ml). The layer was washed with brine (300 ml), dried over anhydrous Na2SO4, filtered, concentrated and Si Purified by O2 column chromatography (5:1 hexane / EtOAc) to obtain a colorless An oil was obtained (6.33 g, 61.3% yield). MS ESI m / z calculated for C 18 H 27 O7S [M+H] + , 3 89.1556, found 389.2809.

[0323] Example 32: tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate Synthesis of acetone [ka]

[0324] tert-Butyl 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoate To a solution of 5.80 g (14.93 mmol, 1.0 eq) in anhydrous DMF (20 ml) Then, NaN3 (5.02 g, 77.22 mmol, 5 eq) was added. The mixture was stirred at room temperature. The mixture was stirred overnight. Water (120 ml) was added and extracted with EtOAc (350 ml). The organic layer was washed with water (3 x 150 ml) and brine (150 ml), and Drying, filtering, and eluting with SiO2 column chromatography (5:1 hexane / EtOAc) ) to give a colorless oil (3.73 g, 69.6%). MS ESI m / z calcd for C 11 H 22 O3N4Na [M+H] + 260.1532, found 260.2259.

[0325] Example 33: tert-Butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate Synthesis of acetone [ka]

[0326] tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (0. Dissolve 18g (0.69mmol) in MeOH (3.0ml, containing 60µl of concentrated HCl). The solution was dissolved and hydrogenated with Pd / C (10 wt%, 20 mg) under a H2 balloon for 30 min. The catalyst was filtered off through a Celite pad and the pad was washed with MeOH. The filtrate was concentrated. A colorless oil was obtained (0.15 g, 93% yield). MS ESI m / z calculated for C 11 H 24 NO4[M+H] + 2 34.16, found 234.14.

[0327] Example 34: Synthesis of 3-(2-(2-azidoethoxy)ethoxy)propionic acid [ka]

[0328] tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (2. 51 g, 9.68 mmol) was dissolved in 1,4-dioxane (30 ml) and heated at room temperature for 10 The mixture was stirred for 35 min and then treated with EtOH (30 ml) and toluene (10 ml). The crude mixture was diluted with ethanol (30 ml) and concentrated under reduced pressure. Silica gel, eluted with methanol (5%-10%) and 1% formic acid in methylene chloride The product was purified by filtration to give the title compound (1.63 g, 83% yield). ESI MS m / z C7H 12 N3O4[ MH] - , calcd. 202.06, found 202.30.

[0329] Example 35: 2,5-Dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy) Synthesis of (ethoxy)propanoate [ka]

[0330] 3-(2-(2-azidoethoxy)ethoxy)propionic acid (1.60 g, 7.87 m mol) in dichloromethane (30 ml), ) and EDC (3.60 g, 18.75 mmol) were added with stirring. After 8 hours, TL C analysis revealed the reaction was complete, and the reaction mixture was concentrated and diluted with ethyl acetate (5% Purification on silica gel eluting with 10% methylene chloride gave the title compound (1.93g, yield 82%). ESI MS m / z C 11 H 17 N4O6[M+H] + , calcd. 301.11, found 3 01.20.

[0331] Example 36: 2,5-Dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy)methyl) Synthesis of ethoxyethoxypropanoate [ka]

[0332] 3-(2-(2-azidoethoxy)ethoxy)propionic acid (4.50g, 18.21 To a dichloromethane solution (80 ml) of NHS (3.0 g, 26.08 mmol), 1) and EDC (7.60 g, 39.58 mmol) were added. After 8 hours, TLC analysis showed The reaction was found to be complete, and the reaction mixture was concentrated and diluted with ethyl acetate (5% to 10%). ) in methylene chloride to give the title compound (5.3 8g, yield 86%). ESI MS m / z C 13 H 20 N4O7[M+H] + , calcd. 345.13, found 345.30.

[0333] Example 37: (14S,17S)-1-azido-17-(2-(tert-butoxy)- 2-Oxoethyl)-14-(4-(tert-butoxycarbonyl)-amino)butyl )-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane Synthesis of -18-acid [ka]

[0334] A mixture of DMA (70 ml) and 0.1 M NaH2PO4 (50 ml, pH 7.5) (S)-2-((S)-2-amino-6-((tert-butoxycarbonyl)amino) Hexanamido)-4-(tert-butoxy)-4-oxobutanoic acid (2.81g) , 6.73 mmol) to 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2 -Azidoethoxy)ethoxy)-ethoxy)propanoate (3.50g, 10.17g) The mixture was stirred for 4 hours, evaporated in vacuo and diluted with methylene chloride containing 0.5% acetic acid. The elution was carried out on silica gel using a mixture of methanol (5% to 15%) in ethylene as the eluent. Purification gave the title compound (3.35 g, 77% yield). ESI MS m / z C 28 H5 1N6O 11 [M+H] + ,calcd 647.35, found 647.80.

[0335] Example 38: (14S,17S)-tert-butyl 1-azido-14-(4-((te rt-Butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl)furan (phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16 Synthesis of -diazanonadecane-19-oate [ka]

[0336] (14S,17S)-1-azido-17-(2-(tert-butoxy)-2-oxo Ethyl)-14-(4-((tert-butoxycarbonyl)-amino)butyl)-12 ,15-Dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18- Acid (3.30 g, 5.10 mmol) and (4-aminophenyl)methanol (0.75 g, 6.09) in DMA solution (25 ml), EDC (2.30 g, 11.97 mmol) The mixture was stirred overnight, evaporated in vacuo, and the methanol containing methylene chloride was added. Purification on silica gel using a mixture of ethanol (5%-8%) gave the title compound (3.1%). 8g, yield 83%). ESI MS m / z C 35 H 58 N7O 11 [M+H] + , calcd. 752.41, found 752.85.

[0337] Example 39: (14S,17S)-tert-butyl 1-amino-14-(4-(ter t-Butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl)phenyl (Nyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13,16- Synthesis of diazanonadecane-19-oate [ka]

[0338] In a hydrogenation bottle, (14S,17S)-tert-butyl 1-azido-14-(4-( (tert-butoxycarbonyl)amino)butyl)-17-((4-(hydroxymethyl (phenyl)phenyl)carbamoyl)-12,15-dioxo-3,6,9-trioxa-13 16-diazanonadecan-19-oate (1.50 g, 1.99 mmol) in THF To the solution (35 ml) was added Pd / C (200 mg, 10% Pd, 50% wet). The mixture was shaken under 1 atm H2 overnight and filtered through Celite (filter aid). Filter and concentrate the filtrate to give the title compound (1.43 g, 99% yield) which is suitable for further purification. Used immediately in the next step without further purification. ESI MS m / z C 35 H 60 N5O 11 [M+H] + , calcd. 726.42, Found 726.70.

[0339] Example 40: (S)-15-azido-5-isopropyl-4,7-dioxo-10,13 Synthesis of -dioxa-3,6-diazapentadecano-1-oic acid [ka]

[0340] A mixture of DMA (50 ml) and 0.1 M NaH2PO4 (50 ml, pH 7.5) (S)-2-(2-amino-3-methylbutanamido)acetic acid (Val-Gly) (1 0.01g, 5.80mmol) to 2,5-dioxopyrrolidin-1-yl 3-(2-( 2-Azidoethoxy)ethoxy)propanoate (1.90 g, 6.33) was added. The mixture was stirred for 4 hours, evaporated under reduced pressure and diluted with methylene chloride containing 0.5% acetic acid as eluent. Purification on silica gel using a mixture of ethylene and methanol (5% to 15%) was carried out. The compound was obtained (1.52 g, 73% yield). ESI MS m / z C 14 H 26 N5O6[M+H] + , calcd. 360.1 8, found 360.40.

[0341] Example 41: (S)-2,5-dioxopyrrolidin-1-yl 15-azido-5-isopropyl propyl-4,7-dioxo-10,13-dioxa-3,6-diazapentadecan-1- Synthesis of oate [ka]

[0342] (S)-15-Azido-5-isopropyl-4,7-dioxo-10,13-dioxa- Dichloromethane of 3,6-diazapentadecan-1-oic acid (1.50 g, 4.17 mmol) NHS (0.88 g, 7.65 mmol) and EDC (2.60 g) were added to the solution (40 ml). (13.54 mmol) was added with stirring. After 8 hours, TLC analysis showed that the reaction Upon completion, the reaction mixture was concentrated and the eluent was ethyl acetate (5% 2 Purification on silica gel using a mixture of methylene chloride containing 0% HCl gave the title compound (1.48g, yield 78%). ESI MS m / z C 18 H 29 N6O8[M+H] + , calcd. 457.20, found 4 57.50.

[0343] Example 42: Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic acid [ka]

[0344] 4-aminobutanoic acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) A solution of 40 ml of CbzCl in HO was cooled to 0°C and CbzCl (16.1 g, 95 mmol) was added. After 1 hour, the reaction was warmed to room temperature and stirred for 3 hours. The THF was removed under vacuum and the pH of the aqueous solution was adjusted to 1.5 by adding 6N HCl. Extraction with ethyl acetate was performed, and the organic layer was washed with brine, dried, and concentrated to give the title compound. (16.4g, yield 92%). MS ESI m / z calcd for C 12 H 16 NO5[M+H] + 238.10, found 2 38.08.

[0345] Example 43: tert-butyl 4-(((benzyloxy)carbonyl)amino)butanoate Synthesis of ethene [ka]

[0346] 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4g, 69.2 A solution of t-BuOH (15.4 g, 208 mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 ml ), DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) ) was added. After stirring at room temperature overnight, the reaction was filtered and concentrated. The residue was dissolved in ethyl acetate. The mixture was dissolved in ethyl acetate, washed with 1N HCl and brine, and dried over Na2SO4. The title compound was obtained by chromatography (10-50% EtOAc / hexane). The product was obtained (7.5 g, 37% yield). MS ESI m / z calculated for C 16 H 23 NO4Na [M+Na] + 316.16 , found 316.13.

[0347] Example 44: Synthesis of tert-butyl 4-aminobutanoate [ka]

[0348] tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate(5 60 mg, 1.91 mmol) was dissolved in MeOH (50 ml) and Pd / C catalyst (10 times The catalyst was mixed with 100 mg of methylcellulose (100% by weight) and then hydrogenated at room temperature (1 atm) for 3 hours. Filtration and removal of all volatiles in vacuo afforded the title compound (272 mg, 90% yield). %). MS ESI m / z calculated for C8H 18 NO2[M+H] +160.13, found 160.13.

[0349] Example 45: Di-tert-butyl 3,3'-(benzylazanediyl)dipropanoate Synthesis of To [ka]

[0350] Phenylmethanamine (2.0 ml, 18.29 mmol, 1.0 eq) and tert - a mixture of butyl acrylate (13.3 ml, 91.46 mmol, 5.0 eq), The mixture was refluxed at 80°C overnight and then concentrated. The crude product was purified by SiO2 column chromatography ( 20:1 hexanes / EtOAc) to give the title compound as a colorless oil (5.10 g, yield 77%). ESI MS m / z: calculated for C 21 H 34 NO4[M+H] + 364.2, found 364.2. 1 H NMR (400MHz, CDCl3) δ7.38-7.21 (m, 5H), 3.58 (s, 2H), 2.76 (t, J=7.0 Hz, 4H), 2.38 (t, J=7.0Hz, 4H), 1.43 (s, 17H).

[0351] Example 46: Synthesis of di-tert-butyl 3,3'-azanediyldipropanoate [ka]

[0352] In a hydrogenation bottle, di-tert-butyl 3,3'-(benzylazanediyl)diproline Panoate (1.37 g, 3.77 mmol, 1.0 equiv) in MeOH (10 ml) To the mixture was added Pd / C (0.20 g, 10% Pd / C, 50% wet). Shake overnight under H2 atmosphere and then filter through a pad of Celite. The filtrate was concentrated and The title compound was obtained as a colorless oil (1.22 g, 89% yield). ESI MS m / z: calculated for C1 4H 28 NO4[M+H] + 274.19, found 274.20.

[0353] Example 47: tert-butyl 4-(2-(((benzyloxy)carbonyl)amino) Synthesis of propanamido)butanoate [ka]

[0354] At 0 °C, tert-butyl 4-aminobutanoate (1.00 g, 6.28 mmol, 1.0 eq) and ZL-alanine (2.10 g, 9.42 mmol, 1.5 eq) In a 50 ml aqueous DCM solution, HATU (3.10 g, 8.164 mmol, 1.3 eq ) and TEA (2.6 ml, 18.8 mmol, 3.0 eq) were added. The reaction was stirred at 0°C. The mixture was stirred for 10 minutes, then allowed to warm to room temperature and stirred overnight. The mixture was diluted with DCM and diluted with water and salt. Washed with water, dried over anhydrous Na2SO4, and then purified by SiO2 column chromatography (10: 3 petroleum ether / ethyl acetate) to give the title compound as a colorless oil ( 1.39g, yield 61%). ESI MS m / z: calcd for C 19 H 29 N2O5Na [M+H] + 387.2, found 387.2.

[0355] Example 48: Synthesis of tert-butyl 4-(2-aminopropanamido)butanoate [ka]

[0356] tert-Butyl 4-(2-(((benzyloxy)carbonyl)amino)propanol (mido)butanoate (1.39g, 3.808mmol, 1.0eq) in MeOH To the mixture (12 ml), Pd / C (0.20 g, 10 wt %, 10% wet) was added. The mixture was shaken for 2 hours, then filtered through Celite (filter aid), concentrated, and the standard The title compound was obtained as a light yellow oil (0.838 g, 95% yield). ESI MS m / z: calcd. for C 11 H 23 N2O3[M+H] + 231.16, found 231.15.

[0357] Example 49: 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propionic acid Synthesis of [ka]

[0358] At ro...

Claims

1. Bis-linked compounds of formula (I): 【Chemical 1】 During the ceremony, 【Chemistry 2】 represents a single bond; 【Chemistry 3】 is optionally either a single or double bond or absent; n and m １ are independently 1 to 20; Z １ and Z ２ The cell-binding molecule in parentheses linked to the binds to, complexes with, or reacts with a portion of a cell population for which It is a molecule / drug. The cell binding agent / molecule is an immunotherapeutic protein, an antibody, a single chain antibody; antibody fragments that bind to target cells; monoclonal antibodies; single-chain monoclonal antibodies; or target Monoclonal antibody fragments that bind to target cells; chimeric antibodies; chimeric antibodies that bind to target cells fragments; domain antibodies; domain antibody sections that bind to target cells; antibody-mimicking adnects DARPins; lymphokines; hormones; vitamins; growth factors; colony-stimulating factors or a nutrient transport molecule; transferrin; a binding peptide having four or more amino acids; or Antibodies, proteins, albumin, polymers, dendrimers, liposomes, nanoparticles, vesicles or a small cell-binding molecule or binding ligand that binds to the (viral) capsid; The cytotoxic molecules in parentheses are drugs / molecules / drugs for therapy or targets for immunotherapy. functional molecules to enhance the binding or stabilization of proteins / molecules or cell-binding agents; Monitoring cell surface receptor binding ligands, or inhibition of cell proliferation, or cell-associated molecule activity for detection, or research; or analogs or prodrugs thereof, or pharmaceutical acceptable salts, hydrates or hydrated salts, or crystal structures, or optical isomers, racemates diastereomeric immunotherapeutic compounds, enantiomers or enantiomers of chemotherapy compounds an antimer, an antibody (probody) or an antibody (probody) fragment; or an siRNA or or DNA molecules; or cell surface-binding ligands; or tubulysins, calicheamicins , auristatin, maytansinoid, CC-1065 analogue, morpholinodoxorubicin amines, taxanes, cryptophycins, amatoxins, epothilones, eribulin, geldane Isin, duocarmycin, daunomycin, methotrexate, vindesine, vinc Listine, and benzodiazepine dimers (pyrrolobenzodiazepines (PBD), tomamates) isine, indolinobenzodiazepine, imidazobenzothiadiazepine, or oxazolidinone and analogs or prodrugs of therapeutic drugs containing benzodiazepines (including dimers of dibenzodiazepines). the law of nature; X and Y are independently the same or different and are disulfides, thioethers, or thioesters. , peptides, hydrazones, ethers, esters, carbamates, carbonates, amines ( secondary, tertiary, or quaternary), imines, cycloheteroalkanes, heteroaromatic, alkane represents a functional group that is linked to a cytotoxic drug via an oxime or amide bond; X and Y are independently standing NH; NHNH; N(R １ ); N(R １ ); N(R ２ ); O; S; S - S, O - NH 、O-N(R １ )、CH ２ -NH、CH ２ -N(R １ )、CH=NH、CH=N(R １ ) 、S(O)、S(O ２ )、P(O)(OH)、S(O)NH、S(O ２ )NH、P(O) (OH)NH、NHS(O)NH、NHS(O ２ )NH、NHP(O)(OH)NH、N (R １ )S(O)N(R ２ )、N(R １ )S(O ２ )N(R ２ )、N(R １ )P(O)( OH)N(R ２ )、OS(O)NH、OS(O ２ )NH、OP(O)(OH)NH、C( O)、C(NH)、C(NR １ )、C(O)NH、C(NH)NH、C(NR １ )NH、 OC(O)NH、OC(NH)NH;OC(NR １ )NH、NHC(O)NH;NHC( NH)NH;NHC(NR １ )NH、C(O)NH、C(NH)NH、C(NR １ )NH 、OC(O)N(R １ )、OC(NH)N(R １ )、OC(NR １ )N(R １ )、NHC <h2 style=";text-align:left;direction:ltr">(O)N(R<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> )、NHC(NH)N(R<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> )、NHC(NR<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> )N(R<h2 style=";text-align:left;direction:ltr"> １ <h2 style=";text-align:left;direction:ltr"> )、N(R<h2 style=";text-align:left;direction:ltr"> １ )C(O)N(R １ )、N(R １ )C(NH)N(R １ )、N(R １ )C(NR １ )N( R １ ); or C １ -C ６ Alkyl, C ２ -C ８ Alkenyl, heteroalkyl, alkylsilyl cycloalkyl, or heterocycloalkyl; C ３ -C ８ aryl, Ar-alkyl, Heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl or heteroaryl; Z １ and Z ２ are independently linked to the same or different cell-binding molecules by disulfides, Ethers, esters, thioethers, thioesters, peptides, hydrazones, carbamate esters, carbonates, amines (secondary, tertiary, or quaternary), imines, cycloheteroalkanes, a heteroaromatic, alkyloxime, or amide bond-forming functional group; Z １ and Z ２ independently have the following structures: C(O)CH, C(O)C, C(O)CH ２ , ArC H ２ 、C(O)、NH;NHNH;N(R １ );N(R １ )N(R ２ );O;S;S-S 、O-NH、O-N(R １ )、CH ２ -NH、CH ２ -N(R １ )、CH=NH、CH= N(R １ )、S(O)、S(O ２ )、P(O)(OH)、S(O)NH、S(O ２ )NH 、P(O)(OH)NH、NHS(O)NH、NHS(O ２ )NH、NHP(O)(OH )NH、N(R １ )S(O)N(R ２ )、N(R １ )S(O ２ )N(R ２ )、N(R １ ) P (O)(OH)N (R ２ ),OS(O)NH、OS(O ２ )NH、OP(O)(OH) NH、C(O)、C(NH)、C(NR １ )、C(O)NH、C(NH)NH、C(NR １ )NH、OC(O)NH、OC(NH)NH;OC(NR １ )NH、NHC(O)NH ;NHC(NH)NH;NHC(NR １ )NH、C(O)NH、C(NH)NH、C(N R １ )NH、OC(O)N(R １ )、OC(NH)N(R １ )、OC(NR １ )N(R １ ),NHC(O)N(R １ ), NHC (NH)N (R １ ),NHC(NR １ )N (R １ ) 、N(R １ )C(O)N(R １ )、N(R １ )C(NH)N(R １ )、N(R １ )C(N R １ ) N (R １ ); or C １ -C ８ Alkyl, C ２ -C ８ Heteroalkyl, alkylcyclo cycloalkyl, or heterocycloalkyl; C ３ -C ８ Aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; L １ and L ２ are independently the same or different, O; NH; S; NHNH; N(R ３ ); N (R ３ ) N (R ３ ') ; C １ -C ８ Alkyl, amide, amine, imine, hydrazine, or hydrazone; C ２ -C ８ Heteroalkyl, alkylcycloalkyl, ether, es esters, hydrazones, ureas, semicarbazides, carbazides, alkoxyamines, alkoxy amines, urethanes, amino acids, peptides, acyloxyamines, hydroxamic acids, or Heterocycloalkyl; C ３ -C ８ Aryl, Ar-alkyl, heterocycle, carbocycle, cyclo alkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; 1 to 8 amino acids; ２ CH ２ ) ｐ OR ３ , or (OCH ２ -CH(CH ３ ) ) ｐ OR ３ , or NH(CH ２ CH ２ O) ｐ R ３ , or NH(CH ２ CH (CH ３ ) O) ｐ R ３ , or N[(CH ２ CH ２ O) ｐ R ３ ]-[(CH ２ CH ２ O) ｐ 'R ３ '],also (OCH ２ CH ２ ) ｐ COOR ３ , or CH ２ CH ２ (OCH ２ CH ２ ) ｐ COOR ３ wherein p and p' are independently from 0 to about 5000 to R is a selected integer or a combination thereof; ３ and R ３ ' is independently H; C １ -C ８ Alkyl; C ２ -C ８ Heteroalkyl, alkylcycloalkyl, or hetero cycloalkyl; C ３ -C ８ Aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkoxy C alkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or C ２ -C ８ an ester, ether, or amide; or 1 to 8 amino acids; or a group of the formula (OC H ２ CH ２ ) ｐ Or (OCH ２ -CH(CH ３ )) ｐ Polyethyleneoxy having wherein p is an integer from 0 to about 5000, or a combination thereof; Or L １ and L ２ are independently 6-maleimidocaproyl (MC), maleimidocaproyl (MC), propanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenyl arylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PA B), 4-thio-pentanoic acid ester (SPP), 4-(N-maleimidomethyl)cyclo Hexane-1-carboxylic acid ester (MCC), (4-acetyl)aminobenzoic acid (SI AB), 4-thio-butyric acid ester (SPDB), 4-thio-2-hydroxysulfonyl- butyric acid ester (2-sulfo-SPDB), or 1 to 8 natural or unnatural amino acids comprising one or more natural or non-natural peptide linker components having the following structure: Or, L １ and L ２ are independently composed of self-immolative components, peptide units, hydrazone bonds, dimers, and a sulfide, ester, oxime, amide, or thioether bond, The unit contains a para-aminobenzylcarbamoyl (PAB) group, a 2-aminoimidazole-5 -methanol derivatives, heterocyclic PAB analogs, β-glucuronides, and o- or p-amino Aromatic compounds electronically similar to benzyl acetals; or containing any of the following structures: ： 【Chemistry 4】 In the formula, ( ＊ ) atoms labeled with an additional spacer or releasable linker unit, cell X is a binding site for a toxic agent and / or a cell adhesion molecule (CBA); １ , Y １ , Z ２ , and Z ３ are independently NH, O or S; Z １ are independently H, NHR １ , OR １ , S.R. １ , COX １ R １ wherein X １ and R １ is defined as above; v is 0 or 1. Ru;U １ are independently H, OH, C １ -C ６ Alkyl, (OCH ２ CH ２ ) ｎ , F, Cl, Br、I、OR ５ 、SR ５ 、NR ５ R ５ ’、N=NR ５ 、N=R ５ 、NR ５ R ５ ’、NO ２ 、SOR ５ R ５ '、SO ２ R ５ 、SO ３ R ５ 、OSO ３ R ５ 、PR ５ R ５ '、POR ５ R ５ ’, PO ２ R ５ R ５ ’, OPO (OR ５ )(OR ５ ’), or OCH ２ PO (OR ５ ) (OR ５ '), wherein R ５ and R ５ ' are each independently H, C １ -C ８ Al kill; C ２ -C ８ alkenyl, alkynyl, heteroalkyl, or amino acid; C ３ - C ８ aryl, heterocycle, carbocycle, cycloalkyl, heterocycloalkyl, heteroarabinan alkyl, alkylcarbonyl, or glycoside group; or a medicinal cation salt; Or, L １ and L ２ independently have a non-self-immolative linker component comprising one of the following structures: do: 【Chemistry 5】 【change】 In the formula, ( ＊ ) atoms labeled with an additional spacer or releasable linker unit, cell X is a binding site for a toxic agent and / or a cell adhesion molecule (CBA); １ , Y １ , U １ , R ５ , and R ５ ' is as defined above; r is 0 to 100; m and n are each independently Standing is 0-6; Or, L １ and L ２ independently contain at least one bond that can be cleaved under physiological conditions. pH-unstable, acid-unstable, base-unstable, oxidatively unstable, metabolically unstable, biochemical a chemically labile or enzyme labile bond, which has one of the following structures: Here is a concatenation: -(CR) ５ R ６ ) ｍ (A) ｒ (CR) ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｔ -、-(CR ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (A) ｒ (OCH) ２ CH ２ ) ｔ -、-(A]) ｒ -(CR) ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｔ -、-(CR ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｒ -(Aa) ｔ -、-(CR ５ R ６ ) ｍ (CR) ７ =R ８ )(CR ９ R １０ ) ｎ (A) ｔ (OCH) ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (NR) １１ CON) (a) ｔ (CR) ９ R １０ ) ｎ -(OCH ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (A) ｔ ( NR １１ CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (OC) O)(Aa) ｔ (CR) ９ R １０ ) ｎ -(OCH ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (O) CNR ７ )(Aa) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (C) (A) ｔ -(CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (NR) １１ CON ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (O) (A) ｔ (CR) ９ R １０ ) ｎ -(OCH ２ CH ２ ) ｒ -、-(CR ５ R ６ ) ｍ (OCNR) ７ )(Aa) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-(C R ５ R ６ ) ｍ (C) (A) ｔ (CR) ９ R １０ ) ｎ -(OCH ２ CH ２ ) ｒ -、-(C R ５ R ６ ) ｍ -phenyl-CO(Aa) ｔ (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｍ -centre Lyl-CO(Aa) ｔ (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｍ -oxazolyl-CO( Aa) ｔ (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｍ -thiazolyl-CO(Aa) ｔ (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｍ -Thienyl-CO(CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｔ -imidazolyl-CO-(CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｔ -morpholino- CO(Aa) ｔ - (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｔ -piperazino-CO(Aa) ｔ - (CR ７ R ８ ) ｎ -, - (CR ５ R ６ ) ｔ -N-methylpiperazino-CO(Aa) ｔ - (CR ７ R ８ ) ｎ -, - (CR ５ R) ｍ -(Aa) ｔ Phenyl-, -(CR ５ R ６ ) ｍ -(Aa) ｔ Frill-,-(CR ５ R ６ ) ｍ -oxazolyl (Aa) ｔ -, - (CR ５ R ６ ) ｍ -thiazolyl (Aa) ｔ -, - (CR ５ R ６ ) ｍ -thienyl-(Aa ) ｔ -, - (CR ５ R ６ ) ｍ -imidazolyl (Aa) ｔ -, - (CR ５ R ６ ) ｍ -Morpho Rino (Aa) ｔ -, - (CR ５ R ６ ) ｍ -piperazino- (Aa) ｔ -, - (CR ５ R ６ ) ｍ -N-methylpiperazino- (Aa) ｔ -, -K (CR ５ R ６ ) ｍ (Aa) ｒ (CR ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｔ -、-K(CR ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (A) ｒ ( OCH ２ CH ２ ) ｔ -、-K(Aa) ｒ -(CR) ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｔ -、-K(CR ５ R ６ ) ｍ (CR) ７ R ８ ) ｎ (OCH) ２ CH ２ ) ｒ (A) ｔ -、-K(CR ５ R ６ ) ｍ -(CR) ７ =R ８ )(CR ９ R １０ ) ｎ (A) ｔ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (NR １１ CO)(Aa) ｔ (CR ９ R １０ ) ｎ ( OCH ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (A) ｔ (NR) １１ CR) ９ R １ ０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (O) (A) ｔ (CR) ９ R １０ ) ｎ -(OCH ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (OCNR) ７ )(Aa) ｔ ( CR ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (C) (A) ｔ - (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (NR) １１ CO)( (A) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (O) (A) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ (OCN) R ７ )(Aa) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K-(CR ５ R ６ ) ｍ (C) (A) ｔ (CR) ９ R １０ ) ｎ (OCH) ２ CH ２ ) ｒ -、-K(CR ５ R ６ ) ｍ -phenyl-CO(Aa) ｔ (CR ７ R ８ ) ｎ -, -K-(CR ５ R ６ ) ｍ -Frill-C O(Aa) ｔ - (CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｍ -oxazolyl-CO(Aa ) ｔ (CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｍ -thiazolyl-CO(Aa) ｔ (CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｍ -Thienyl-CO(CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｔ -imidazolyl-CO-(CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｔ - Morpholi No-CO(Aa) ｔ - (CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｔ -piperazino-CO( Aa) ｔ - (CR ７ R ８ ) ｎ -, -K (CR ５ R ６ ) ｔ -N-methylpiperazino-CO( Aa) ｔ - (CR ７ R ８ ) ｎ -, -K (CR ５ R) ｍ -(Aa) ｔ Phenyl-, -K-( CR ５ R ６ ) ｍ -(Aa) ｔ Frill-, -K (CR ５ R ６ ) ｍ -oxazolyl (Aa) ｔ -, -K (CR ５ R ６ ) ｍ -thiazolyl (Aa) ｔ -, -K (CR ５ R ６ ) ｍ -Thienyl -(Aa) ｔ -, -K (CR ５ R ６ ) ｍ -imidazolyl (Aa) ｔ -, -K (CR ５ R ６ ) ｍ -morpholino-(Aa) ｔ -, -K (CR ５ R ６ ) ｍ -piperazino- (Aa) ｔ G- , -K(CR ５ R ６ ) ｍ -N-methylpiperazino (Aa) ｔ -, During the ceremony, m, Aa, m, and n are as defined above; t and r are independently 0 to 100; R ３ , R ４ , R ５ , R ６ , R ７ and R8 is a halogen atom; H; halide; C １ -C ８ Alkyl; C ２ -C ８ Aryl, alkenyl, alkynyl, ether, ester, amine or amide, optionally selected from one or more halides, CN, NR １ R ２ , C.F. ３ , OR １ , aryl, heterocycle, S(O)R １ , S.O. ２ R １ , -CO ２ H, -SO ３ H, - OR １ , -CO ２ R １ , -CONR １ , -PO ２ R １ R ２ , -PO ３ , -POH, or P(O)R １ R ２ R ３ is replaced by; KONR １ 、-SS-、-C(=O)-、-C(=O)NH-、-C(=O)--、- C=NH-O-, -C=N-NH-, -C(=O)NH-NH-, O, S, Se, B, H et (C ３ -C ８ a heterocyclic or heteroaromatic ring having Petite; Or, L １ and L ２ independently comprises one of the following hydrophilic structures: 【Chemistry 6】 During the ceremony, 【Chemistry 7】 is the binding site; X ２ , X ３ , X ４ , X ５ , or X ６ are independently NH; NHNH; N(R ３ ); N(R ３ ) N (R ３ ');O;S;C １ -C ６ Alkyl; C ２ -C ６ Heteroalkyl, alkyl cycloalkyl, or heterocycloalkyl; C ３ -C ８ Aryl, Ar-alkyl, heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl; or 1 to 8 amino acids, wherein R ３ and R ３ ' is independently H;C １ -C ８ Alkyl; C ２ -C ８ Heteroalkyl, alkylcyclo alkyl, or heterocycloalkyl; C ３ -C ８ aryl, Ar-alkyl, heterocycle, Carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heterocyclic ring or C ２ -C ８ an ester, ether, or amide; or a group of the formula (OCH ２ CH ２ ) ｐ Or (OCH ２ CH (CH ３ )) ｐ It is a polyethyleneoxy unit having wherein p is an integer from 0 to about 5000, or a combination thereof; Furthermore, the X, Y, and L １ , L ２ , Z １ , or Z ２ is independent of one or more of the following components: It can be configured as: 【Chemistry 8】 【change】 【change】 and L- or D-, natural or non-natural peptides containing 1 to 20 amino acids; During the ceremony, The bond in the center of the atom can bond with any of the adjacent carbon atoms. The wavy lines indicate where you can connect within another bond; Or X, Y, L １ , L ２ , Z １ , or Z ２ may not exist independently, but L １ and Z １ , or L ２ and Z ２ cannot exist simultaneously.

2. prepared from a labile bis-linked compound containing a cytotoxic molecule of formula (II): The two or more residues of the cell-binding molecule may be simultaneously or sequentially linked to form formula (I). A conjugate compound of formula (I) according to claim 1 which can be reacted with: 【Chemistry 9】 During the ceremony: 【Chemistry 10】 represents a single bond; 【Chemistry 11】 is optionally either a single bond, a double bond, or a triple bond, or is optionally not present It doesn't have to be; 【Chemistry 12】 represents a triple bond, Lv １ and Lv ２ Both are absent; Cytotoxic molecules in parentheses, m １ , X, Y, L １ , L ２ , Z １ , and Z ２ is claim 1 and The same definition; Lv １ and Lv ２ The thiols, amines, and carboxylates on the same or different cell-binding molecules Represents a leaving group capable of reacting with a carboxylic acid, selenol, phenol, or hydroxyl group. Lv １ and Lv ２ are OH; F; Cl; Br; I; nitrophenol; N-hydroxy Thiosuccinimide (NHS); phenol, dinitrophenol, pentafluorophenol phenol, tetrafluorophenol; difluorophenol; monofluorophenol; Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol 1-Hydroxybenzotriazole; Tosylate; Mesylate; 2-Ethyl 5-phenyl-3'-oxazolium-3'-sulfonate, by itself or with other anhydrides Anhydrides formed: acetic anhydride or formyl anhydride; or EDC (N-(3-dimethylethyl) (dicyclohexylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl- N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl 1-N'-(2-morpholinoethyl)carbodiimide meso-p-toluenesulfonate ( CMC, or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TB TU(O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronate tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzyl) (benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (benzotriazol-1-yloxy)tripyrrolidine phosphonium hexafluorophosphate (PyBOP), diethylcyanophosphonate DEPC, chloro-N,N,N',N'-tetramethylformamidinium hexahydrate Fluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3- Triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (H ATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3] Triazolo[4,5-b]pyridin-1-ium-3-oxide hexafluorophosphine 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (HDMA), phosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamide Dinium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl 1-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, -(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) carbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium hexamethyl Trifluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyl) (dimethylaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphine O-(benzotriazol-1-yl)-N,N,N',N'-bis ... (Tetramethylene)uronium hexafluorophosphate (HBPyU), N-Benzyl N-cyclohexylcarbodiimide (with or without polymer linkage), dipyrrolidone Lysino(N-succinimidyloxy)-carbenium hexafluorophosphate (H SPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCIU) ), 2-chloro-1,3-dimethylimidazolinium tetrafluoroborate (CIB) , (benzotriazol-1-yloxy)dipiperidinocarbenium hexafluorophosphate sulfate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N, N,N',N'-Tetramethyluronium tetrafluoroborate (TCTU), bromo Tris(dimethylamino)phosphonium hexafluorophosphate (BroP), pro Pyrphosphonic anhydride (PPACA, T3P®), 2-morpholinoethyl isopropyl cyanide (MEI), N,N,N',N'-tetramethyl-O-(N-succinimidyl ) Uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridine Dinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyl] phenylamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate ( TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl Methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethylmorpholinium chloride thyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N ,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1 , 1'-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl)a di-tert-butyl azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate ( DBAD), diisopropyl azodicarboxylate (DIAD), or diethyl Peptide bond reaction selected from azodicarboxylate (DEAD) or Mitsunobu an intermediate molecule formed by a condensation reagent for the reaction; １ and Lv ２ teeth , by itself or by other C １ -C ８ It can be an anhydride formed with an acid anhydride. ru; Or, Lv １ and Lv ２ are independently halides (fluoride, chloride, bromide, iodide), methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoro trifluoromethylsulfonyl (triflate), trifluoromethylsulfonate, nitrophen N-succinimidyl hydroxyl (NHS), phenoxyl; dinitrophenoxy Sil; Pentafluorophenoxyl, Tetrafluorophenoxyl, Trifluorophenoxyl Xyl, difluorophenoxyl, monochlorophenoxyl, pentachlorophenoxyl , 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl Chlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy 2-ethyl-5-phenyloxazolium-3'-sulfonyl, phenyloxadiazolium 2-ethyl-5-phenylisoxazolium -yl, phenyloxadiazol-yl (ODA), oxadiazol-yl, unsaturated Carbon (carbon-carbon, carbon-nitrogen, carbon-sulfur, carbon-phosphorus, sulfur-nitrogen, phosphorus-nitrogen, acid carbon-nitrogen or carbon-oxygen double or triple bond), or one of the following structures: You can choose: 【Chemistry 13】 【change】 During the ceremony, X １ ' is F, Cl, Br, I, or Lv ３ and X ２ ' is O, NH, N(R １ ), or CH ２ and R ３ are independently H, aromatic, heteroaromatic, or one or more H atoms are —R １ ,- Halogen, -OR １ , -SR １ , -NR １ R ２ , -NO ２ , -S(O)R １ , -S(O) ２ R １ , or -COOR １ is an aromatic group independently substituted by Lv ３ are F, Cl, Br, I, nitrophenol; N-hydroxysuccinimide (NH S); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol phenol; difluorophenol; monochlorophenol; pentachlorophenol; dichlorophenol; tetrachlorophenol; 1-hydroxybenzoate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzoate; Tosylate; Mesylate; 2-Ethyl-5-phenylisoxazole a leaving group selected from azolium-3'-sulfonates; R １ and R ２ is H, C １ -C ８ Alkyl, C ２ -C ８ Alkenyl, heteroalkyl, alkylcycloalkyl, or heterocycloalkyl; C ３ -C ８ Aryl, Ar-Alkyl Heterocycle, carbocycle, cycloalkyl, heteroalkylcycloalkyl, alkylcarbocycle aryl, or heteroaryl, or C ２ -C ８ an ester, ether, or amide; or is a peptide containing 1 to 8 amino acids; or is a peptide of the formula (OCH ２ CH ２ ) ｐ Or (OCH ２ CH (CH ３ )) ｐ where p is from 0 to about 500. It can be an integer up to 0, or a combination thereof.

3. prepared from a labile bis-linked compound conjugated to a cell-binding molecule of formula (III): The two or more functional groups of the cytotoxic molecule may be simultaneously or sequentially bonded to form the compound of formula (I): A conjugate compound of formula (I) according to claim 1 which can be reacted with: 【Chemistry 14】 During the ceremony, m １ 、n、 【Chemistry 15】 , cell-binding agents / molecules, L １ , L ２ , and Z ２ has the same definition as in claim 1; X' and Y' are independently reacted with the residue group of a cytotoxic agent simultaneously or sequentially to form is a functional group capable of forming X and Y, wherein X and Y are as defined in formula (I). Be righteous; X' and Y' are disulfide substituents, maleimides, haloacetyls, alkoxyamines , azide, ketone, aldehyde, hydrazine, amino, hydroxyl, carboxylate , imidazole, thiol, or alkyne; or N-hydroxysuccinimide ester, p-nitrophenyl ester, dinitrophenyl ester, pentafluorophenyl esters, pentachlorophenyl esters; tetrafluorophenyl esters; difluorophenyl esters Fluorophenyl ester; Monofluorophenyl ester; Pentachlorophenyl ester , dichlorophenyl ester, tetrachlorophenyl ester, or 1-hydroxy Benzotriazole esters; triflates, mesylates, or tosylates; 2-ethoxybenzotriazole ethyl-5-phenylisoxazolium-3'-sulfonate; pyridyl disulfide, or nitropyridyl disulfide; maleimide, haloacetate, acetylenedicarboxylic carboxylic acid group, carboxylic acid halide (fluoride, chloride, bromide, or iodide) or is one of the following structures: 【Chemistry 16】 【change】 During the ceremony, X １ ' is F, Cl, Br, I, or Lv ３ and X ２ ' is O, NH, N(R １ ), or CH ２ and R ３ and R ５ is H, R １ , aromatic, heteroaromatic, one or several H atoms independently -R １ , -halogen, -OR １ , -SR １ , -NR １ R ２ , -NO ２ , -S(O)R １ , -S(O) ２ R １ , or -COOR １ is an aromatic group substituted with Lv ３ are methanesulfonyl (mesyl), toluenesulfonyl (tosyl), trifluoro Trifluoromethyl-sulfonyl (triflate), trifluoromethylsulfonate, nitrophen Noxyl, N-succinimidyloxy (NHS), phenoxyl; dinitrophenoxy pentafluorophenoxyl, tetrafluorophenoxyl, trifluorophenoxyl Sil, difluorophenoxyl, monofluorophenoxyl, pentachlorophenoxyl , 1H-imidazol-1-yl, chlorophenoxyl, dichlorophenoxyl, trichlorophenoxyl chlorophenoxyl, tetrachlorophenoxyl, N-(benzotriazol-yl)oxy Sil, 2-ethyl-5-phenylisoxazolium-yl, phenyloxadiazole- yl (ODA), oxadiazol-yl, or condensation reagents for Mitsunobu reaction is a leaving group selected from the intermediate molecule １ and R ２ is defined above. There is something.

4. Cytotoxic molecules and cell-binding compounds prepared from a labile bis-linked molecule of formula (IV): The molecules may be reacted independently, simultaneously, or sequentially to form the compound of formula (I).

2. A conjugate compound of formula (I) according to claim 1, which can: 【Chemistry 17】 During the ceremony, 【Chemistry 18】 , m １ , L １ , L ２ , Z １ , and Z ２ has the same definition as in claim 1; Lv １ and Lv ２ has the same definition as in claim 2, and X' and Y' have the same definition as in claim 3. It is righteous.

5. The following formulas (I-a), (I-b), (I-c), (I-d), (I-e), and (If) , (I-g), (I-h), (I-i), (I-j), (I-k), (I-m), (I- n), (I-o), (I-p), (I-q), (I-r), (I-s), (It), ( The compound of formula (I) of claim 1 having the structures represented by Iu), (Iv), and (Iw). Conjugate compounds: 【Chemistry 19】 【change】 【change】 【change】 During the ceremony, X ７ and Y ７ are independently CH, CH ２ ,NH,O,S,NHNH,N(R １ ), and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. This means that 【Chemistry 20】 , X, Y, R １ , n, L １ , and L ２ has the same definition as in claim 1; the cytotoxic agent is The cytotoxic molecule is the same as that described in claim 1.

6. Formulas (II-a), (II-b), (II-c), (II-d), (II-e), (II -f), (II-g), (II-h), (II-i), (II-j), (II-k), ( II-m), (II-n), (II-o), (II-q), (II-r), (II-s) , (II-t), (II-u), (II-v), (II-w), (II-x), (II- y), (II-z), (II-a1), (II-a2), (II-a3), and (II- a4) a compound of formula (II) according to claim 2, having the structure: 【Chemical 21】 【change】 【change】 During the ceremony, X ７ and Y ７ are independently CH, CH ２ ,NH,O,S,NHNH,N(R １ ), and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. This means that 【Chemical 22】 , cytotoxic agents, R １ , X, Y, n, L １ , L ２ , Lv １ , and Lv ２ claims 1 and 2 is the same definition as

7. The compounds represented by the following formulae (III-a), (III-b), (III-c), (III-d), and (II I-e), (III-f), (III-g), (III-h), (III-i), (II I-j), (III-k), (III-l), (III-m), (III-n), (II I-o), (III-p), (III-r), (III-s), (III-t), (II The compound according to claim 3, having a structure represented by (I-u), (III-v), and (III-w). The compound of formula (III) 【Chemical 23】 【change】 【change】 【change】 During the ceremony, X ７ and Y ７ are independently CH, CH ２ ,NH,O,S,NHNH,N(R １ ), and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. means that it can be done; １ , X', Y', n, L １ , and L ２ is the same definition as in claims 1 and 2. It is righteous.

8. The following formulas (IV-a), (IV-b), (IV-c), (IV-d), and (IV-e): (IV-f), (IV-g), (IV-h), (IV-i), (IV-j), (IV-k ), (IV-m), (IV-n), (IV-o), (IV-p), (IV-q), (IV -r), (IV-s), (IV-t), (IV-u), (IV-v), (IV-w), ( IV-x), (IV-y), (IV-z), (IV-a1), (IV-a2), (IV- The compound of formula (IV) according to claim 4, having a structure represented by (IV-a3), and (IV-a4). Compound: 【Chemistry 24】 【change】 During the ceremony, X ７ and Y ７ are independently CH, CH ２ ,NH,O,S,NHNH,N(R １ ), and N; the chemical bond between the two atoms can connect either of the two adjacent atoms. This means that 【Chemistry 25】 , R １ , X', Y', n, L １ , and L ２ has the same definition as in claims 1 and 2.

9. ： A pair of thiols from the interchain disulfides of the cell-binding agent is reacted with dithiothreitol (D TT), dithioerythritol (DTE), dithiolbutylamine (DTBA), L- Glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 2 -mercaptoethylamine (β-MEA), or / and β-mercaptoethanol (β-M 2. The conjugate of claim 1, wherein the conjugate is reduced by a reducing agent selected from the group consisting of 2-ME, 2-ME, 2-ME.

10. 2. The conjugate compound of claim 1, wherein the cytotoxic molecule is selected from the following: (1) A chemotherapeutic agent selected from the group consisting of: a) an alkylating agent selected from the group consisting of: nitrogen mustard; chlorambucil; chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifos Famide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobromide Nititol, melphalan, mitolactol, pipobroman, nobembrine, fenesterol thiamin, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-10 65 and adozelesin, carzelesin, bizelesin, or synthetic analogs thereof; Duoka Lumycin and its synthetic analogs, KW-2189, CBI-TMI, or CBI dimer; Benzodiazepine dimer or (pyrrolobenzodiazepine (PBD) dimer, dimers of indolinobenzodiazepines, dimers of imidazobenzothiadiazepines dimers, or dimers of oxazolidinobenzodiazepines; carmustine, lomustine, Nitrosoureas, including chlorozotocin, fotemustine, nimustine, and ranimustine Alcohols including busulfan, treosulfan, improsulfan and piposulfan Cisulfonate; triazene or dacarbazine; carboplatin, cisplatin, oxaliplatin Platinum-containing compounds, including saliplatin; aziridines, benzodopa, carboquone, methole Dopa or uredopa; ethyleneimines, as well as altretamine and triethylenemelamine , triethylene phosphoramide, triethylene thiophosphoramine, and trimethylol methyl methylmelamines, including lamin; b) Plant alkaloids selected from the group consisting of vincristine, vinblastine, vincristine ... Vinca alkaloids, including vindecine, vinorelbine, and navelbine; paclitaxel, Taxoids including cetaxel and its analogues: DM1, DM2, DM3, DM4 , DM5, DM6, DM7, maytansine, ansamitocin and their analogs Maytansinoids; cryptophycins (cryptophycin 1 and cryptophycin 8) including the group consisting of epothilones, eluterobins, discodermolide, bri Ostatins, dolostatins, auristatins, tubulysins, cephalostatins Pancratistatin; Sarcodictyin; Spongestatin; c) DNA topoisomerase inhibitors selected from the group consisting of: 9-aminocamptothecin camptothecin, crisnatol, daunomycin, etoposide, etoposide phosphate , irinotecan, mitoxantrone, novantrone, retinoic acid (or retinols) , teniposide, topotecan, 9-nitrocamptothecin, or RFS 2000 Hypodophyllins; or mitomycins and their analogues; d) an antimetabolite selected from the group consisting of: {[Antifolates: (methotrexate, trimethoprim, Rexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid DHFR inhibitors, including folic acid analogs); IMP dehydrogenase inhibitors (mycofenases); ribonucleotide reductase (including ribonucleotides, tiazofurin, ribavirin, and EICAR); inhibitors (including hydroxyurea, deferoxamine)]; [pyrimidine analogues: uracil Antibody analogues: (ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda) ), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine , 5-fluorouracil, floxuridine, and raltitrexed (Tomudex) Cytosine analogs (including cytarabine, cytosine arabinoside, fludarabine); Purine analogues: (azathioprine, fludarabine, mercaptopurine, thiamiprine, thiamine Contains folic acid supplements, folic acid}; e) A hormone therapy agent selected from the following group: {receptor antagonist: [antiestrogen: ( megestrol, raloxifene, tamoxifen); LHRH agonists: (including Antiandrogens: (including cerelin, leuprolide acetate); Antiandrogens: (bicalutamide, flutamide , calsterone, dromostanolone propionate, epithiostanol, goserelin, Euprolide, mepitiostane, nilutamide, testolactone, trilostane, and other androgen inhibitors)]; retinoids / deltoids: [vitamin D3 analogs: (CB 1093, EB1089, KH1060, cholecalciferol, ergocalciferol Photodynamic therapy agents: (verteporfin, phthalocyanine, photosensitizer Pc4 , demethoxy-hypocrelin A); cytokines: (interferon alpha, interferon including interferon gamma, tumor necrosis factor (TNF), and TNF domain-containing human proteins ］｝； f) a kinase inhibitor selected from the group consisting of: BIBW2992 (anti-EGFR / Erb 2) Imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib , E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), buff etinib (INNO-406), bosutinib (SKI-606), cabozantinib, bis Modegib, Iniparib, Ruxolitinib, CYT387, Axitinib, Tivozanib, So Rafenib, bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab Bu, ispinesib; g) Olaparib, Niraparib, Iniparib, Talazoparib, Veliparib, CEP972 2 (Cephalon), E7016 (Eisai), BGB-290 (BeiGene ), or 3-aminobenzamide lyase (PARP) inhibitors; h) Antibiotics selected from the following group: enediyne antibiotics (calicheamicins, calicheamicin γ1, δ1, α1 and β1; dynemicin A and deoxydynemicin dynemicins including esperamicin, kedarcidin, C-1027, maduropeptin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotics chromophores), aclacinomycins, actinomycin , anthramycin, azaserine, bleomycins, cactinomycin (cactin omycin, carabicin, carminomycin, carzinophycin Phosphorus; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazomethane 5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, Anomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxol Bicine, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin Mycophenolic acid, nogalamycin, olivomycins, peplomycin, pot-au-feu Iromycin, puromycin, querramycin, rhodolubicin, streptonigrin , streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; i) Polyketides (acetogenins), bullatacin and bullatacinone; gemcitabine, Poxomicins (including carfilzomib), bortezomib, thalidomide, lenalidomide Do, pomalidomide, tosedostat, zybrestat, PLX4032, STA-9 090, Stimuvax, Allovectin-7, Zygeba, Proven Di, Elvoy, isoprenylation inhibitors and lovastatin, dopaminergic neurotoxins and 1 -methyl-4-phenylpyridine ion, cell cycle inhibitors (selected from staurosporine Actinomycins (including the group consisting of actinomycin D and dactinomycin) ), bleomycins (bleomycin A2, bleomycin B2, peplomycin anthracyclines (daunorubicin, doxorubicin (adriamycin), The group consisting of isin, idarubicin, epirubicin, pirarubicin, and zorubicin. ), mitoxantrone, MDR inhibitor or verapamil, Ca ２＋ ATP inhibitors or thapsigargin Galgin, histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat) Stat, valproic acid, mocetinostat (MGCD0103), belinostat, PC I-24781, entinostat, SB939, resminostat, gibinostat, Includes the group consisting of AR-42, CUDC-101, sulforaphane, and trichostatin A ); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfide Filament, Salinosporamide A, antiadrenal drugs (aminoglutethimide, mitotane, trilostridium including the group consisting of: aceglatone; aldophosphamide glycoside; aminolevulin Acid; Amsacrine; Arabinoside, Bestravsil; Bisantrene; Edatrexate; Defofamine, demecolcine, diazicon, elfornithine (DFMO), elfo Micin; elliptinium acetate, etocluzid; gallium nitrate; gacytosine; hydroxyurea ibandronate, lentinan, lonidamine, mitoguazone, mitoxatrone, mopi Damor; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Podophyllin Acid; 2-ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Rhizoxane Syn; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicon; 2,2', 2''-trichlorotriethylamine; trichothecenes (T2 toxin, verrucarin A, including the group consisting of roridin A, and anguidine; urethane, siRNA, antisense Pharmaceuticals; (2) Anti-autoimmune disease drugs: Cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine acetaminophen, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (ambucil, Cinonide, betamethasone, budesonide, hydrocortisone, flunisolide, fluticasone Propionate ester, fluocortodanazol, dexamethasone, triamcinolone cetonide, beclomethasone dipropionate), DHAE, etanercept hydroxychloroquine, infliximab, meloxicam, methotrexate, mycobacterium phenolate mofetil, prednisone, sirolimus, tacrolimus; (3) Anti-infective drugs: a) Aminoglycosides: amikacin, astromycin, gentamicins (nechima) Isin, Sisomycin, Isepamicin), Hygromycin B, Kanamycins (Ami Kashin, Arbekacin, Bekanamycin, Dibekacin, Tobramycin), Neomycin (framycetin, paromomycin, ribostamycin), netilmicin, specti nomycin, streptomycin, tobramycin, verdamycin; b) Amphenicols: azidamphenicol, chloramphenicol, florphenicol Nicole, thiamphenicol; c) Ansamycins: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem / cilasta tin, meropenem, panipenem; e) Cephems: carbacephem (loracarbef), cefacetrile, cefaclor, Cephradine, cefadroxil, cephalonium, cephaloridine, cephalothin or cephalosporin Phalosporin, cephalexin, cephaloglycin, cefamandole, cephapirin , cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cef Capen, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil , cephalosporins, ceftezole, cefuroxime, cefixime, cefdinir, cefdito Ren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cef Operazon, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin Cefpiramide, cefpirome, cefpodoxime, cefprozil, Fukinom, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ftizoxime, ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cef Cefoxitin, cefotetan, cefmetazole, oxacephem (flo moxef, latamoxef); f) Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin) ), teicoplanin (dalbavancin), ramoplanin; g) Glycylcyclines: tigecycline; h) β-lactamase inhibitors: penams (sulbactam, tazobactam), clavams (clavams), Lavranic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics (C D.A.); k) Macrolides: azithromycin, cethromycin, clarithromycin, zirconium Thromycin, erythromycin, flurithromycin, josamycin, ketolide ( Telithromycin, cethromycin), midecamycin, miocamycin, oleandoma Rifamycins (rifampicin, rifampin, rifabutin, rifapentin) rokitamycin, roxithromycin, spectinomycin, spiramycin , tacrolimus (FK506), troleandomycin, telithromycin; l) Monobactams: aztreonam, tigemonam; m) Oxazolidinones: Linezolid; n) Penicillins: amoxicillin, ampicillin, pivampicillin, hetacillin, Campicillin, methampicillin, talampicillin, azidocillin, azlocillin, benzyl benzathine benzylpenicillin, benzathine phenoxymethylpenicillin , clometocillin, procaine benzylpenicillin, carbenicillin (calindacillin) , cloxacillin, dicloxacillin, epicillin, flucloxacillin, mecillinam ( pivmecillinam), mezlocillin, methicillin, nafcillin, oxacillin, penamecillin penicillin, phenethicillin, phenoxymethylpenicillin, piperacillin, propyl Cillin, sulbenicillin, temocillin, ticarcillin; o) Polypeptides: bacitracin, colistin, polymyxin B; p) Quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, Linafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin Syn, Floxacin, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepaflo flucloxacin, carbafloxacin, levofloxacin, lomefloxacin, marbofloxacin fluoxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, Tafloxacin, sparfloxacin, temafloxacin, tosufloxacin, torovaf Loxacin; q) Streptogramins: Pristinamycin, Quinupristin / Dalfopristin ； r) Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethoxazole azole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim , trimethoprim-sulfamethoxazole (cotrimoxazole); s) steroidal antibacterial agents: selected from fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, chromocycline Phosphorus, demeclocycline, lymecycline, meclocycline, methacycline, minocycline Cycline, oxytetracycline, penimepicycline, rolitetracycline, tetracycline tracycline, glycylcyclines (including tigecycline); u) Other types of antibiotics: annonacin, arsphenamine, bactoprenol inhibitors agent (bacitracin), DADAL / AR inhibitor (cycloserine), dictyostatin, Discodermolide, eleutherobin, epothilone, ethambutol, etoposide, Alopenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole ru, mupirocin, mycolactone, NAM synthesis inhibitor (fosfomycin), nitrofuran Toxin, paclitaxel, platensimycin, pyrazinamide, quinupristin / dalphos Pristine, rifampin (rifampin), tazobactamcinidazole, uvaricin; (4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41(E) fluvirtide), PRO140, CD4 (ibalizumab); b) Integrase inhibitors: raltegravir, elvitegravir, globoidnan A; c) Maturation inhibitors: Bevirimat, Vivicon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: abacavir, acyclovir, adefovir, amphotericin Doxovir, apricitabine, brivudine, cidofovir, clevudine, dexerbucitabine bin, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir vir, famciclovir, fluorouracil (5-FU), 3'-fluoro substituted 2',3 '-deoxynucleoside analogues (3'-fluoro-2',3'-dideoxythymidine ( FLT) and 3'-fluoro-2',3'-dideoxyguanosine (FLG) including ), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), L-nucleosides (the group consisting of β-L-thymidine and β-L-2'-deoxycytidine) including penciclovir, rasvir, ribavirin, stampidine, stavudine set (d 4T), taribavirin (viramidine), telbivudine, tenofovir, trifluridine, Lacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: amantadine, ateviridin, capravirine, diarylpyrimidin (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efaviridine Virenz, foscarnet (phosphoryl formate), imiquimod, interferon alpha, Robinson Lidl, rhodenosine, methisazone, nevirapine, NOV-205, peginterferon alpha , podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), Romantadine; g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir , fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, Tonavir, Saquinavir, Telaprevir (VX-950), Tipranavir; h) Other antiviral drugs: Abzyme, Arbidol, Calanolide A, Seragenin, Cyanovirin-N, diarylpyrimidine, epigallocatechin gallate (EGCG), phosphatidylcholine Carnet, Griffithsin, taribavirin (viramidine), hydroxyurea, KP- 1461, Miltefosine, Pleconaril, Portmanteau inhibitors, Ribavirin, Selicin Crib; (5) 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、 １７ ７ Lu, ２１１ At, or ２１３ Radioisotopes (radioactive nuclides); (6) Chromophore molecules capable of absorbing UV light, fluorescent light, IR light, near-IR light, and visual light. xanthophores, erythrophores, iridophores, leucophores, melanophores, chelophores, and fluorophores classes or a subclass, wherein the phosphor is a fluorescent chemical that re-emits light with light, a visible light Sensitive molecules, light-emitting molecules, luminescent molecules, luciferin compound classes or subclasses lases, visible light transmitting molecules, light emitting molecules, luminescent molecules, luciferin compounds; Non-protein organic fluorophores: xanthene derivatives (fluorescein, rhodamine, oligonucleotides) cyanine derivatives (including cyanine, ink, and ink); These include thiacarbocyanines, merocyanines, oxacarbocyanines, and thiacarbocyanines. Squalene derivatives and ring-substituted squaraines (Seta, SeTau, and Squa) re dyes); naphthalene derivatives (including dansyl and prodan derivatives); coumarin Derivatives: Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole) and benzoxadiazoles); anthracene derivatives (DRAQ5, DRAQ7 anthraquinones, including CyTRAK Orange; pyrene derivatives ( Scade Blue); Oxazine derivatives (Nile Red, Nile Blue, Cresyl Violet) Acridine derivatives (including proflavine, acridine oleate, and oxazine 170) (including acridine yellow). Arylmethine derivatives (auramine, crystallized including olet and malachite green). Tetrapyrrole derivatives (porphine, phthalocyanine any analogs and derivatives of the following fluorophore compounds: CF dyes , DRAQ and CyTRAK probes, BODIPY, Alexa Fluor, DyL Light Fluor, Atto and Trancey, FluoProbes, Abber ior dyes, DY and MegaStokes dyes, SulfoCy dyes, HiLyte Fluor, Seta, SeTau and Square dyes, Quasar and Cal F Lour dye, SureLight dye (APC, RPEPerCP, phycobilisome ), APC, APCXL, RPE, BPE, allophycocyanin (APC), Aminocouma Phosphorus, APC-Cy7 conjugate, BODIPY-FL, Cascade Blue, Cy2, Cy3 , Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX , hydroxycoumarin, Lissamine rhodamine B, Lucifer Yellow, methoxycoumarin , NBD, Pacific Blue, Pacific Orange, PE-Cy5 Conjugate, PE-Cy7 conjugate, PerCP, R-phycoerythrin (PE), Red613 , Seta-555-Azide, Seta-555-DBCO, Seta-555-NHS , Seta-580-NHS, Seta-680-NHS, Seta-780-NHS, Seta-APC-780, Seta-PerCP-680, Seta-R-PE-67 0, SeTau380-NHS, SeTau405-maleimide, SeTau405-N HS, SeTau425-NHS, SeTau647-NHS, Texas Red, TRI TC, TruRed, X-rhodamine, 7-AAD (7-aminoactinomycin D, C G selective), acridine orange, chromomycin A3, CyTRAK orange (red excitation (wake / dark), DAPI, DRAQ5, DRAQ7, ethidium bromide, Hoechst 3325 8, Hoechst 33342, LDS751, mithramycin, propidium iodide (PI) , SYTOX Blue, SYTOX Green, SYTOX Orange, Thiazole Orange, TO-PRO: Cyanine monomer, TOTO-1, TO-PRO-1, TOTO-3, TO -PRO-3, YOseta-1, YOYO-1; Fluorescent compounds containing the following compounds or derivatives thereof Photopigments: DCFH (2'7'dichlorodihydro-fluorescein, oxidized form), DHR (di Hydrorhodamine 123, oxidized form, photocatalytic oxidation), Fluo-3 (AM ester, pH > 6), Fluo-4 (AM ester, pH 7.2), Indo-1 (AM ester, low / High calcium (Ca ２＋ )), SNARF (pH 6 / 9), allophycocyanin (APC ), AmCyan1 (tetramer, Clontech), AsRed2 (tetramer, Clontech), Aza mi Green (monomer), azurite, B-phycoerythrin (BPE), cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP"), EBFP, EBFP2, ECFP, EGFP (weak dimer), Emerald (weak dimer), EYFP (weak dimer), GFP (S65A mutant), GFP (S65C mutant), GFP (S65L mutant) variant), GFP (S65T mutant), GFP (Y66F mutant), GFP (Y66H mutant) GFP (Y66W mutant), GFP ｕｖ , HcRed1, J-Red, Katyusi Kusabira Orange (monomer, MBL), mCFP, mCherry, m Citrine, Midrishi Cyan (dimer, MBL), mKate (Tag FP635, monomer), mKeima-Red (monomer), mKO, mOrange, m Plum, mRaspberry, mRFP1 (monomer), mStrawberry, m TFP1, mTurquoise2, P3 (phycobilisome complex), Peridin in Chlorophyllin (PerCP), R-phycoerythrin (RPE), T- Sapphire, TagCFP (dimer), TagGFP (dimer), TagRFP ( dimer), TagYFP (dimer), tdTomato (tandem dimer), Topaz, TurboFP602 (dimer), TurboFP635 (dimer), TurboGFP (dimer), TurboRFP (dimer), TurboYFP635 (dimer), Vina s, native GFP, YPet, Zs Green 1 (tetramer), Zs Yellow 1 (tetramer). (7) A cell-binding ligand or receptor agonist that can be selected from the following: folic acid-derived Conductors; Glutamic acid urea derivatives; Somatostatin and its analogues (Octreotide (San dostatin) and lanreotide (somatrin); phonamide; pituitary adenylate cyclase-activating peptide (PACAP) (PAC1); Vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VPAC2); melanin Cell-stimulating hormone (α-MSH); cholecystokinin (CCK) / gastrin receptor bombesin (Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 / gastrin-releasing peptide (GRP); neurotensin receptor Receptor ligands (NTR1, NTR2, NTR3); substance P (NK1 receptor) ligand Neuropeptide Y (Y1-Y6); RGD (Arg-Gly-Asp), NGR (Asn-Gly-Asp) rg), dimeric and multimeric cyclic RGD peptides (selected from cRGDfVc), TAA SGVRSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ligands) and homing peptides including F3 peptides; cell-penetrating peptides (C PPs); follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are involved in testosterone production as well. Luteinizing hormone-releasing hormone (LH), which acts by targeting LH RH) agonists and antagonists, and gonadotropin-releasing hormone (GnRH) ) agonists, buserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)- Leu-Arg-Pro-NHEt), gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 ) , goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-AzGly-NH2), histidine Phosphorus (Pyr-His-Trp-Ser-Tyr-D-His(N-benzyl)-Leu-Arg-Pro-NHEt), leuprolide (Py r-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt, nafarelin (Pyr-His-Trp-Ser-Tyr-2N al-Leu-Arg-Pro-Gly-NH 2 ), Triptorelin (Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro- Gly-NH 2 ), nafarelin, deslorelin, abarelix (Ac-D-2Nal-D-4-chloroPhe-D- 3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropyl-Pro-DAla-NH 2 ), Cetroleri (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-Cit-Leu-Arg-Pro-D- Ala-NH 2 ), Degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-4-amido Phe(L-hydroorotyl)-D-4-amioPhe(carbamoyl)-Leu-isopropylLys-Pro-D-Ala-NH 2 ), and ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-Tyr-D-(N (9,N10-diethyl)-homoArg-Leu-(N9,N10-diethyl)-homoArg-Pro-D-Ala-NH 2 ) Peptide hormones selected from the group consisting of Toll-like receptor (TLR) ligands, C-type lectins, and Nod-like receptor (NLR) ligands. receptors (PRRs); calcitonin receptor agonists; integrin receptors and their receptors Container subtype (α Ｖ β １ 、α Ｖ β ３ 、α Ｖ β ５ 、α Ｖ β ６ 、α ６ β ４ 、α ７ β １ 、α Ｌ β ２ , α ＩＩｂ β ３ ) agonists (GRGDSPK, cyclo (RGDfV) (L1) and its derivatives [cyclo(-N(Me)R-GDfV), cyclo(-N(Me)R-GDfV), (R-Sar-DfV), cyclo(RG-N(Me)D-fV), cyclo(RGD-N( Me)f-V), cyclo(RGDf-N(Me)V-) (silengitide)]; antibodies (derivatives of VHH (camel Ig)); domain antibodies (dAb, VH or VL domains) derivatives), bispecific T cell induction (Bite, bispecific antibody); biaffinity retargeting tetravalent tandem antibodies (TandAb, dimerized tandem antibodies); specific antibodies); anticalins (derivatives of lipocalin); adnectins (10th F N3 (fibronectin); designed ankyrin repeat proteins (DARPins) ; avimer; EGF receptor and VEGF receptor agonist. (8) Pharmaceutically acceptable salts, acids, derivatives, hydrates, or hydrates of any of the above drugs. salts; or crystal structures; or optical isomers, racemates, diastereomers, or enantiomers Mah.

11. The cytotoxic molecule is a chromophore molecule, and the interaction and / or function of the cell-binding molecule or for detecting, monitoring, or studying the interaction of said conjugate with a target cell. The conjugate compound of claim 1 .

12. The cytotoxic molecule is polyalkylene glycol (poly(ethylene glycol) (PE Gs), poly(propylene glycol), ethylene oxide or propylene oxide or a copolymer thereof, wherein the conjugate, when administered to a mammal, The conjugate compound of claim 1 is used to extend the half-life of the cell-binding molecule in combination with thing.

13. The cytotoxic molecule may be a cell-binding ligand, a cell receptor agonist, or a cell receptor-binding and the conjugate is a compound for delivery to malignant cells or for modulating a desired immune response. Target conductors / determiners to stimulate or co-stimulate or alter signal transduction pathways The conjugate compound of claim 1, which is used for a director.

14. The cytotoxic molecule is selected from the group consisting of tubulysins, calicheamicins, auristatins, Maytansinoids, CC-1065 analogs, daunorubicin and doxorubicin compounds , taxanoids (taxanes), cryptophycins, epothilones, benzodiazepines Dimers of pyrrolobenzodiazepines (PBD), tomaymycin dimers, anthraquinone dimers, Mycin dimer, indolinobenzodiazepine dimer, imidazobenzothiadiazepine dimers of benzodiazepines, or dimers of oxazolidinobenzodiazepines, and their derivatives ), calicheamicins and enediyne antibiotics, actinomycins, amanitin , azaserine, bleomycin, epirubicin, tamoxifen, idarubicin , dolastatins / auristatins (monomethyl auristatin E, MMAE, MMA F, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP), and derivatives thereof. lumycins, geldanamycins, methotrexate , thiotepa, vindesines, vincristine, hemiasterides nazumamides, microginins ns), radiosumins, alterobactins obactins, microsclerodermicins ns), theonellamides, esperamicins (esp eramicins), siRNA, miRNA, piRNA, nucleases, and / or or pharmaceutically acceptable salts, acids, and / or analogs or derivatives thereof of any of the above molecules. or crystal structure; or optical isomer, racemate, diastereomer 3. The conjugate according to claim 1 or 2, wherein the conjugate is selected from the group consisting of 1 and 2, and an enantiomer thereof. body compound.

15. The cell binding agent / molecule may be an antibody, a protein, a probody, a nanobody, a vitamin ( including acids), peptides, polymer micelles, liposomes, lipoprotein drug carriers, nano Particulate drug carriers, dendrimers, and molecules coated with cell-binding ligands, or combinations thereof 4. The conjugate compound of claim 1 or 3, wherein the conjugate compound is selected from a combination of:

16. The cell-binding molecule / agent may be an antibody, an antibody-like protein, a full-length antibody (polyclonal antibody, monoclonal antibodies, dimers, multimers), or multispecific antibodies (bispecific antibodies, trispecific antibodies) a monoclonal antibody, ... antibody fragments, monoclonal antibodies, single-chain monoclonal antibodies, and monoclonal antibodies that bind to target cells. monoclonal antibody fragments, chimeric antibodies, chimeric antibody fragments that bind to target cells, domain antibodies, target Domain antibody fragments that bind to target cells, resurfaced antibodies, single-chain resurfaced antibodies, target cells Resurfaced antibody fragments, humanized antibodies or resurfaced humanized antibodies, single chain Humanized antibodies or humanized antibody fragments that bind to target cells, anti-idiotypic (anti-Id) antibodies antibody, CDR, dimer, trimer, tetramer, miniantibody, probody, pro Body fragments, small immune proteins (SIPs), lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules, large molecular weight proteins, and antibodies or large molecular weight 3. The nanoparticle or polymer modified with a protein according to claim 1 or 3.

16. The conjugate compound according to claim 15.

17. The cell binding agent / molecule may bind to tumor cells, virus-infected cells, microbial-infected cells, parasitic-infected cells, or the like. cells, autoimmune disease cells, activated tumor cells, bone marrow cells, activated T cells, affected B cells, or melanocytes, or expressing one or more of the following antigens or receptors: Claims 1, 3, 5, 7, 10, 11, 12, 13, and 14, which can target cells 17. The conjugate compound according to claim 16, wherein: CD2, CD2R, CD3, CD3gd, CD 3e, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD1 0, CD11a, CD11b, CD11c, CD12, CD12w, CD13, CD14 , CD15, CD15s, CD15u, CD16, CD16a, CD16b, CD17, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24 , CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 , CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40 , CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD4 6、CD47、CD47R、CD48、CD49a、CD49b、CD49c、CD49 e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55、CD 56、CD57、CD58、CD59、CD60、CD60a、CD60b、CD60c 、CD61、CD62E、CD62L、CD62P、CD63、CD64、CD65、C D65s、CD66、CD66a、CD66b、CD66c、CD66d、CD66e、 CD66f、CD67、CD68、CD69、CD70、CD71、CD72、CD73 、CD74、CD74、CD75、CD75s、CD76、CD77、CD78、CD7 9、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、C Dw84、CD85、CD86、CD87、CD88、CD89、CD90、CD91、 CD92、CDw92、CD93、CD94、CD95、CD96、CD97、CD98 、CD99、CD99R、CD100、CD101、CD102、CD103、CD10 4、CD105、CD106、CD107、CD107a、CD107b、CD108、 CD109、CD110、CD111、CD112、CD113、CDw113、CD1 14、CD115、CD116、CD117、CD118、CD119、CDw119、 CD120a、CD120b、CD121a、CD121b、CDw121b、CD12 2、CD123、CDw123、CD124、CD125、CDw125、CD126、 CD127、CD128、CDw128、CD129、CD130、CD131、CDw 131、CD132、CD133、CD134、CD135、CD136、CDw136 、CD137、CDw137、CD138、CD139、CD140a、CD140b、 CD141、CD142、CD143、CD144、CD145、CDw145、CD1 46、CD147、CD148、CD149、CD150、CD151、CD152、C D153、CD154、CD155、CD156a、CD156b、CDw156c、C D157, CD158a、CD158b、CD159a、CD159b、CD159 c、CD160、CD161、CD162、CD162R、CD163、CD164、C D165、CD166、CD167、CD167a、CD168、CD169、CD17 0、CD171、CD172a、CD172b、CD172g、CD173、CD174 、CD175、CD175s、CD176、CD177、CD178、CD179、CD 180、CD181、CD182、CD183、CD184、CD185、CD186、 CDw186、CD187、CD188、CD189、CD190、CD191、CD1 92、CD193、CD194、CD195、CD196、CD197、CD198、C Dw198、CD199、CDw199、CD200、CD200a、CD200b、C D201、CD202、CD202b、CD203、CD203c、CD204、CD2 05、CD206、CD207、CD208、CD209、CD210、CDw210、 CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw 218b、CD220、CD221、CD222、CD223、CD224、CD225 、CD226、CD227、CD228、CD229、CD230、CD231、CD2 32、CD233、CD234、CD235a、CD235ab、CD235b、CD2 36、CD236R、CD238、CD239、CD240、CD240CE、CD24 0D、CD241、CD242、CD243、CD244、CD245、CD246、C D247、CD248、CD249、CD252、CD253、CD254、CD256 、CD257、CD258、CD261、CD262、CD263、CD265、CD2 66、CD267、CD268、CD269、CD271、CD273、CD274、C D275、CD276(B7-H3)、CD277、CD278、CD279、CD28 0、CD281、CD282、CD283、CD284、CD289、CD292、CD w293、CD294、CD295、CD296、CD297、CD298、CD299 、CD300a、CD300c、CD300e、CD301、CD302、CD303、 CD304、CD305、CD306、CD309、CD312、CD314、CD31 5、CD316、CD317、CD318、CD319、CD320、CD321、CD 322、CD324、CDw325、CD326、CDw327、CDw328、CDw 329、CD331、CD332、CD333、CD334、CD335、CD336、 CD337, CDw338, CD339, 4-1BB, 5AC, 5T4 (trophoblast glycoproteins) protein, TPBG, 5T4, Wnt activation inhibitory factor 1 or WAIF1), adenocarcinoma antigen, A GS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, α integrin, αvβ6, aminopeptidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toki Syn protective antigen, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, anthrax Bacteria, BAFF (B-cell activating factor), BCMA, B-lymphoma cells, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MU C16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (C-C 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), clumping factor A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte colony stimulating factor 1 receptor, CD115), phage 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, Dabiga Tran, 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), E GFR, EGFRII, EGFRvIII, endoglin, endothelin B receptor, end Dotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), E. coli, ETV6-AML, FAP (fibroblast activation protein α), FCGR1, α -fetoprotein, fibrin II, beta chain, fibronectin ectodomain B, FOL R (folate receptor), folate receptor α, folate hydrolase, respiratory syncytial virus Fos-related antigen 1 F 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, GP10 0, GPNMB (transmembrane protein NMB), GUCY2C (guanylate cyclase 2C) , guanylate cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase Ze-C receptor, heat-stable enterotoxin receptor (hSTAR), heat shock proteins Protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor) Condition 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (stem cell growth factor / scatter factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6 / E7, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype , IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, Influenza hemagglutinin, IgE, IgE Fc region, IGHE, 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, L-12, IL-13, IL-1 5, IL-17, IL-17A, IL-18, IL-19, IL-20, IL-21, I including L-22, IL-23, IL-27, or IL-28. ), IL-31RA, IL GF2 (insulin-like growth factor 2), integrin (α4, α ＩＩＩｂ β ３ , αvβ3, a ４ b ７ 、α5β1、α6β4、α7β7、αIΙβ3、α5β5、αvβ5), インタ -feron gamma-inducible protein, ITAGA2, ITGB2, KIR2D, Kappa I g, LCK, Le, legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-associated antigen 1) , CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, L TA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE- 3, MAGEA1, MAGEA3, MAGE4, ​​MART1, MCP-1, MIF (Mac phage migration inhibitory factor or glycosylation inhibitory factor (GIF), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (mesothelin), MUC1 (mucin 1 , cell surface-associated (MUC1) or Polymorphic epithelial mu cin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotaxis chemotactic protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 ( Transmembrane 4-domain subfamily A), MYCN, myelin-associated glycoprotein, Myos Tatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (A SG-22ME), NGF, neuronal apoptosis-regulating proteinase 1, NOGO-A, N itch 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, Page 4, PAP, anti-(N-glycolylneuraminic acid) Latope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, programmed Cell death protein 1), PDGF-Rα, (platelet-derived growth factor receptor α), PDGFR -β, PDL-1, PLAC1, PLAP-like testicular alkaline phosphatase, platelet-derived component Long factor receptor β, sodium phosphate cotransporter, PMEL17, polysialic acid, protein Prostate cancer, PS (phosphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein, RHD (Rh polypeptide) 1 (RhPI), Rhesus factor, RANKL, Ph oC, Ras mutant, RG55, ROBO4, respiratory syncytial virus, RON, ROR1, sarcoma metastasis Translocation breakpoint, SART3, sclerostin, SLAMF7 (SLAM family Member 7), selectin P, SDC1 (syndecan 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 1 7, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn , TAG-22 (tumor-associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane Common proteins: TEM1 (tumor epithelial marker 1), TENB2, tenascin-C (TN-C ), TGF-α, TGF-β (transforming growth factor β), TGF-β1, TG F-β2 (transforming growth factor β2), Tie (CD202b), Tie2, TIM-1 (CDX-014), TN, TNF, TNF-α, TNFRSF8, TNFR SF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF1 3B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblast glycoprotein protein), TRAIL-R1 (tumor necrosis-inducing ligand receptor 1), TRA ILR2 (Death Receptor 5 (DR5)), a tumor-associated calcium signal transducer -2. Tumor-specific glycosylation of MUC1, TWEAK receptor, and TYRP1 (glycoprotein Quality 75), TRP-2, tyrosinase, VCAM-1, VEGF, VEGF-A, VEGF F-2, VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or any Cells expressing any insulin growth factor receptor, or any epidermal growth factor receptor.

18. The tumor cells of claim 17 are selected from the group consisting of lymphoma cells, myeloma cells, renal cells, breast cancer cells, prostate cancer cells, and the like. Adenocarcinoma cells, ovarian cancer cells, colon cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, non-small Lung cancer cells, testicular cancer cells, or cells that grow and divide uncontrollably, promoting a cancer-causing pace The cell is selected from any cell that

19. The cytotoxic molecule is a chromophore molecule, and the conjugate compound of formula (I) is Ac01, Ac02, Ac03, Ac04, Ac05, Ac06, and Ac07 The conjugated compound of claim 1 selected from: 【Chemical 26】 During the ceremony, 【Chemical 27】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; R １２ and R １２ 'OH, NH ２ , N.H.R. １ , N.H.N.H. ２ ,NHNHCOOH,O -R １ -COOH、NH-R 1 -COOH、NH-(Aa) ｎ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH (CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH SO ３ H、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、R １ -NHSO ３ H、 NH-R １ -NHSO ３ H、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、NH (CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、R １ -NHPO ３ H ２ 、R １ -OP O ３ H ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OPO ３ H ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、OR １ -NHPO ３ H ２ 、NH-R １ -NHPO ３ H ２ , NH-Ar-COOH, NH-Ar-NH ２ where p is 0 to 5000; , Aa is an amino acid; R １ , L １ and L ２ has the same definition as in claim 1.

20. wherein the cytotoxic molecule is a tubulysin analog and the conjugate compound of formula (I) is The following T01, T02, T03, T04, T05, T06, T07, T08, T09, T 10, and T11, ​ 【change】 During the ceremony, 【Chemical 29】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; R １２ は、OH、NH ２ 、NHR １ 、NHNH ２ 、NHNHCOOH、O-R １ -CO OH、NH-R 1 -COOH、NH-(Aa) ｎ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NR １ R １ '、NHOH、NHOR １ 、O(CH ２ CH ２ O) ｐ C H ２ CH ２ COOH、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH-Ar-C OOH、NH-Ar-NH ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、NH (CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、R １ -NHSO ３ H、NH-R １ -N HSO ３ H、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、OR １ 、R １ -NHPO ３ H ２ 、R １ -OPO ３ H ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OPO ３ H ２ 、OR １ -NHPO ３ H ２ 、NH -R １ -NHPO ３ H ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ S) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ OH、N H(CH ２ CH ２ S) ｐ CH ２ CH ２ OH, NH-R １ -NH ２ 、 or NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ where Aa is 1 to 8 amino acids ； n and m １ are independently 1 to 20; p is 1 to 5000; R １ , R １ ', R ２ , R ３ , and R ４ are independently H, C １ -C ８ Linear or branched alkyl alkyl, amide, or amine; C ２ -C ８ aryl, alkenyl, alkynyl, heteroaryl Aryl, heteroalkyl, alkylcycloalkyl, ester, ether, heterocyclo alkyl or acyloxylamine; or a peptide containing 1 to 8 amino acids, or is the formula (OCH ２ CH ２ ) ｐ Or (OCH ２ CH (CH ３ )) ｐ Polyethyleneoxy units, where p is an integer from 1 to about 5000; The two R's: １ R ２ , R ２ R ３ , R １ R ３ , or R ３ R ４ is alkyl, aryl, hexamethyl forming a 3- to 8-membered ring of a heteroaryl, heteroalkyl, or alkylcycloalkyl group; can be; X ３ is H, CH ３ , C.H. ２ CH ３ , C ３ H ７ , or X １ 'R １ ', where X １ ' is NH, N(CH ３ ), NHNH, O, or S, and R １ ' is H or C １ -C ８ linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcyclo alkyl, acyloxyamine; R ３ ' is H or C １ -C ６ is a straight chain or branched alkyl; Z ３ ,H,COOR １ , N.H. ２ , N.H.R. １ , OR １ , CONHR １ , NHCOR １ , OCOR １ 、OP(O)(OM １ )(OM ２ ), OCH ２ OP(O)(OM) １ )(OM ２ ), OSO ３ M １ , R １ , or O-glycosides (glucosides, galactosides, mannosides, Glucuronosides / glucuronides, arosides, fructosides), NH-glycosides, S-glycosides Lycoside, or CH ２ -glycoside; M １ and M ２ are each independently H, Na , K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and L １ and L ２ has the same definition as in claim 1.

21. wherein the cytotoxic molecule is a calicheamicin analog and the conjugate compound of formula (I) is 2. The conjugated compound of claim 1, wherein the compound is selected from the following structures C01 and C02: 【Chemistry 30】 During the ceremony, 【Chemical 31】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; p is 1 to 5000; R １ , L １ , and L ２ has the same definition as in claim 1.

22. The cytotoxic molecule is a maytansinoid analog, and the conjugate compound is y01, My02, My03, My04, My05, and My06 structures 10. The conjugated compound of claim 1: 【Chemical 32】 【change】 【Chemical 33】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; p is 1 to 5000; R １ , L １ , and L ２ has the same definition as in claim 1.

23. The cytotoxic molecule is a taxane analog, and the conjugate compound is Tx01: The conjugated compound of claim 1, selected from the structures of Tx02 and Tx03: 【Chemical 34】 【Chemistry 35】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; R １ , L １ , and L ２ has the same definition as in claim 1.

24. the cytotoxic molecule is a CC-1065 analog and / or a duocarmycin analog, , wherein the conjugate compound is selected from the following structures CC01, CC02, and CC03:

10. The conjugated compound of claim 1: 【Chemical 36】 During the ceremony, mAb is an antibody; Z ３ は、H、PO(OM １ )(OM ２ ), SO ３ M １ ,H ２ PO(OM) １ )(OM ２ )、CH ３ N(CH ２ CH ２ ) ２ NC(O)-、O(CH ２ CH ２ ) ２ NC(O)-、R １ or a glycoside; 【Chemical 37】 is either a single or double bond or is absent; X １ , X ５ , Y １ , and Y ２ are independently O, NH, NHNH, NR ５ , S, C(O) O, C(O)NH, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S 、OC(O)N(R １ )、N(R １ )C(O)N(R １ )、CH、C(O)NHNHC( O), and C(O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; R １ , L １ , and L ２ has the same definition as in claim 1.

25. the cytotoxic molecule is daunorubicin or a doxorubicin analog, The items are Da01, Da02, Da03, Da04, Da05, Da06, and Da07.

2. The conjugated compound of claim 1, wherein the conjugated compound is selected from the structures: ​ 【change】 During the ceremony, 【Chemical Formula 39】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and R １２ はOH、NH ２ 、NHR １ 、NHNH ２ 、NHNHCOOH、O-R １ -COO H、NH-R １ -COOH、NH-(Aa) ｎ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ O) ｐ C H ２ CH ２ NH ２ 、NR １ R １ '、NHOH、NHOR １ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH-Ar-CO OH、NH-Ar-NH ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、NH( CH ２ CH ２ O) ｐ CH ２ CH ２ NH-SO ３ H、R １ -NHSO ３ H、NH-R １ -N HSO ３ H、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、NH(CH ２ CH ２ O) ｐ CH ２ -CH ２ NHPO ３ H ２ 、OR １ 、R １ -NHPO ３ H ２ 、R １ -OPO ３ H ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OPO ３ H ２ 、OR １ -NHPO ３ H ２ 、N HH-R １ -NHPO ３ H ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ NH ２ 、NH(C H ２ CH ２ S) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ OH、 NH(CH ２ CH ２ S) ｐ CH ２ CH ２ OH, NH-R １ -NH ２ 、 or NH(CH ２ C H ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ where Aa is 1 to 8 amino acids ； p is 1 to 5000; mAb is an antibody; n and m １ are independently 1 to 20; R １ , L １ , and L ２ has the same definition as in claim 1.

26. ： the cytotoxic molecule is an auristatin or dolastatin analog and the conjugated compound is selected from the group consisting of Au01, Au02, Au03, Au04, Au 05, Au06, Au07, Au08, Au09, Au10, Au11, Au12, and 2. The conjugated compound of claim 1, selected from the structure of Au13: 【Chemistry 40】 【change】 During the ceremony, 【Chemistry 41】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and R １２ は、OH、NH ２ 、NHR １ 、NHNH ２ 、NHNHCOOH、O-R １ -CO OH、NH-R 1 -COOH、NH-(Aa) ｎ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NR １ R １ '、NHOH、NHOR １ 、O(CH ２ CH ２ O) ｐ C H ２ CH ２ COOH、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH-Ar-C OOH、NH-Ar-NH ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、NH (CH ２ CH ２ O) ｐ CH ２ CH ２ NH-SO ３ H、R １ -NHSO ３ H、NH-R １ - NHSO ３ H、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、NH(CH ２ CH ２ O) ｐ CH ２ -CH ２ NHPO ３ H ２ 、OR １ 、R １ -NHPO ３ H ２ 、R １ -OPO ３ H ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OPO ３ H ２ 、OR １ -NHPO ３ H ２ 、 NH-R １ -NHPO ３ H ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ NH ２ 、NH( CH ２ CH ２ S) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ NH) ｐ CH ２ CH ２ OH , NH(CH ２ CH ２ S) ｐ CH ２ CH ２ OH, NH-R １ -NH ２ , or NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ where Aa is 1 to 8 amino acids Yes; p is 1-5000; mAb is an antibody; n and m １ are independently 1 to 20; p is 1-5000; R １ , R ２ , R ３ , and R ４ are independently H; C １ -C ８ linear or branched alkyl, Aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, ester, ether or 1 to 8 alkyl, amide, amine, heterocycloalkyl, or acyloxylamine; or a peptide containing an amino acid of the formula (OCH ２ CH ２ ) ｐ Or (OCH ２ CH ( CH ３ )) ｐ where p is an integer from 1 to about 5000. 。 The two R's: １ R ２ , R ２ R ３ , R １ R ３ , or R ３ R ４ is alkyl, aryl, hexamethyl forming a 3- to 8-membered ring of a heteroaryl, heteroalkyl, or alkylcycloalkyl group; can be; X ３ is H, CH ３ , or X １ 'R １ ', where X １ ' is NH, N(CH ３ ) , NHNH, O, or S, and R １ ' is H or C １ -C ８ a linear or branched alkyl group of Aryl, heteroaryl, heteroalkyl, alkylcycloalkyl, acyloxya Min; R ３ ' is H or C １ -C ６ is a straight chain or branched alkyl; Z ３ ', H, COOR １ , N.H. ２ , N.H.R. １ , OR １ , CONHR １ , NHCOR １ 、OCOR １ 、OP(O)(OM １ )(OM ２ ), OCH ２ OP(O)(OM) １ )(OM ２ ), OSO ３ M １ , R １ , or O-glycosides (glucosides, galactosides, mannosides , glucuronosides / glucuronides, arosides, fructosides), NH-glycosides, S- Glycoside, or CH ２ -glycoside; M １ and M ２ are each independently H, N a, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and L １ and L ２ has the same definition as in claim 1.

27. the cytotoxic molecule is a dimer of a benzodiazepine analog, and the conjugate compound is Below PB01, PB02, PB03, PB04, PB05, PB06, PB07, PB0 8, PB09, PB10, PB11, PB12, PB13, PB14, PB15, PB1 6, PB17, PB18, PB19, PB20, PB21, and PB22 2. The conjugated compound of claim 1, 【Chemistry 42】 【change】 【change】 【change】 During the ceremony, 【Chemistry 43】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; L １ , L ２ , Z １ , and Z ２ has the same definition as in claim 1; R １ , R ２ , R ３ , R １ ', R ２ ', and R ３ ' are independently H; F; Cl; ═O; ═ S;OH;SH;C １ -C ８ Straight or branched alkyl, aryl, alkenyl, heteroaryl Aryl, heteroalkyl, alkylcycloalkyl, ester (COOR ５ Or -OC( O)R ５ ), ether (OR ５ ), amide (CONR ５ ), carbamate (OCONR ５ ), amine (NHR ５ , N.R. ５ R ５ '), heterocycloalkyl, or acyloxyl Min (-C(O)NHOH, -ONHC(O)R ５ ); or 1 to 8 natural or non-natural amino acids a peptide containing an amino acid, or a peptide of the formula (OCH ２ CH ２ ) ｐ Or (OCH ２ CH (CH ３ )) ｐ where p is an integer from 1 to about 5000. The two R's: １ R ２ , R ２ R ３ , R １ R ３ , R １ 'R ２ ', R ２ 'R ３ ', or R １ ' R ３ ' is independently alkyl, aryl, heteroaryl, heteroalkyl, or alkyl; The cycloalkyl group can form a 3- to 8-membered ring; X ２ and Y ２ are independently N, CH ２ , or CR ５ where R ５ is H, OH, NH ２ , NH(CH ３ ), NHNH ２ , COOH, SH, OZ ３ , SZ ３ , or C １ -C ８ linear or branched alkyl, aryl, heteroaryl, heteroalkyl, alkylcyclo alkyl, acyloxyamine; Z ３ はH、OP(O)(OM １ )(OM ２ ), OCH ２ OP(O)(OM) １ )(OM ２ ), OSO ３ M １ , or O-glycosides (glucosides, galactosides, mannosides, glucosides) Glucuronides / glucuronides, alosides, fructosides), NH-glycosides, S-glycosides Do or CH ２ -glycoside; M １ and M ２ are each independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ is.

28. The cytotoxic molecule is an amanitin analogue, and the conjugate compound is Am01: The conjugated compound of claim 1, selected from the structures Am02, Am03, and Am04. thing: 【Chemical 44】 During the ceremony, 【Chemistry 45】 may optionally be a single or double bond, or may optionally be absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody, preferably a monoclonal antibody; n and m １ are independently 1 to 20; R ７ , R ８ , and R ９ are independently H, OH, OR １ , N.H. ２ , N.H.R. １ , C １ -C ６ alkyl or absent; Y ２ Yes, O, O ２ , N.R. １ , NH or absent; R １０ は、CH ２ 、O、NH、NR １ 、NHC(O)、NHC(O)NH、NHC(O )O、OC(O)O、C(O)、OC(O)、OC(O)(NR １ )、(NR １ )C(O ) (NR １ ), C(O)R １ or is absent; R １１ は、OH、NH ２ 、NHR １ 、NHNH ２ 、NHNHCOOH、O-R １ -CO OH、NH-R １ -COOH、NH-(Aa) ｎ COOH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ OH、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH ２ 、NR １ R １ ’、O(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、N H(CH ２ CH ２ O) ｐ CH ２ CH ２ COOH、NH-Ar-COOH、NH-Ar-N H ２ 、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHSO ３ H、NH(CH ２ CH ２ O) ｐ C H ２ CH ２ NHSO ３ H、R １ -NHSO ３ H、NH-R １ -NHSO ３ H、O(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ 、NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH PO ３ H ２ 、OR １ 、R １ -NHPO ３ H ２ 、R １ -OPO ３ H ２ 、O(CH ２ CH ２ O ) ｐ CH ２ CH ２ OPO ３ H ２ 、OR １ -NHPO ３ H ２ 、NH-R １ -NHPO ３ H ２ , or NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NHPO ３ H ２ where Aa is 1 to 8 amino acids; n and m １ are independently 1 to 20; p is 1 to 5000; R １ , L １ , and L ２ has the same definition as in formula (I); L １ , L ２ , R １ , Z １ , and Z ２ has the same definition as in claim 1.

29. The cytotoxic molecule is a polyalkylene glycol analogue, and the conjugate compound is 13. The compound according to claim 1 or 12, wherein the compound is selected from the structures Pg01, Pg02, and Pg03 below: Conjugate compounds of: 【Chemistry 46】 During the ceremony, 【Chemistry 47】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; p is 1 to 5000; R １ and R ３ are independently H, OH, OCH ３ , C.H. ３ , or O.C. ２ H ５ and L １ and L ２ has the same definition as in claim 1.

30. The cytotoxic molecule is a cell-binding ligand or a cell receptor agonist and its analogs. The conjugate compound is LB01 (folic acid conjugate), LB02 ( LB03 (PMSA ligand conjugate), LB04 (PMSA ligand conjugate), Ligand conjugate), LB05 (somatostatin conjugate), LB06 (somatostatin conjugate) LB07 (octreotide, somatostatin analogue conjugate), LB08 (lanreotide LB09 (vapreotide (Sanvar), somatostatin analog conjugate) tostatin analogue conjugate), LB10 (CAIX ligand conjugate), LB11 (CAIX ligand conjugate), LB12 (gastrin-releasing peptide receptor (GRPr), MBA conjugate LB13 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH co- LB14 (luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand) conjugate), LB15 (GnRH antagonist, abarelix conjugate), LB16 ( LB17 (cobalamin, VB12 analog conjugate), LB17 (cobalamin, VB12 analog conjugate), LB18 (α ｖ β ３ Integrin receptor, cyclic RGD pentapeptide conjugate), LB19 (hetero-bivalent peptide-ligand conjugate for VEGF receptors), LB20 (neurome) Bombesin B conjugate), LB21 (bombesin conjugate for G protein-coupled receptors), LB 22 (TLR for Toll-like receptor) ２ conjugate), LB23 (for androgen receptor Meno), LB24 (α ｖ Cilengitide / cyclo(-RGD) for integrin receptors fV)-) conjugate), LB23 (fludrocortisone conjugate), LB25 (rifabutin LB26 (rifabutin analog conjugate), LB27 (rifabutin analog conjugate) LB28 (fludrocortisone conjugate), LB29 (dexamethasone conjugate) ), LB30 (fluticasone propionate conjugate), LB31 (beclomethasone dipropionate) LB32 (triamcinolone acetonide conjugate), LB333 (prednisolone conjugate) LB34 (prednisolone conjugate), LB35 (methylprednisolone conjugate) LB36 (betamethasone conjugate), LB37 (irinotecan analogue conjugate), LB 38 (crizotinib analogue conjugate), LB39 (bortezomib analogue conjugate), LB40 (carfilzomib analog conjugate), LB41 (carfilzomib analog conjugate), LB LB42 (luprolide analogue conjugate), LB43 (triptorelin analogue conjugate), LB44 ( Clindamycin conjugate), LB45 (liraglutide analogue conjugate), LB46 (semag lucid analogue conjugate), LB47 (retapamulin analogue conjugate), LB48 (indigo LB49 (vinblastine analogue conjugate), LB50 (lixisen LB51 (osimertinib analogue conjugate), LB52 (nucleotidinib analogue conjugate) LB53 (erlotinib analog conjugate), and LB54 (rapamycin analog conjugate).

10. The conjugate compound of claim 1, selected from the group consisting of: 【Chemistry 48】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 During the ceremony, 【Chemistry 49】 is optionally either a single or double bond or absent; X １ and Y １ are independently O, NH, NHNH, NR ５ , S, C(O)O, C(O)N H, OC(O)NH, OC(O)O, NHC(O)NH, NHC(O)S, OC(O)N (R １ ), N(R １ )C(O)N(R １ ), CH, C(O)NHNHC(O), and C( O)NR １ and mAb is an antibody; n and m １ are independently 1 to 20; X ３ はCH ２ 、O、NH、NHC(O)、NHC(O)NH、C(O)、OC(O)、 OC(O)(NR ３ ), R １ , N.H.R. １ , N.R. １ , or C(O)R １ or Does not exist; X ４ はH、CH ２ 、OH、O、C(O),C(O)(NH、C(O)N(R). １ ),R １ 、 NHR １ , N.R. １ , C(O)R １ or C(O)O; X ５ is H, CH ３ , F, or Cl; M １ and M ２ are each independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and R ６ is 5'-deoxyadenosyl, Me, OH, or CN; L １ , L ２ , R １ , R １ ', R ２ , Z １ , and Z ２ has the same definition as in claim 1.

31. The cytotoxic molecule may be DNA, RNA, mRNA, small interfering RNA (siRNA), or , microRNA (miRNA), or PIWI-interacting RNA (piRNA), 2. The conjugated compound of claim 1, wherein the conjugated compound is selected from the following structures of SI-1: Compound: 【Chemistry 50】 During the ceremony, mAb、m １ 、n、X １ 、L １ 、L ２ 、Z １ 、Z ２ 、 【Chemistry 51】 has the same definition as in claim 1; 【Chemistry 52】 is a single strand of DNA, RNA, mRNA, siRNA, miRNA, or piRNA, or is double-stranded; Y is O, S, NH, or CH ２ is.

32. Claims 1, 3, 5, 7, 10, 11, 12, 13, 14, 15, 16, 17, 19, 2 Any of 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 10. The conjugate of claim 1, wherein the cell-binding molecule / agent is an IgG antibody, a monoclonal antibody, an antibody, or an IgG antibody-like protein, and the conjugate is between the light and heavy chains, the upper disulfide bond between the two heavy chains, and the lower disulfide bond between the two heavy chains. It is specifically conjugated with a pair of thiols generated by reduction of the disulfide bond at the side of the Conjugates having the structures ST1, ST2, ST3, ST4, ST5, or ST6 below. body: 【Chemistry 53】 【change】 During the ceremony, Z １ 、Z ２ 、X、Y、L １ 、L ２ 、 【Chemical 54】 , m １ and the cytotoxic molecule have the same definitions as in claim 1.

33. The cytotoxic molecules containing the same or different bis-linkers are sequentially conjugated to cell-binding molecules. or the cytotoxic molecules comprising the same or different bis-linkers comprise cell-binding molecules. When added stepwise to the conjugation reaction mixture, the conjugation molecules bind to different binding sites on the cell-binding molecule. The cytotoxic molecule and m １ 33. The conjugate of claim 32, wherein:

34. The cytotoxic agent is selected from the group consisting of tubulysins, maytansinoids, taxanoids (taxanoids), and the like. compounds), CC-1065 analogs, daunorubicin and doxorubicin compounds, indoles Carboxamides, benzodiazepine dimers, pyrrolobenzodiazepine (PBD) dimers tomaymycin dimers, anthramycin dimers, indolinobenzodiazepines Dimers, imidazobenzothiadiazepine dimers, oxazolidinobenzodiazepine dimers dimers, calicheamicins and enediyne antibiotics, actinomycin, amanitin amatoxins, azaserines, bleomycins, epirubicin, eribulin, Moxifen, idarubicin, dolastatins, auristatins (monomethyl auristatins) Auristatin E, MMAE, MMAF, Auristatin PYE, Auristatin TP, Auristatin Tatins 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP) and their analogs including analogs), duocarmycins, geldanamycins (geldanamycin ns) or other HSP90 inhibitors, centanamycin , methotrexates, thiotepa, vindesines, vincristines, hemiasterins , nazumamides, microginins ), radiosumins, streptonigtin nigtin, SN38 or other analogs or camptothecin n), metabolites of alterobactins, microscleroderma, Lumicins (microsclerodermins), theonelamides (theone llamides), esperamicins, PNU-159 682, and their analogs and derivatives, as well as pharmaceutically acceptable salts, acids, etc. of the above drugs. , derivatives, hydrates or hydrated salts, crystal structures, optical isomers, racemates, diastereomers or enantiomers; or the cytotoxic molecule / compound of claim 10, 34. The conjugate of claim 32 or 33.

35. ： 3. A compound of formula (II) according to claim 2, having the following structure: 【Chemistry 55】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】

36. 103, 105, 113, 117, 120, 127, 129, and 131 of the following structures: 、１３３、１３５、１４０、１４２、１５０、１５２、１６９、１７７、１８６、１９０ , 197, 217a, 217b, 217c, 217d, 217e, 217f, 223a, 223b, 223c, 223d, 223e, 223f, 245a, 245b, 245c, 245d, 245e, 245f, 255, 303a, 303b, 303c, 303d, 3 03e, 303f, 312a, 312b, 312c, 316a, 316b, 316c, 3 16d, 316e, 316f, 320a, 320b, 320c, 325a, 325b, 3 25c, 340a, 340b, 340c, 342a, 342b, 342c, 356, 38 4, 386, 393, 395a, 395b, 397, 399a, 399b, 399c, 4 ０１、４０４、４０７、４１１、４１３、４１６、４１９、４２１、４２４、４４１、４ 49, 452, 457, 461, 465, A-3a, A-4a, A-5a, B-3a, B -6a, B-9a, B-12a, B-15a, B-18a, B-19a, B-20a, B -21a, B-22a, B-23a, B-24a, B-25a, B-26a, B-28a , C-3a, C-4a, D-1a and D-2a, ) A conjugate according to 【Chemical 56】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 where m is 0-20 unless otherwise indicated in the formula; mAb, m １ , and n is a claim This is the same definition as 1.

37. Therapeutically effective amounts of claims 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, and 19 、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、３１、３２、 37. The conjugate compound of any one of claims 33, 34, or 36, and a pharmaceutically acceptable salt thereof; a carrier, diluent, or excipient, or a combination thereof, or a pharmaceutical composition for the treatment or prevention of an infectious disease.

38. Claims 1, 5, 10, 11, 12, 13, and 14, each having a concentration of 0.1 g / L to 300 g / L; １５、１６、１７、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２ Conjugates of any one or more of 9, 30, 31, 32, 33, 34, or 36; a buffer at a concentration of 10 mM to 500 nM and a pH of 4.5 to 7.5; 0% to 15% of one or more polyols (fructose, mannose, maltose, lactose, sugar, arabinose, xylose, ribose, rhamnose, galactose, glucose, Sugar, trehalose, sorbose, melezitose, raffinose, mannitol, xylitol Toluitol, erythritol, maltitol, lactitol, erythritol, threitol , sorbitol, glycerol, or L-gluconic acid and its metal salts; 0 to 1.0% surfactant [polysorbate (polysorbate 20, polysorbate 4 0, Polysorbate 65, Polysorbate 80, Polysorbate 81, or Polysorbate poloxamer (including poloxamer 188, poly(ethylene oxide)-poly (propylene oxide), or poloxamer 407, or polyethylene-polypropylene glycol, etc.); Triton; Sodium dodecyl sulfate (SDS); Sodium lauryl sulfate thorium; sodium octylglycoside; lauryl, myristyl, linoleyl, or is stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl Linoleyl-, myristyl-, or cetyl-betaine; Lauroamidopropyl Propyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, pa Lumidopropyl- or isosteamidopropyl-betaine (lauromidopropyl); Myristamidopropyl-, palmidopropyl-, or isosteamidopropyl-dimethyl Amines; sodium methyl cocoyl taurate or disodium methyl oleyl taurate Dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine , and cocoamphoglycinic acid; or "MONAQUAT" ＴＭ Series (Isosteari ethylimidonium ethosulfate); polyethyl glycol, polypropyl glycol Licor, and ethylene and propylene glycol copolymer (Pluronics, PF68 ) selected from the group consisting of 0-5 mg / ml of antioxidants (selected from ascorbic acid and / or methionine) )； 0-2 mM chelating agent (chosen from EDTA or EGTA); 0-5% preservatives (benzyl alcohol, octadecyl dimethyl benzyl ammonium chloride) Um, hexatonium chloride, benzalkonium chloride, benzethonium chloride, phenol, Butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, Tetradecyl, resorcinol, cyclohexanol, 3-pentanol, or m-cresol selected from: 0-15% free amino acids; and / or An isotonicity agent (mannitol) to control the osmolality of the final composition to about 250-350 mOsm. ethanol, sorbitol, sodium acetate, potassium chloride, sodium phosphate, potassium phosphate trisodium citrate, or NaCl); 38. The pharmaceutical composition of claim 37, comprising:

39. In vials, bottles, prefilled syringes, or prefilled auto-injector syringes, 39. The pharmaceutical composition of claim 37 or 38, which is maintained in liquid or lyophilized solid form.

40. In vitro, in vivo, or ex vivo x vivo) have a cell-killing activity according to claims 1, 5, 10, 11, 12, 13, and 14. 、１５、１６、１７、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、 29, 30, 31, 32, 33, 34, or 36, or claim 37 or 38. A form of pharmaceutical composition according to claim 38.

41. chemotherapeutic agents to synergistically treat or prevent cancer, autoimmune diseases, or infectious diseases Concomitant administration with other conjugates, such as radiotherapy, immunotherapy, autoimmune disease drugs, anti-infective drugs, or other conjugates 39. The pharmaceutical composition of claim 37 or 38,

42. The synergist of claim 41 is selected from one or more of the following drugs: abatacept acetaminophen / hydrochloride (Orencia), abiraterone acetate (Zytiga®), Locodon, aducanumab, adalimumab, ADXS31-142 (Advaxis'), ADX S-HER2 (Advaxis'), afatinib dimaleate (Gilotrif®), Alemtuzumab (Campath®), alitretionin (Panretin®) , ado-trastuzumab em (Kadcyla®), amphetamine mixed salts (Amphetamine amine / dextroamphetamine, or adderall XR), anastrozole (Arimidex ( Registered trademark), aripiprazole, atazanavir, atezolizumab (MPDL3280A), atotsumab (ATV), Rubastatin, axitinib (Inlyta®), avelumab, belinstat ( Beleodaq™), bevacizumab (Avastin®), cabazitaxel (Jevtan a®), cabozatinib (Cometriq™), bexarotene (Targretin®), (Blincyto™), blinatumomab (Blincyto™), bortezomib (Velcade® ), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), (trademark), budesonide, budesonide / formoterol, buprenorphine, capecitabine , carfilzomib (Kyprolis®), celecoxib, ceritinib (LDK378 / Zyk adia), cetuximab (Erbitux®), cyclosporine, cinacalcet, Rizotinib (Xalkori®), Cosentyx, CTL0 19. Dabigatran, dabrafenib (Tafinlar®), daratumab (Darzalex®) ), darbepoetin alfa, darunavir, imatinib mesylate (Gleevec®), da Satinib (Sprycel®), denileukin diftitox (Ontak®) ), denosumab (Xgeva®), Depakote, dexlansoprazole le, dexmethylphenidate, dexamethasone, Dignitana Dignica p Cooling System, dinutuximab (Unituxin™), doxycycline Clin, duloxetine, duvelisib, elotuzumab, emtricitabine / rilpibili emtricitabine / tenofovir / efavirenz , enoxaparin, enzalutamide (Xtandi®), epoetin alfa, erlotinib Nib (Tarceva®), esomeprazole, eszopiclone, etanercept, Everolimus (Afinitor®), exemestane (Aromasin®), Verolimus (Afinitor®), ezetimibe, ezetimibe / simvastatin, febuxostat Nofibrate, filgrastim, fingolimod, fluticasone propionate, flu Ticazone / salmeterol, fulvestrant (Faslodex®), Gadiva (Ga Zyva), gefitinib (Iressa®), glatiramer, goserelin acetate (Zo ladex), icotinib, imatinib (Gleevec), ibritumomab tiuxetan (Zevali n®), ibrutinib (Imbruvica™), idelalisib (Zydelig®), (trademark), infliximab, iniparib, insulin aspart, insulin detemir insulin glargine, insulin lispro, interferon beta-1a, interferon eron beta 1b, lapatinib (Tykerb®), ipilimumab (Yervoy®) )), ipratropium bromide / salbutamol, Ixazomi (Ninlaro®), Kanuma, lanreotide acetate (Somatuline® Depot), Lenalid lenaliomide (Revlimid®), lenvatinib mesylate (Lenvima®) (trademark), letrozole (Femara®), levothyroxine, lidocaine Cain, linezolid, liraglutide, lisdexamfetamine, LN-144 (Lion Bio tech), MEDI4736 (AstraZeneca, Celgene), memantine, methylphenidate , metoprolol, mekinist, modafinil, mometasone, nilotinib (Tasigna (Registered Trademark), niraparib, nivolumab (Opdivo®), ofatumumab (Arzer®), a®), obinutuzumab (Gazyva™), olaparib (Lynparza™) ), olmesartan, olmesartan / hydrochlorothiazide, omalizumab, omega-3 fat acid ethyl ester, oseltamivir, oxycodone, palbociclib (Ibrance® (Trademark), palivizumab, panitumumab (Vectibix®), panobinostat (Fa rydak®), pazopanib (Votrient®), pembrolizumab (Keytr uda®), pemetrexed (Alimta), pertuzumab (Perjeta™), pulmonary Streptococcus pneumoniae conjugate vaccine, pomalidomide (Pomalyst®), pregabalin, Pros caVax, propranolol, quetiapine, rabeprazole, radium-223 chloride (Xofigo®), raloxifene, raltegravir, ramucirumab (Cyramza®), Registered Trademark), ranibizumab, regorafenib (Stivarga®), rituximab (Rituxan®), rivaroxaban, romidepsin (Istodax®), Savastatin, ruxolitinib phosphate (Jakafi™), salbutamol, sevelamer , sildenafil, siltuximab (Sylvant™), sitagliptin, sitagliptin metformin, solifenacin, solanezumab, sorafenib (Nexavar (registered trademark) (trademark), sunitinib (Sutent®), tadalafil, tamoxifen, tafin Tafinlar, talazoparib, telaprevir, temsirolimus (Torisel) Registered Trademark), tenofovir / emtricitabine, testosterone gel, thalidomide (Im munoprin, Talidex), tiotropium bromide, toremifene (Fareston®), Trametinib (Mekinist®), trastuzumab, tretinoin (Vesanoid ( Registered trademark), ustekinumab, valsartan, veliparib, vandetanib (Caprelsa Registered Trademark), vemurafenib (Zelboraf®), venetoclax, vorinos tat (Zolinza®), divaflibercept (Zaltrap®), and Zolinza® Starbucks, as well as their analogs, derivatives, and pharmaceutically acceptable salts, A carrier, diluent or excipient, or a combination thereof.

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