Novel linkers and their use in specific conjugation of biomolecules and drugs

Abstract

A linker for linking two drugs and a cell-binding agent is provided. Cell-binding agent-drug conjugate compounds are provided, such as the following structure 127: where mAb is an antibody. TIFF2026041725000161.tif50151

Description

[Technical Field]

[0001] The present invention provides a method for linking two drugs per conjugate by cross-linking a pair of thiols on a cell-binding molecule. Novel conjugates for specific conjugation of compounds, particularly cytotoxic agents, with cell-binding molecules. The present invention also relates to the preparation of conjugates by first modifying drug molecules with these conjugates, and Reacting with cell-binding molecules or first modifying cell-binding molecules with these conjugates, and reacting the cell-binding molecule-drug (cell) with the drug molecule in a specific manner. The present invention also relates to a method for preparing the toxic agent conjugate. [Background technology]

[0002] Chemotherapy drugs usually cannot distinguish between normal and malignant cells, so therapeutic concentrations Therefore, it is likely to cause side effects that limit the tolerated dose below the clinically effective dose. In contrast, immunotherapy, usually in the form of monoclonal antibodies, is a major challenge. They can specifically bind to certain proteins or molecules in cells, leaving normal cells unharmed. Therefore, compared to chemotherapy drugs, they have fewer side effects and a larger therapeutic window. Monoclonal antibodies (mAbs) have the following advantages: 1) Further identification of cancer cells in the immune system (Villaruz, LC et al, 2014, Transl Lung Cancer Res, 3, 2-14; Camacho, LH 2015 Cancer Med 4, 661-72);2) Blockade of growth signals (Dillman, RO 2011, Cancer Biother Ra diopharm, 26, 1-64; Ferris, RL et al 2010, J Clin Oncol, 28, 4390-9);3) Blood Arrest of angiogenesis (Arrillaga-Romany, I., et al, 2014, Expert Opin Investig Drugs, 23 , 199-210); 4) Radiation exposure to cancer cells (Chapuy, B. et al, 2007, Biotechnol J. 2, 1435-43);5) Delivery of chemotherapeutic agents to cancer cells (Chari RJ 2008 Acc Chem Res . 41, 98-107; Mullard A. 2013, Nature Reviews Drug Discovery 12, 329-332; Zhao, RJ 2012, J. Med. Chem., 55, 766-782;6) Enzyme delivery to cancer cells (Francis R. J. et al, 2002, Br. J. Cancer 87, 600-7) One of these applications is antibody-drug conjugates (ADCs) and There are also chemotherapy drugs called chemotherapeutic drugs that are delivered to cancer cells, which combine the cytotoxic effects of anticancer drugs. The antibody has excellent targeting ability, leaving normal cells largely unaffected and targeting cancer cells. This has been a hugely popular approach over the past 20 years, enabling drug delivery and targeting. In particular, the US FDA's 2011 "Adcetris" tuximab vedotin) and 2013's Kadcyla (ad-transtuzumab Since the approval of chemotherapeutic drug (chemotherapy) emtansine, antibody-drug conjugates (ADCs) have emerged as promising targeted cancer treatments. Applications of this approach have proliferated, with most major pharmaceutical and biotechnology companies using it. (Chari, R. et al, Angew. Chem., Int. Ed. 2014, 53, 3796-3827; Sie vers, EL et al. Annu Rev Med. 2013, 64, 15-29; Mehrling, T. Future Oncol, 201 5, 11, 549). According to www.clinictrails.gov, there are currently 50 ADC drugs in clinical trials. That's all.

[0003] First-generation ADCs, including Kadcyla and Adcetris, are compounds that bind to natural receptors on antibodies. Nonselective conjugation of cytotoxic agents to the amino groups of natural lysines or to the thiol groups of the internal chains of cysteines. IgG1 antibodies contain 50 surface-exposed lysine residues and 8 lysine residues. Because of the range of cysteine ​​residues, this non-selective conjugation results in substantially All regions can be optionally cross-linked with cytotoxic agents to increase the drug content per antibody. A diverse population of ADCs with a wide distribution of activity (DAR) is formed (Wang, L., et al. 2005 Protein Sci. 14, 2436; Hamblett, KJ, et al. 2004 Clin. Cancer Res. 10, 7063) Therefore, some undesirable ADC subpopulations may have short circulating half-lives, low efficacy, This may lead to increased potential off-target toxicity and broader in vivo pharmacokinetics (Ha mblett, KJ et al, Clin. Cancer Res. 2004, 10, 7063-7070; Adem, YT et al, B ioconjugate Chem. 2014, 25, 656-664; Boylan, NJ Bioconjugate Chem., 2013, 24 , 1008-1016; Strop, P., et al 2013 Chem. Biol. 20, 161-167). Classical conjugation is difficult to achieve batch-to-batch consistency in the production of ADCs and requires diligent Manufacturing capacity may be required (Wakankar, mAb, 2011, 3, 161-172).

[0004] Therefore, biotechnology companies and academic institutions are seeking new methods for site-specific ADC conjugation. There is a strong focus on establishing reliable methods. There are several approaches for the preparation of site-selective ADCs (Panowski, S, 2 014, mab 6, 34). They include unpaired cysteines, e.g., THI from Genentech. Introduction of a designed reactive cysteine ​​residue called OMAB (Junutula, JR, et al. 2 010 Clin. Cancer Res. 16, 4769; Junutula, JR, et al 2008 Nat Biotechnol. 26, 925-32; U.S. Patents 8,309,300; 7,855,275; 7,521,541; 7,723,485, WO2008 / 141044), Streptoverticillium mobaraense transglutaminase (mTG) (Strop, P., Bioconjugate Chem., 2014, 25, 855-862; Strop, P., et al., 2013, Chem. Biol. 20, 161-167; US Patent 8871908 Rinat-Pfizer) or microbial transglutaminase (MTG ase) (Dennler, P., et al, 2014, Bioconjug. Chem. 25, 569-578; U.S. Application 201301 89287, Innate Pharma; U.S. Patent 7,893,019, Bio-Ker Srl (IT) Inserted glutamine tag, introduction of thiol fucose (Dennler, P., et al., 2014 Bioconjugate Ugate Chemistry 25, 569; Okeley, NM, et al 2013 Bioconjugate Chem. 24, 1650) , introduction of unnatural amino acids by mutagenesis (Axup, JY, et al., 2012, Proc. Natl. Acad. Sci. USA, 2012). Sci. 109, 16101-16106; Zimmerman, ES, et al., 2014, Bioconjug. Chem. 25, 351 -361; Wu, P., et al, 2009 Proc. Natl. Acad. Sci. 106, 3000-3005; Rabuka, D., et al, 2012 Nat. Protoc. 7, 1052-67; U.S. Patent 8,778,631 and U.S. Patent Application 20100184135, WO2010 / 081110, Sutro Biopharm; WO2006 / 069246, 2007 / 059312, U.S. Patent No. 7,332,571, 7 ,696,312, 7,638,299 Ambrx; WO2007 / 130453, U.S. Patents 7,632,492 and 7,829,659, Allozy ne), introduction of selenocysteine ​​into antibodies (Hofer, T., et al 2009, Biochemistry 48, 12 047-12057; U.S. Patent 8,916,159, US National Cancer Institute), Formylglycine Formylation of cysteines located in the CXPXR consensus sequence by the formation enzyme (FGE) Conversion to glycine (FGly) (Drake, PM, et al., 2014, Bioconjug. Chem. 25, 13 31-1341. Carrico; Isaac S. et al. U.S. Patents 7,985,783; 8,097,701; 8,349,910, U.S. Patents Patent applications 20140141025, 20100210543, Redwood Bioscience), and galactosyl and silyl Glucoengineering the introduction of sialic acid using aryltransferase (Zhou, Q., et al 2014, Bioconjug. Chem., 25, 510-520, U.S. Patent Application 20140294867, Sanofi-G These above mentioned methods produce a nearly homogenous product profile. However, they require reoptimization of the antibody engineering process and cell culture conditions. Additionally, the genetic coding of unnatural amino acids has a significant impact on the cost of ADC products. The expression yields for these genes have usually not been high enough to be promising (Tian, ​​F., et al., 2014). 014, Proc. Natl. Acad. Sci. USA 111, 1766-71). In addition, The ADCs obtained using this method have been shown to have limited stability in the circulation, leads to the premature cleavage of the cytotoxic payload before it reaches the tumor site (Junutula, J.R. ., et al 2008, Nat. Biotechnol. 26, 925-32).

[0005] The disulfide bond structures of the four subclasses of IgG antibodies were known in the 1960s. (Milstein C. Biochem J., 1966, 101: 338 - 351; Pink JR, Milstein C. Nature 1 967, 214:92-94; Frangione B, Milstein C. Nature 1967, 216:939 - 941; Pink JR, Mi lstein C. Nature 1967, 216:941 -942; Frangione B, et al. Biochem J. 1968, 106,1 5 - 21; Frangione B, Milstein C. J Mol Biol 1968; 33:893 - 906; Edelman GM, et a l. Proc Natl Acad Sci USA 1969; 63:78 -85; Frangione B, et al. Nature 196, 221:1 45-148, Spiegelberg, HL et al. Biochemistry, 1975, 10, 2157-63). Disulfide The bond structure is important for the structure, stability, and biological function of the IgG molecule. Among the four subclasses of antibodies, IgG1, IgG2, IgG3, and IgG4, IgG contains a total of 12 intrachain disulfide bonds; each disulfide bond is linked to an individual I The two heavy chains are of variable number, IgG1 and IgG Two disulfide bonds connect the hinge region in IgG4, four in IgG2, and 11 in IgG3 The light chain of IgG1 has a cysteine ​​residue at the end of the light chain and at the fifth cysteine ​​of the heavy chain. The heavy chain is connected to the IgG1 heavy chain by a disulfide bond between the IgG1 heavy chain and the IgG2 heavy chain. For IgG3 and IgG4, the last cysteine ​​residue of the light chain and the third cysteine ​​of the heavy chain It is linked to the heavy chain by a disulfide bond between the ribosomal amino acid residues (Liu, H. and May, K. ., 2012, mAbs 4, 17-23). ​​Experimental reduction, different alkylation reactions, and LC-MS analysis revealed The sensitivity ranking of disulfide bonds in human IgG1 antibodies (Liu, H, et al. An al. Chem., 2010, 82, 5219-5226), interchain disulfide bonds are more strongly bonded than intrachain disulfide bonds. The disulfide bond between the light and heavy chains is more susceptible to reduction than the two heavy chains. The disulfide bond between the two heavy chains was more sensitive than the disulfide bond between the two heavy chains. The upstream disulfide bond is more sensitive than the downstream one. Furthermore, the disulfide bond in the CH2 domain is the most sensitive to reduction. The disulfide bonds in the VL, CL, VH, and CH1 domains were The disulfide bond in the CH3 domain has similar and moderate sensitivity, while the At least it is susceptible to reduction (Liu, H, et al Anal. Chem., 2010, 82, 5219-5226 ).

[0006] Based on the greater sensitivity of interchain disulfide bonds in human IgG1 antibodies, multiple Institutions and companies have developed bromo- or dibromomaleimides, known as next-generation maleimides (NGMs). mide (Schumacher, FF, et al 2014, Org. Biomol. Chem. 12, 7261-7269; UCL Cancer Institute; applying bis-alkyl reagents via a three carbon bridge (Bade scu, G., et al., 2014, Bioconjug. Chem. 25, 1124-1136., WO2013 / 190272, WO2014 / 06 4424 PolyTherics Ltd); Disubstituted Heteroaromatic Ring Bridges (U.S. Patent Application No. 2015 / 0105539, Conco rtis system); or by dimaleimide as a crosslinker (WO2014 / 114207) Chemically specific conjugation by re-crosslinking reduced interchain disulfide bonds of antibodies We also adopted a strategy to conjugate both the drug and the antibody for a long period of time. For this purpose, bromomaleimide and dibromomaleimide linkers were used (WO2014 / 009774, PCT / However, these above-mentioned bridges are composed of a pair of disulfide bonds. It is designed to conjugate only one cytotoxic agent, making it more accessible for conjugation. Since the number of reduced disulfide bonds is limited (approximately 2 pairs), Most of the time, they only produce ADCs with a DAR (drug per antibody) of less than 2. are.

[0007] One of the major problems with ADCs is the number or amount of cytotoxic compounds that ultimately reach the tumor. A favorable DAR of 3 or more is of great importance for improving the ADC therapeutic index. This is an important factor (Epenetos, AA et al, Cancer Res., 1986, 46, 3183-3191; Chari, R. V. Acc. Chem. Res., 2008, 41, 98-107, Zhao, RY 2011 J. Med. Chem. 54, 3606-3 623). [Prior art documents] [Patent documents]

[0008] [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. 8,871,908 [Patent Document 7] US Patent Publication No. 20130189287 [Patent Document 8] U.S. Patent No. 7,893,019 [Patent Document 9] U.S. Patent No. 8,778,631 [Patent Document 10] US Patent Publication No. 20100184135 [Patent Document 11] International Publication No. WO2010 / 081110 [Patent Document 12] International Publication No. WO2006 / 069246 [Patent Document 13] International Publication No. 2007 / 059312 [Patent Document 14] U.S. Patent No. 7,332,571 [Patent Document 15] U.S. Patent No. 7,696,312 [Patent Document 16] U.S. Patent No. 7,638,299 [Patent Document 17] International Publication No. WO2007 / 130453 [Patent Document 18] U.S. Patent No. 7,632,492 [Patent Document 19] U.S. Patent No. 7,829,659 [Patent Document 20] U.S. Patent No. 8,916,159 [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] US Patent Publication No. 20140141025 [Patent Document 25] US Patent Publication No. 20100210543 [Patent Document 26] US Patent Publication No. 20140294867 [Patent Document 27] International Publication No. WO2013 / 190272 [Patent Document 28] International Publication No. WO2014 / 064424 [Patent Document 29] US Patent Publication No. 2015 / 0105539 [Patent Document 30] International Publication No. WO2014 / 114207 [Patent Document 31] International Publication No. WO2014 / 009774 [Non-patent literature]

[0009] [Non-Patent Document 1] 2014, Transl Lung Cancer Res, 3, 2-14 [Non-patent document 2] 2015, Cancer Med 4, 661-72 [Non-patent document 3] 2011, Cancer Biother Radiopharm, 26, 1-64 [Non-patent document 4] 2010, J Clin Oncol, 28, 4390-9 [Non-Patent Document 5] 2014, Expert Opin Investig Drugs, 23, 199-210 [Non-patent document 6] 2007, Biotechnol J. 2, 1435-43

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[0010] Therefore, we used 2 or more per linker to achieve a higher DAR (≥4). Not only can drugs be conjugated, but also overloaded TCEP or DTT reduction The antibody surface is selectively reduced to form interchain disulfide bonds, which are generated by the activating agent. The present invention discloses novel disulfur bridge linkers that can be crosslinked again. Excess reduced thiol groups not utilized by the bridge linker are reconstituted as disulfides. To form the bond, an oxide, e.g., dehydroascorbic acid, is added to the end of the conjugate. It can be recombined (regenerated) with acid (DHAA) or Cu(II). Compared to cyclohexyl-linked ADCs, this reduced disulfide bond re-bridging results in a more stable Furthermore, ADCs with a stable or longer half-life can be obtained. The open-chain succinimide ring linker is significantly more potent than the mono-thiol-maleimide conjugated ADC. This has resulted in improved in vitro stability, improved PK exposure, and improved efficacy (Tumey, L. N, et al, 2014, Bioconjug. Chem. 25, 1871-80; Lyon, R. P, et al. 2014, Nat. Biotechnol 32, 1059-62), the latter of which is capable of delivering payloads via a retro-Michael-type reaction of maleimide conjugates. (Shen, B. Q, et al, 2012, Nat Biotechnol. 30, 184-9; Tumey , L. N, et al., 2014 Bioconjug Chem. 25, 1871-80), 2,3-disubstituted succinic acid groups, or is a 2-monosubstituted or 2,3-disubstituted fumaric or maleic acid group (trans (E The bridge linkers of the present invention containing cis(Z)- or cis(Z)-butenedicarboxylic acid groups have been Compared to non-hydrolyzed bromo- or dibromo-maleimide conjugates tested in the laboratory, There is little loss.

[0011] Thus, the methods of the present invention are useful for immunoconjugates carrying drugs, particularly combinations of different drugs. and can be used for simultaneous and specific delivery to specific target sites, The therapeutic drug molecules in pharmaceuticals are highly homogeneous and consistent from lot to lot. The main advantages of the system are: simultaneous targeted delivery of multiple drugs that act synergistically on targeted malignant cells; Different stages of the cell cycle can be targeted to increase the number of target cells exposed to a particular drug or effect. combining drugs that act at the same time; minimizing exposure to non-target cells, tissues, or organs; This involves precisely controlling the payload-drug ratio of the compound, leading to a homogeneous final product. Furthermore, the bridge linkers of the present invention enable the homogeneous production of specific ADCs in a simple manner. do. [Means for solving the problem]

[0012] The present invention relates to a 2,3-disubstituted succinic acid group or a 2-monosubstituted or 2,3-disubstituted succinic acid group. maleic acid group or maleic acid group (trans (E)- or cis (Z)-butenedicarboxylic acid group) ) to provide a linker for linking two drugs to a cell binding agent (e.g., an antibody). A preferred formula for the cell-binding molecule-linker-drug conjugate can be represented as follows: . [ka]

[0013] where Cb is a cell binding agent, LCb is a cell binding agent, L is a succinic acid group, fumaric acid group, Drug1 and Drug2 are drug molecules. where n is an integer of 1 to 30, and two S (sulfur) atoms from Cb are linked to L in a bridging manner. , covalently binds two or more drugs. Advantages of applying the linker to cell molecule-drug conjugates a) the covalent binding of a reduced pair of disulfides to a cell-binding agent, particularly an antibody; b) maintaining the stability of the conjugate by (re)crosslinking the conjugate; allowing for the conjugation of cytotoxic agents / drugs to specific sites, e.g., intrachain sites of IgG antibodies, This results in a homogeneous production of ADC.

[0014] In one embodiment of the present invention, the linker is represented by formula (I). [ka]

[0015] During the ceremony, [ka] represents any single bond, [ka] represents a single bond or a double bond.

[0016] U and U' represent the same or different leaving groups which may be substituted by a thiol. Such leaving groups include, but are not limited to, halides (e.g., fluoride, chloride, etc.). methanesulfonyl (mesyl), p-toluenesulfonyl ( Tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonyl nate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; di Nitrophenols; pentafluorophenols, tetrafluorophenols, difluorophenols Phenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol chlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl 5-phenylisoxazolium-3'-sulfonate, or for the Mitsunobu reaction It is an intermediate molecule produced by a condensation reagent.

[0017] [ka] represents a single bond, U and U' are not both H, [ka] represents a double bond, either U or U' can be H, but at the same time do not have.

[0018] Z1 and Z2 are disulfides, ethers, esters, thioethers, thioesters, Peptides, hydrazones, carbamates, carbonates, amines (secondary, tertiary or quaternary ), imine, cycloheteroalkane, heteroaromatic ring, alkoxide, or amide bond These are the same or different functional groups that can react with a cytotoxic agent to form a cytotoxic group.

[0019] R1 and R2 are the same or different and may be absent, a linear alkyl group having 1 to 6 carbon atoms, Branched or cycloalkyl, straight-chain, branched or cycloalkenyl having 3 to 6 carbon atoms or alkynyl, ester, ether or amide having 1 to 6 carbon atoms, or a group having the structural formula ( OCH2CH2) p (p is an integer of 0 to about 1000), or It is a combination of these.

[0020] Additionally, R1 and R2 each covalently bond X1 or X2 to Z1 or Z2. It is a chain of atoms selected from C, N, O, S, Si, and P, and preferably has a length of 0 to 500 The atoms used to form R1 and R2 are alkylene, alkenylene, alkynylene, alkynyl ... quinylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, Hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine amines, alkoxyamines, urethanes, amino acids, peptides, acyloxyamines, or All chemically related compounds that form hydroxamic acids, or combinations thereof They may also be joined by the method.

[0021] X1 and X2 are independently selected from NH, N(R3), O, S, or CH2; R3 is H, straight-chain alkyl having 1 to 6 carbon atoms, branched or cycloalkyl having 3 to 6 carbon atoms, straight-chain , branched or cycloalkenyl or alkynyl, esters having 1 to 6 carbon atoms, ethers aryl or amide, or structural formula (OCH2CH2) p (p: 0 to approximately 1000) integer) polyethyleneoxy units, or a combination thereof.

[0022] In another aspect, the present invention provides a cell-binding agent-drug conjugate of formula (II), which is The cell-binding molecule Cb reacts with the drugs Drug 1 and Drug 2 at the ends of the cross-linker. There are. [ka]

[0023] wherein Cb represents a cell binding agent, preferably an antibody.

[0024] The brackets indicate a linker conjugated to a pair of sulfur atoms of the cell-binding agent. The sulfur atom is preferably reacted with a reducing agent such as DTT and / or TCEP. This results in a pair of thiols being reduced from the interchain disulfide bonds of the cell-binding agent.

[0025] "Drug 1" and "Drug 2" are disulfides, thioethers, thioesters, Peptides, hydrazones, ethers, esters, carbamates, carbonates, cyclohexenes The alkane, heteroaromatic ring, alkoxide, or amide may be used to connect the aryl group to the aryl group via a bridge linker. The present invention represents the same or different cytotoxic agent linked to the cell-binding agent.

[0026] n is 1 to 30.

[0027] [ka] R1, R2, X1, and X2 are the same as those described above in formula (I).

[0028] In a further aspect, the present invention provides a modified cell-binding agent of formula (III): where the cell-binding agent Cb binds to a pair of thiols generated by reduction of the disulfide bond. Therefore, the cross-linked linker has functional groups Z1 and Z2 that can react with the drug. do. [ka]

[0029] During the ceremony, [ka] Cb, Z1, Z2, n, R1, R2, X1, and X2 are the same as those in formula (I) and formula (II). This is the definition.

[0030] In a further aspect, the present invention provides a modified drug of formula (IV), wherein the drug "D Drug 1 and Drug 2 reacted with the linker of formula (I) and were still present in the cells. A 2,3-substituted succinic group or a 2-monosubstituted succinic group capable of reacting with a pair of sulfur atoms of the linking agent. Substituted or 2,3-disubstituted fumaric or maleic groups (trans (E)- or cis (Z)-) )-butenedicarboxylic acid) groups. [ka]

[0031] During the ceremony, [ka] Drug1, Drug2, U, U', R1, R2, X1, and X2 are compounds represented by formula (I) and formula ( This is the same definition as II).

[0032] The present invention further relates to a method for producing the cell-binding molecule-drug conjugate of formula (II), Drug 1 and Drug 2 are linked to the cell-binding molecule via the bridge linker.

[0033] The present invention also relates to a method for producing a modified cell-binding molecule of formula (III), comprising: The linking molecule is reacted with said bridge linker of formula (I).

[0034] The present invention also relates to a method for preparing a modified drug of formula (IV), wherein said drug is a compound of formula (I) It reacts with the bridge linker. [Brief explanation of the drawings]

[0035] [Figure 1] The synthesis of a polyethylene glycol-containing bridge linker and its application in conjugating drugs to antibodies via amide bonds is presented. [Figure 2] The synthesis of a polyethylene glycol-containing bridge linker and its application in conjugating antibodies to drugs via amide bonds is presented. [Figure 3] The synthesis of a polyethylene glycol-containing bridge linker and its application in conjugating drugs to antibodies via oxime bonds is presented. [Figure 4] The synthesis of a polyethylene glycol-containing bridge linker and its application in conjugating two drugs to an antibody via a hydrazone bond are presented. [Figure 5] The synthesis of a polyethylene glycol-containing bridge linker and its application in conjugating two different drugs per linker to an antibody via an amide bond is presented. [Figure 6] The synthesis of a bridge linker and its application in conjugating two different drugs per linker to an antibody via a posterior amide bond is presented. [Figure 7] The synthesis of peptide- or polyethylene glycol-containing bridge linkers and their application in the conjugation of two (different) drugs to an antibody via a hydrazone bond are presented. [Figure 8] 1 shows the synthesis of conjugable analogs of MMAE, tubulysin, and PBD cytotoxic agents. [Figure 9] 1 shows the synthesis of conjugable analogs of PBD, MMAF, and tubulysin D cytotoxic agents. [Figure 10] The synthesis of a cell-binding molecule-tubulysin analogue conjugate via a bridge linker is shown. [Figure 11] 1 shows the synthesis of antibody conjugates with both a PBD dimer analog and a tubulysin B analog per linker, or with both MMAE and a tubulysin D analog per linker. [Figure 12] 1 shows the synthesis of antibody conjugates with both a PBD dimer analog and an MMAF analog per linker, or with both a PBD dimer and a tubulysin B analog per linker. [Figure 13] The synthesis of antibody conjugates with both maytansinoid analogs and tubulysin B analogs per linker is shown. [Figure 14] The synthesis of antibody conjugates with both a maytansinoid analog and a PBD dimer analog per linker, or two tubulysin B analogs per linker, is shown. [Figure 15] The synthesis of antibody conjugates containing polyethylene glycol and both two MMAF analogs per linker or two tubulysin B analogs per linker is shown. [Figure 16]This figure shows the antitumor efficacy of conjugates 127, 129, and 142 compared with T-DM1 when administered intravenously at a single dose of 3 mg / kg in a human gastric tumor N87 cell model. All four conjugates did not cause animal weight loss (top panel). Animals in the control group were sacrificed on day 37 due to tumor volumes exceeding 1500 mm3; they were too lethargic. Three compounds, 127, 129, and 142, outperformed T-DM1: all 6 / 6 animals in the compound 127 and 129 groups had no measurable tumors from days 13 to 60 (the end of the experiment). All 6 / 6 animals in the compound 142 group had no measurable tumors by day 21, and 2 / 6 animals had measurable tumors by day 48, but tumor growth was still inhibited beyond day 55. In contrast, T-DM1 at a dose of 3 mg / kg inhibited tumor growth for approximately 28 days, but failed to completely eradicate the tumors. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition "Alkyl" refers to an aliphatic group having 1 to 8 carbon atoms in the chain, which may be straight or branched. "Branched" refers to a hydrocarbon group having one or more lower alkyl groups attached to a linear alkyl group. The alkyl group has a methyl, ethyl, or propyl group attached to it. Specific examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, and t-butyl. n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclo Hexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl 2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl butyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4-dimethylpentyl, 2 -methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl, and isooctyl. The C1-C8 alkyl group may be unsubstituted and may have one or more It may be substituted with a substituent (but is not limited to the following substituents). C1-C8 alkyl, -O-(C1-C8 alkyl), aryl, -C(O)R', - OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O )N(R')2, -NHC(O)R', -SR', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN wherein R' is independently selected from C1 to C8 alkyl and aryl. .

[0037] "Halogen atom" refers to a fluorine, chlorine, bromine, or iodine atom, and includes bromine and chlorine atoms. Children are preferred.

[0038] "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:

[0039] "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]. C3-C8 carbocycles include cyclopropyl, cyclobutyl, Cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexyl Cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cyclo Heptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctyl Including, but not limited to, cutadienyl.

[0040] 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 or substituted with one or more of the following substituents, but not limited to: That is, the substituents are not limited to these, but may be -C1 to C8 alkyl groups. 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, -halo -N, -NH, -NH(R'), -N(R'), and -CN, and each R' is independently selected from C1-C8 alkyl and aryl.

[0041] "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.

[0042] "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.

[0043] 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:

[0044] "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-).

[0045] 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.

[0046] "Aryl" or Ar refers to an alkyl 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 The term refers to a group in which one or several carbons, preferably 1, 2, 3, or 4 carbon atoms on the aromatic group are It refers to those replaced by O, N, Si, Se, P, or S, preferably O, S, and N. The term aryl or Ar also refers to the group in which one or more H atoms are independently replaced by -R', -halogen. , -OR', or -SR', -NR'R'', -N=NR', -N=R', -NR'R' ', -NO2, -S(O)R', -S(O)2R', -S(O)2OR', -OS(O) 2OR', -PR'R'', -P(O)R'R'', -P(OR')(OR''), -P Replaced by (O)(OR')(OR'') or -OP(O)(OR')(OR'') The R' and R'' are independently H, alkyl, alkenyl, alkylene, alkynyl ... is aryl, heteroalkyl, aryl, arylalkyl, carbonyl, or a pharmaceutical salt thereof. .

[0047] 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 ysics, 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, tetrahydropyranyl, dihydropyranyl, tetrahydropyranyl, Dropyridyl, dihydropyridyl, tetrahydropyridinyl, dihydrothiopyranyl, Zepanyl, as well as condensed systems resulting from condensation with a phenyl group are included.

[0048] The term "heteroaryl" or aromatic heterocycle refers to a 5- 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

[0049] "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.

[0050] "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:

[0051] "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, but are not limited to, 2-benzimidazoline. These include 2-furylmethyl, 2-furylethyl, and the like.

[0052] Examples of "hydroxy protecting groups" include, but are not limited to, methoxymethyl ether, 2-Methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether p-Methoxybenzyl ether, Trimethylsilyl ether, Triethylsilyl ether ter, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenyl Nylmethylsilyl ether, acetate ester, substituted acetate ester, pivaloate, benzoate These include methylsulfonate, methanesulfonate, and p-toluenesulfonate.

[0053] 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, but are not limited to, halo genides (e.g., chlorides, bromides, and iodides), methanesulfonyl (mesyl), p -toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and and trifluoromethylsulfonates.

[0054] 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.

[0055] "Pharmaceutically" or "pharmaceutically acceptable" means that the corresponding compound or compound composition is suitable for are harmful, allergic or produce other adverse reactions when administered to animals or humans in an appropriate manner; It refers to the inability to do so.

[0056] 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.

[0057] Pharmaceutically acceptable auxiliary materials include any carrier, diluent, adjuvant or excipient, e.g. , preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, These include antibacterial agents, antifungal agents, isotonic agents, and absorption delaying agents. It is common practice to add these auxiliary materials to drug components that have the desired properties. It is reasonable to add auxiliary materials to the active pharmaceutical ingredient, unless the auxiliary materials are incompatible with the active pharmaceutical ingredient. It can be said that in order to achieve good results, active auxiliary materials are added to the drug components. Good too.

[0058] In the present invention, the term "pharmaceutical salt" refers to a salt derivative of the compound of the present invention. By suitable modification, the compounds of the present invention can be formed into the corresponding acid or alkali salts. Possible salts include the conventional non-toxic salts or quaternary ammonium salts, which are not intended to be limiting unless otherwise specified. The compound can be prepared by using a non-toxic inorganic or organic acid corresponding to the compound. Examples of the acid include hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, and the organic acid Acids include acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzoic acid, Toluene sulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluene sulfonate Acids that are used in pharmaceutically acceptable salts include carboxylic acids, oxalic acids, fumaric acids, and lactic acids. Other salts include trometamol, meglumine, and pyrroleethanol. ammonium salts such as ammonium salts, and sodium, potassium, calcium, zinc, magnesium, etc. Contains metal salts of

[0059] 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. .

[0060] "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.

[0061] The novel conjugates disclosed herein utilize bridge linkers. The bridge linkers and their synthesis methods are shown in Figures 1 through 15.

[0062] cross-linked body Similar to the preparation of drug conjugates to cell-binding molecules of the present invention, the synthetic pathways for obtaining crosslinkers are The pathways are shown in Figures 1-15. The bridge linker has two components: a) 2,3-disubstituted succinic acid. or 2-monosubstituted or 2,3-disubstituted fumaric groups; or 2-monosubstituted or 2,3-disubstituted fumaric groups ,3-disubstituted maleic acid, which reacts with a pair of thiols to form thioether bonds. and b) groups capable of reacting with a drug. However, disulfides, maleimides, haloacetyls, aldehydes, ketones, azides, and amino acids are not included. Alkoxyamines, hydrazides, ethenesulfonyl, acyl halides (acidic halogen 2,3-disubstituted comonomers include 2,3-disubstituted comonomers, ... or 2-monosubstituted or 2,3-disubstituted fumaric acid; or 2-monosubstituted or The bridging substituents of 2,3-disubstituted maleic acids are the same as those of these 2,3-disubstituted succinic acids, or mono- or 2,3-disubstituted fumaric or maleic acids with amides, esters, or direct condensation with amine, alcohol, or thiol groups to form thioester bonds The synthesis of these bridge linkers is shown in Figures 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, and 15.

[0063] Preferably, the bridge linker is a compound of formula (I): [ka]

[0064] During the ceremony, [ka] represents any single bond, [ka] represents a single bond or a double bond.

[0065] [ka] represents a single bond, U and U' are not both H, [ka] represents a double bond, either U or U' can be H, but at the same time do not have.

[0066] a 2,3-disubstituted succinic acid group, or a 2-monosubstituted or 2,3-disubstituted fumaric acid group, or The component may be a 2-monosubstituted or 2,3-disubstituted maleic acid. [ka] can react with a pair of sulfur atoms of the cell-binding agent. reducing agents, such as dithiothreitol (DTT) and dithioerythritol (DTE) , L-glutathione (GSH) and tris(2-carboxyethyl)phosphine (TCE P), or / and β-mercaptoethanol (β-ME, 2-ME), as a cell-binding agent It is a pair of thiols reduced from the interchain disulfide bonds of .

[0067] U and U′ are the same or different leaving groups optionally substituted by thiol; Such leaving groups include, but are not limited to, halides (e.g., fluoride, chloride, etc.). methanesulfonyl (mesyl), p-toluenesulfonyl ( Tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfonyl nate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; di Nitrophenols; pentafluorophenols, tetrafluorophenols, difluorophenols Phenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol chlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl 5-phenylisoxazolium-3'-sulfonate, or for the Mitsunobu reaction It is an intermediate molecule produced by a condensation reagent.

[0068] Z1 and Z2 are disulfides, thioethers, thioesters, peptides, and hydrazones. , ethers, esters, carbamates, carbonates, amines (secondary, tertiary or quaternary ), imine, cycloheteroalkane, heteroaromatic ring, alkoxide, or amide bond These are the same or different functional groups that can react with a cytotoxic agent to form a cytotoxic group.

[0069] R1 and R2 are the same or different and may be absent, a linear alkyl group having 1 to 6 carbon atoms, Branched or cycloalkyl, straight-chain, branched or cycloalkenyl having 3 to 6 carbon atoms alkynyl, ester, ether or amide having 1 to 6 carbon atoms, structural formula (OCH2 CH2) p (p: an integer of 0 to about 1000) polyethyleneoxy unit or structure Formula (OCH2(CH3)CH2) p (p is an integer of 0 to about 1000) polypropylene oxy units, or combinations thereof.

[0070] Additionally, R1 and R2 each covalently bond X1 or X2 to Z1 or Z2. It is a chain of atoms selected from C, N, O, S, Si, and P, and preferably has a length of 0 to 500 The atoms used to form R1 and R2 are alkylene, alkenylene, alkenylene, and alkylene. Alkynylene, ether, polyoxyalkylene, ester, amine, imine, polyamine , hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxya amines, alkoxyamines, urethanes, amino acids, peptides, acyloxyamines, or or any chemically related compound that forms a hydroxamic acid, or a combination thereof. The bonding may be performed in a similar manner.

[0071] X1 and X2 are independently selected from NH, N(R3), O, S, or CH2; R3 is H, straight-chain alkyl having 1 to 6 carbon atoms, branched or cycloalkyl having 3 to 6 carbon atoms, straight-chain , branched or cycloalkenyl or alkynyl, esters having 1 to 6 carbon atoms, ethers aryl or amide, or structural formula (OCH2CH2) p (p: 0 to approximately 1000) integer) polyethyleneoxy units, or a combination thereof.

[0072] In another embodiment, R1, R2, and R3 are each a cell surface binding molecule and / or a conjugate. a chain of atoms selected from C, N, O, S, Si, and P covalently bonded to the attached drug; The atoms used to form the bridge linker can be alkylene, alkenyl, olefins, alkynylenes, ethers, polyoxyalkylenes, esters, amines, imines, poly amines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, arsenides koxyamine, alkoxyamine, urethane, amino acid, acyloxyamine, or Combining in all chemically related ways, such as forming hydroxamic acids, or many others Additionally, the atoms forming the linker (L) may be saturated or unsaturated. or may be a radical, or may be a cycloalkane, cyclic Divalent ring structures including ethers, cyclic amines, arylenes, heteroarylenes, etc. It is understood that the rings may be cyclized together to form the ring.

[0073] Examples of functional groups Z1 and Z2 that allow for the attachment of a cytotoxic agent include, but are not limited to: , disulfides, thioethers, thioesters, peptides, hydrazones, esters, cations Groups that can be linked via a bamate, carbonate, alkoxide or amide bond Such functional groups include, but are not limited to, thiol, disulfide, Do, amino, carboxy, aldehyde, ketone, maleimide, haloacetyl, hydrazine , alkoxyamino, and / or hydroxy groups.

[0074] Examples of functional groups Z1 and Z2 that can react with the amine terminus of a drug / cytotoxic agent include: Examples include, but are not limited to, N-hydroxysuccinimide esters, p-nitrophenyl esters, esters, dinitrophenyl esters, pentafluorophenyl esters, carboxylic acid chlorides It can be a compound or a carboxylic acid anhydride capable of reacting with the thiol terminal. Examples include, but are not limited to, pyridyl disulfide, nitropyridyl disulfide, Maleimide, haloacetic acid, methylsulfone, phenyloxadiazole (ODA), carbo The terminal groups of ketones or aldehydes can be carboxylic acid chlorides and carboxylic acid anhydrides. Reactions that can occur include, but are not limited to, amines, alkoxyamines, hydroxyamines, and the like. It can be an azide or acyloxylamine. The functional groups may be, but are not limited to, alkynes. An example is shown below.

[0075] [ka]

[0076] In the formula, X1 is F, Cl, Br, I, or Lv; X2 is O, NH, N(R1), or is CH2. R5 and R3 are H, R1, an aromatic ring, a heteroaromatic ring, or one or more H atoms are independently -R1, -halogen, -OR1, -SR1, -NR1R2, -NO 2. Aromatic groups substituted with -S(O)R1, -S(O)2R1 or -COOR1 Lv is nitrophenol; N-hydroxysuccinimide (NHS); phenol; Dinitrophenol; Pentafluorophenol; Tetrafluorophenol; Difluoro Monofluorophenol; Pentachlorophenol; Triflate; Imine Dichlorophenol; Tetrachlorophenol; 1-Hydroxybenzotriazole 2-Ethyl-5-phenylisoxazolium-3'- Sulfonates; acid anhydrides formed with themselves or with other acid anhydrides (e.g., acetic anhydride, Formic anhydride); or intermediate molecule for peptide coupling reactions, or Mitsunobu reactions is a leaving group selected from the intermediates generated by the condensation reagents for

[0077] In a preferred embodiment, R1, R2, and R3 are each a linear alkyl group having 1 to 6 carbon atoms; is the formula (OCH2CH2) p (p=1 to 100) is a polyethyleneoxy unit.

[0078] 2,3-disubstituted succinic acid group, or 2-monosubstituted or 2,3-disubstituted fumaric acid group, or This is a key step in the synthesis of bridge linkers containing 2-monosubstituted or 2,3-disubstituted maleic acid groups. As shown in the following scheme (Ia), the topping may be 2,3-disubstituted succinic acid or 2,3-disubstituted succinic acid. -mono- or 2,3-disubstituted fumaric acids or 2-mono- or 2,3-disubstituted Maleic acid or its derivatives with amines (1° or 2° amines), alcohols, or terminal condensation with other moieties containing thiols.

[0079] [ka]

[0080] wherein X is any of the X groups described in formula (I) as NH, N(R), O, or S. R is R1 and / or R2 as described in formula (I). R3 is a group represented by the formula (I) is the same as defined in (I).

[0081] Lv1 and Lv2 may be the same or independently OH; F; Cl; Br; I; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentaf Fluorophenol; Tetrafluorophenol; Difluorophenol; Monofluorophenol phenol; pentachlorophenol; triflate; imidazole; dichlorophenol ;Tetrachlorophenol;1-hydroxybenzotriazole;Tosylate;Mesylate 2-Ethyl-5-phenylisoxazolium-3'-sulfonate; and acid anhydrides formed with (e.g., acetic anhydride, formic anhydride); or intermediate molecules Condensation reagents for peptide coupling reactions or Mitsunobu reactions The condensation reagent is selected from intermediates such as EDC (N-(3-dimethylamino)methyl) (aminopropyl)-N'-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide) Imide), N,N'-diisopropylcarbodiimide (DIC), N-cyclohexyl- N'-(2-morpholinoethyl)carbodiimide and meso-p-toluenesulfonate (C MC, or CME-CDI), 1,1'-carbonyldiimidazole (CDI), TBT U(O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurony tetrafluoroborate), N,N,N',N'-tetramethyl-O-(1H-benzo[a]thiazo[rho ... (Isotriazol-1-yl)uronium hexafluorophosphate (HBTU), (Be (1-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro Orophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidino Phosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamidinium hexafluoride Fluorophosphate, 1-[bis(dimethylamino)methylene]-1H-1,2,3-trifluoromethyl Triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HA TU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]thiazolinone Triazolo[4,5-b]pyridin-1-ium-3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate Phosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate ( PyCloP), fluoro-N,N,N',N'-bis(tetramethylene)formamidite Nitrium hexafluorophosphate (BTFFH), N,N,N',N'-tetramethyl -S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O- (2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethylthiuronium Tetrafluoroborate (TPTU), S-(1-oxido-2-pyridyl)-N,N, N',N'-Tetramethylthiuronium tetrafluoroborate, O-[(ethoxylated (N,N,N',N'-tetramethyluronium) Trifluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethyl (Imine)aminooxydimethylamino-morpholino-carbenium hexafluorophosphine ate (COMU), O-(benzotriazol-1-yl)-N,N,N',N'-biphenyl (tetramethylene)uronium hexafluorophosphate (HBPyU), N-benzo[a]pyridine Dimethyl-N'-cyclohexylcarbodiimide (with or without polymer linkage), dipyridyl Lolidino(N-succinimidyloxy)-carbenium hexafluorophosphate ( HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyCI) U), 2-chloro-1,3-dimethylimidazolinium tetrafluoroborate (CIB ), (benzotriazol-1-yloxy)dipiperidinocarbenium hexafluoro Phosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N ,N,N',N'-Tetramethyluronium tetrafluoroborate (TCTU), Bro Motris(dimethylamino)phosphonium hexafluorophosphate (BroP), Propylphosphonic anhydride (PPACA, TsP®), 2-morpholinoethyl isopropyl N,N,N',N'-tetramethyl-O-(N-succinimidyl) 2-Bromo-1-ethyl-pyridinium hexafluorophosphate (HSTU) BEP, O-[(ethoxycarbonyl)cyanomethyl] N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- Methylmorpholinium chloride (MMTM, DMTMM), N,N,N',N'-tetramethylmorpholinium chloride Methyl-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) Azodicarboxylate (DCAD), Di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azo dicarboxylate (DEAD).

[0082] Detailed examples of the synthesis of bridge linkers are shown in Figures 1 to 10. Typically, 2,3-disubstituted succinic acid groups or is a 2-monosubstituted or 2,3-disubstituted fumaric acid group, or a 2-monosubstituted or 2,3- The bridging substituent of the disubstituted maleic acid group is a functional group that can react with a desired conjugated drug. is condensed with a linker component comprising:

[0083] Cell-binding agent-drug conjugates The conjugates of the present invention can be represented by the formula: [ka]

[0084] where Cb is a cell-binding molecule, and L is a succinate, fumarate, or maleate group. "Drug1" and "Drug2" are drug molecules, and n is 1 to 30. and two S (sulfur) atoms from Cb are bridgingly linked to L, and Each L is covalently linked to two or more drugs.

[0085] The bridge linker L may be composed of one or more linker moieties. The ingredients include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), and val-citrulline (val-cit or vc), alanine-phenylalanine (ala-p he or af), p-aminobenzyloxycarbonyl (PAB), 4-thiopentanoic acid ester (SPP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester (MCC), (4-acetyl)aminobenzoic acid (SIAB), 4-thiobutyrate ( SPDB), 4-thio-2-hydroxysulfonyl butyrate (2-sulfo-SP DB), one or more repeating units being ethyleneoxy (-CH2CH2O-) units ( Additional linker moieties are known in the art and include: Some of them are described here.

[0086] Examples of the structures of these components, including the linker, are shown below. [ka] TIFF2026041725000023.tif30132

[0087] Preferably, the conjugate is a compound of formula (II): [ka]

[0088] In the formula, Cb represents a cell-binding agent, preferably an antibody, and is bound to a pair of sulfur atoms (thiols). The conjugable thiol atom is generally , dithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH) and tris(2-carboxyethyl)phosphine (TCEP), and / or Paired disulfides on cell-binding molecules by β-mercaptoethanol (β-ME, 2-ME) It can be generated from the reduction of an iodo bond.

[0089] "Drug 1" and "Drug 2" are alkyl, alkylene, alkenylene, alkylene, Nylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine , urethane, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide amides, thioethers, thioesters, carbamates, carbonates, heterocycles, heteroalkoxy groups Cross-linked by alkyl, heteroaromatic, or alkoxyl bonds, or a combination thereof represents the same or a different cytotoxic agent linked to said cell binding agent via a body.

[0090] n is 1 to 30.

[0091] [ka] R1, R2, X1, and X2 are the same as those described above in formula (I).

[0092] As detailed below, "Drug1" and "Drug2" are the most common small molecule drugs. Any of the above may be used, including tubulysins, calicheamicins, auristatins, and methanogens. Itansinoids, CC-1065 analogs, morpholinos, doxorubicins, taxanes benzodiazepine dimers (e.g., pyrrolo Benzodiazepines (PBD or tomaymycin), indolinobenzodiazepines, Contains midazobenzothiadiazepine or oxazolidinobenzodiazepine dimers Examples include, but are not limited to:

[0093] To synthesize the conjugate, the cell-binding molecule is first cleaved by cleaving the disulfide bond in the cell-binding molecule. The resulting pair of free radicals can be modified with the bridge linker of the present invention through reduction of the Thiols are also available as disulfides, maleimides, haloacetyls, azides, 1-ynes, ketones, and acetylenes. Introduce reactive groups Z1 and Z2, including aldehyde, alkoxyamino, or hydrazide. For example, DMA, DMF, ethanol, methanol, acetone, acetonitrile , THF, isopropanol, dioxane, propylene glycol, or ethylenediamine pH 5 with or without the addition of 0-30% of a water-soluble (miscible) organic solvent such as ethanol. The cells can then be reacted with the cross-linker of formula (I) in an aqueous medium at a temperature of 1000°C to 900°C. The reactive group of the toxic agent reacts with the appropriately modified cell-binding molecule, e.g., disulfide. The synthesis of cell-binding agent-drug conjugates linked via bonds involves the dimerization of the modified cell-binding agent. This is achieved by disulfide exchange between the sulfide bond and a drug containing a free thiol group. The synthesis of cell-binding agent-drug conjugates linked via thioethers has been reported using maleimides or Reaction of haloacetyl- or ethylsulfonyl-modified cell-binding agents with drugs containing free thiol groups The synthesis of conjugates bearing acid labile hydrazones can be achieved by methods known in the art. This can be achieved by reaction of the carbonyl group with a hydrazide residue in the linker by (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. Trail et al., Cancer R es., 57; 100-105, 1997). The synthesis of conjugates with triazole bonds has been achieved using click chemistry. Reaction of the 1-yne group in the drug with the azide residue in the linker via a trie (Huisgen cycloaddition) (Lutz, JF. et al, 2008, Adv. Drug Del. Rev. 60, 95 8-970; Sletten, EM et al 2011, Acc Chem. Research 44, 666-676).

[0094] Alternatively, to obtain a modified cell-binding molecule conjugate having the functionality of formula (III), can react with the bridge linkers of the invention conjugated to cell-binding molecules. To obtain cell-binding molecule-drug conjugates via ether bridges, thiol-containing drugs are Maleimide, haloacetyl, or ethylsulfonyl in aqueous buffer solution at pH 5.5-9.0 The modified cell-binding molecule of formula (III) can be reacted with a bridge linker having a substituent. To obtain conjugates with disulfide bridges, thiol-containing drugs are substituted with pyridyldithio residues. A modified bridge linker of formula (III) having a hydroxyl group can undergo disulfide exchange. To obtain modified drugs with ether or thiol ether bonds, hydroxyl or thiol groups are The agent having the formula (I) reacts with halogens, especially hydroxybenzoates, in the presence of a mild base, e.g., pH 8.0-9.5. can be reacted with a modified bridge linker of formula (III) bearing a carboxylic acid alpha halide Drugs containing hydroxyl groups can be prepared in the presence of dehydrating agents such as EDC or DCC to obtain ester crosslinks. condensation with a bridged cross-linker of formula (I) having a carboxyl group, and then The modified crosslinker can be conjugated to a cell-binding molecule via an amide bond. To obtain the conjugate, the drug containing an amino group is reacted with a cell-binding molecule-bridge linker of formula (III) Above, NHS, imidazole, carboxyl ester of nitrophenol; N-hydroxy Thiosuccinimide (NHS); Phenol; Dinitrophenol; Pentafluorophenol phenol; tetrafluorophenol; difluorophenol; monofluorophenol; phenol Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol 1-Hydroxybenzotriazole;Tosylate;Mesylate;2-Ethyl It can be condensed with phenyl-5-phenylisoxazolium-3'-sulfonate.

[0095] The conjugates can be prepared by standard biochemical methods, e.g., Sephadex G25 or Sephac Gel filtration, adsorption chromatography, ion exchange, or permeation chromatography on a ryl S300 column In some cases, small molecules (e.g., 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 chromatography It can be purified by chromatography.

[0096] Modified cell-binding agents / molecules The cell-binding agent modified by reaction with the conjugate of the present invention is preferably represented by formula (III): will be done. [ka]

[0097] formula [ka] Cb, Z1, Z2, n, R1, R2, X1, and X2 have the same definitions as in formulas (I) and (II). It is righteous.

[0098] In a preferred embodiment, Z1 and Z2 are selected from the group consisting of disulfide substituents, maleimides, haloacetates, and the like. Alkoxyamines, azides, ketones, aldehydes, hydrazines, alkynes, N-hydroxybenzoates Hydroxysuccinimide ester, or phenol; dinitrophenol; pentafluoro Fluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol Pentachlorophenol; Triflate; Imidazole; Dichlorophenol; Tetrachlorophenol Trachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; Carvone formed with 2-ethyl-5-phenylisoxazolium-3'-sulfonate Then, Z1 and Z2 are disulfides, thioethers, hydrazones, etc. , amide, alkoxide, carbamate, ester, ether bond, or aromatic ring The modified cell-binding agent can be represented by the formula (II) above, and can react with a cytotoxic agent. As described, they can be prepared via reaction of a bridge linker of formula (I) with a cell-binding agent. This can be done.

[0099] A pair of free thiols on the cell-binding molecule, preferably an antibody, and an alkyne group on the cross-linker To achieve higher conjugation yields, a small proportion of an organic co-solvent may be added to the reaction mixture. Similarly, in order to maintain the solubility of formula (III) in aqueous solution, To modify the cell binding agent, first, a compound of the formula ( The cross-linking reagent (cross-linker) of I) is dissolved in a polar organic solvent miscible with water, such as methanol, ethanol, or the like. Different alcohols such as ethanol, propanol, acetone, acetonitrile, tetrahydrofuran THF, 1,4-dioxane, dimethylformamide (DMF), dimethyl Acetamide (DMA) or dimethyl sulfoxide (DMSO) at high concentrations, e.g. It can be dissolved at 1 to 500 mM, while the pH is 5 to 9.5, preferably 6 to 8. A cell-binding molecule such as an antibody dissolved in an aqueous buffer solution of 5 at a concentration of 1 to 35 mg / ml is The product is treated with 0 equivalents of TCEP or DTT for 20 minutes to 12 hours. After reduction, SEC chromatography is performed. DTT can be removed by chromatographic purification. TCEP can also be used, if desired, in S It can be removed by EC chromatography or can be used in the next reaction without purification. Furthermore, the TCEP reduction can be simultaneously followed by the addition of cell-binding molecules. To achieve cross-linking conjugation of the antibody or other cell-binding agent, reduction of the antibody or other cell-binding agent with TCEP yields the compound of formula This can be carried out together with the cross-linker (I).

[0100] The aqueous solution for the modification of cell-binding agents has a pH between 6 and 9, preferably between 6.5 and 7.5. It can be buffered and contain non-nucleophilic buffer salts useful in these pH ranges. Reagents include phosphate, triethanolamine HCl, HEPES, and MOPS buffers Further, for example, dextrin, sucrose, salt (e.g., NaCl, KCl), etc. The crosslinker of formula (I) can be added to a solution containing a reduced cell-binding molecule. After adding the conjugate, the reaction mixture is incubated at 4°C to 45°C, preferably at ambient temperature. The progress of the reaction can be monitored by a decrease in absorbance at 254 nm or an increase in absorbance at 280 nm. The reaction can be monitored by measuring changes in wavelength, such as red, blue, green, or any other suitable wavelength. After completion of the procedure, the modified cell-binding agent can be isolated by conventional methods, such as gel filtration chromatography. This can be done by either gel or adsorption chromatography.

[0101] 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 (CEMS) The bridge linkers described herein can be used with any drug, with appropriate substituents, Preferably, it has a variety of functional groups that can react with cytotoxic agents, such as amino or hydroxy groups. Modified cell-binding molecules bearing hydroxyl groups are synthesized using N-hydroxysuccinimide (NHS) esters. The modified cell-binding molecule having a thiol substituent can react with a drug having a thiol substituent. It can react with drugs that have a acetyl group or a haloacetyl group. Modified cell-binding molecules with substituents (ketone or aldehyde) are used as hydrazides or alkyl groups. Those skilled in the art can easily determine the available functional groups on the linker and react them with the agent having the hydroxyamine. The linker to be used can be readily determined based on the known reactivity of the groups.

[0102] Modified cytotoxic agents Cytotoxic agents modified by reaction with the bridge linkers of the present invention preferably have the formula (IV): is represented. [ka]

[0103] During the ceremony, [ka] U, U', Drug 1, Drug 2, R1, R2, X1, and X2 are each a group represented by the formula (I) and (I This is the same definition as I).

[0104] The modified drug may have a 2,3-disubstituted succinic acid group or a 2-monosubstituted or 2,3-disubstituted succinic acid group. substituted fumaric acid group, or 2-monosubstituted or 2,3-disubstituted maleic acid group to obtain the modified drug of formula (IV), which is prepared by reacting the linker of formula (I) with the drug. However, in the case of drugs containing thiols, or thioethers, Drugs that bind to cell-binding molecules via cross-linking linkers via ester or disulfide bonds In the case of a drug, preferably, Drug 1 or Drug 2 is first converted to a thioether, a thioester, or to connect with a portion of the R1 or R2 component via a di- or disulfide bond linkage. The synthesized compounds may then be reacted with the compound to form the bridge-linker-modified drug of formula (IV). The R1-Drug1 or R2-Drug2 component is a 2,3-disubstituted succinic acid or a 2-monosubstituted succinic acid. 2-monosubstituted or 2,3-disubstituted fumaric acids, or 2-monosubstituted or 2,3-disubstituted Assembled into maleic acid.

[0105] In the synthesis example, R1-Drug1 or R2-Drug2 having a thioether bond To obtain the desired fraction, thiol-containing drugs are prepared by dissolving maleimide substituents in aqueous buffer at neutral pH. The linker can be reacted with components R1 and R2 having a thioether bond. to obtain a modified drug of formula (IV) having the formula: or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted maleic acid R1-Drug1 or R2-Drug2 having an ether bond can be condensed with carboxylic acid. To obtain the ug2 compartment, drugs with hydroxy groups are reacted with hydroxy groups in the presence of a mild base. reacting the linker component R1 or R2 with halogen, tosylate, or mesylate; and then reacting the compound 2, to obtain a modified drug of formula (IV) having a thioether bond. 3-disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2- It can be condensed with mono- or 2,3-disubstituted maleic acid. To obtain modified drugs of formula (IV) via In the presence of EDC or dicyclohexylcarbodiimide (DCC), The R1-Dr having a thioether bond can be condensed with a linker of formula (I) To obtain the ug1 or R2-Drug2 compartment, thiol-containing drugs are used, such as maleimides, vinyl reacting the R1 and R2 components of the linker with a phenylsulfonyl or haloacetyl group; and subsequently, to obtain a modified drug of formula (IV) having a thioether bond, -disubstituted succinic acid, or 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2-monosubstituted or 2,3-disubstituted It can be condensed with a mono- or 2,3-disubstituted maleic acid section. Amide bond To obtain a modified drug of formula (IV), a drug having an amino group can be similarly reacted with a compound of formula (I The modified drug can be condensed with the carboxyl group on the bridge linker of silica gel. Or alumina column chromatography, crystallization, preparative thin layer chromatography, It can be purified by standard methods such as ion exchange chromatography or HPLC. do.

[0106] Cell-binding agents The cell-binding molecules that make up the conjugates and modified cell-binding molecules of the present invention are useful therapeutically or Any currently known compound that binds, complexes, or reacts with other biologically modified moieties of the cell population. The molecule may be any molecule known or known to exist.

[0107] Cell binding agents include large molecular weight proteins, such as full-length antibodies (polyclonal or monoclonal). monoclonal), dimeric, multimeric, multispecific antibodies (e.g., bispecific antibodies); single-chain antibodies antibody fragments, e.g., Fab, Fab', F(ab')2, Fv [Parham, J.I. Munol. 131, 2895-2902 (1983)], and Fab expression library fragments obtained by the above procedure, anti-idiotypic (anti-Id) antibodies, CDRs, bispecific antibodies a specific antibody, a trispecific antibody, a cancer cell antigen, a viral antigen, a microbial antigen, or a specific antigen; Proteins produced by the immune system that can bind to or exert a desired biological activity epitope-binding fragments of any of the foregoing that immunospecifically bind to a protein; interferon (e.g., peptides; lymphokines, e.g., IL-2, IL-3, I Il-4, IL-5, IL-6, IL-10, GM-CSF, or interferon gamma (I FN-γ; hormones, such as insulin, TRH (thyrotropin-releasing hormone) ), MSH (cell-stimulating hormone), or androgen, estrogen or melanin cell steroid hormones such as follicle-stimulating hormone (MSH); growth factors and colony-stimulating factors, e.g. For example, epidermal growth factor (EFG), granulocyte-macrophage colony-stimulating factor (GM-CSF), Transforming growth factors (TGF), e.g., TGFα, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II), G-CSF, M-CSF, and GM-CSF [Burgess, Immunology Today, 5, 155- 158 (1984)]; Vaccine growth factor (VGF); Fibroblast growth factor (FGF); Small molecular weight proteins, polypeptides, peptides, and peptide hormones, e.g., bombardment sin, gastrin, and gastrin-releasing peptide; platelet-derived growth factor; interleukin and cytokines, e.g., interleukin-2 (IL-2), interleukin- 6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF) F); vitamins such as folic acid; apoproteins and glycoproteins, e.g., transferrin [O'Keefe et al,J.Bio.Chem.260,932-927(19 85)]; carbohydrate-binding proteins such as lectins or lipoproteins; cellular nutrient transport molecules; and Small molecule inhibitors, e.g., inhibitors of prostate-specific membrane antigen (PSMA), small molecule tyrosine kinases TKIs, non-peptides, or other cell-binding molecules or substances, e.g., Active polymers (Dhar, et al., Proc. Natl. Acad. Sci. 200 8,105,17356-61), bioactive dendrimers (Lee, et al., Nat .Biotechnol.2005,23,1517-26;Almutairi,et al;Proc.Natl.Acad.Sci.2009,106,685-90), Nanoparticles (Liong, et al., ACS Nano, 2008, 19, 1309-1 2;Medarova,et al,Nat.Med.2007,13,372-7;J avier,et al,Bioconjugate Chem.2008,19,13 09-12), liposomes (Medinai, et al., Curr. Phar. Des .2004,10,2981-9), viral capsid (Flenniken,et a l,Viruses Nanotechnol.2009,327,71-93) However, the present invention is not limited to these.

[0108] 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.

[0109] 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.

[0110] The monoclonal antibodies used in the present invention are those secreting antibodies with appropriate antigen specificity. Start a monoclonal hybridoma cell culture in a nutrient medium containing hybridoma cells. In the culture, the hybridoma cells release the antibody into the culture medium. The antibody-containing culture supernatant was collected and the conditions and time required for the antibody to be secreted into the culture medium were maintained. This is followed by well-known techniques, such as protein A affinity chromatography, anion exchange chromatography, etc. 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 methods. The antibodies can be isolated by standard methods for protein purification.

[0111] Media useful for the preparation of these compositions are well known in the art and commercially available. Exemplary synthetic media include Dulbecco's Minimum Essential Medium (MIM) DMEM (Dulbecco et al., Virol 8:396 (1959)), 4.5 mg / L glucose, 0-20mM glutamine, 0-20% FBS, ppm amount 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). It is an addition.

[0112] 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 or oncogenic viruses, such as Epstein-Barr virus (EBV, human herpes simplex virus) Also known as pesvirus 4 (HHV-4) or Kaposi's sarcoma-associated virus Transfection with KSHV (see U.S. Patent No. 4,341,761 for details) ;4399121;4427783; 4444887; 4451570; 44669 17;4472500; 4491632; 4493890). Monoclonal antibodies The body may then prepare an anti-receptor peptide or a peptide containing a terminal carboxyl group according to known methods. (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 a polypeptide analog of the anti-receptor polypeptide of a monoclonal antibody. It can be used as an immunogen to prepare polypeptides, either alone or in a cross-linked immunogenic carrier. This can be done.

[0113] 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).

[0114] Monoclonal antibodies obtained from other species (e.g., mice) using hybridoma technology Humanized antibodies are designed to prevent the immune side effects of foreign antibodies on the human body. Among these, the well-known method for humanizing antibodies is The method involves transplantation and remodeling of complementarity determining regions. For more information, see U.S. Patent No. 5,859 , No. 205 and No. 6,797,492; Liu et al., Immunol Rev. 222: 9-27(2008);Almagro et al.,Front Biosci.1;13:16 19-33(2008);Lazar et al., Mol Immunol.44(8):198 6-98(2007);Li et al. Proc.Natl.Acad.Sci.USA.10 3(10):3557-62 (2006), the disclosure of which is incorporated by reference. Fully human antibodies are transgenic antibodies that contain large amounts of human immunoglobulin light and heavy chains. It is prepared by immunizing a feline mouse, rabbit, monkey, or other mammal with an antigen. Using mice as an example, Xenomouse (Abgenix, Inc.), H uMab-Mouse(Medarex / BMS), VelociMouse(Rege For more information, see U.S. Patent Nos. 6,596,541 and 6,207,418. No. 6,150,584, 6,111,166, 6,075,181, 5,922 ,545, 5,661,016, 5,545,806, 5,436,149 and See US Pat. No. 5,569,825. In the course of human therapy, mouse antibody variable region genes and human Immunogenicity of chimeric antibodies produced in humans by integrating human antibody constant region genes is much lower than that of mouse antibodies (Kipriyanov et al., Mol Biote chnol.26:39-60(2004);Houdebine,Curr Opin Biotechnol. 13:625-9 (2002)). The disclosure of said document is incorporated herein by reference. Furthermore, by site-directed mutagenesis of antibody variable regions, Antibody affinity and specificity can be improved (Brannigan et al., Natl. Rep. v Mol Cell Biol.3:964-70(2002);Adams et al., J. Immunol Methods. 231: 249-60 (1999)). By partially replacing the target region, it effectively promotes affinity with immune effector cells. , which can enhance the cytotoxic effect.

[0115] 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.

[0116] 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.

[0117] The use of the charged conjugates of the present invention to treat cancer, autoimmune diseases, and infectious diseases. Antibodies used to bind substances include, but are not limited to: ): 3F8 (anti-GD2 antibody), abagovomab (anti-CA-125 antibody), abciximab ( Anti-CD41 antibody (integrin α-IIb), adalimumab (anti-TNF-α antibody), Dalimumab (anti-EpCAM antibody, CD326), afelimomab (anti-TNF-α); Tuzumab (anti-CD20 antibody), Alacizumab pegol (anti-VEG FR2 antibody), ALD518 (anti-IL-6 antibody), alemtuzumab (also known as Campus, MabCampus (anti-CD52 antibody), Altumomab (anti-CEA antibody), Anatumomab (anti tag-72 antibody), anrukinzumab (IMA-638, anti-IL-13 antibody), apolipoprotein C Ibumab (anti-HLA-DR antibody), arcitumomab (anti-CEA antibody), acelizumab (anti L-selectin (CD62L) antibody), Atlizumab (also known as Tocilizumab) Actemra, RoActemra, anti-IL-6 receptor antibody), atrolimumab (Atorolim umab) (anti-rhesus factor antibody), bapineuzumab (anti-β-amyloid antibody), basilisk Cimab (Simulect, anti-CD25 (IL-2 receptor α chain) antibody), bavituximab (Bavi tuximab (anti-phosphatidylserine antibody), bectumomab (also known as Lymphoma) oScan, anti-CD22 antibody), belimumab (also known as BENLYSTA, LymphoStat-B, anti-BAFF anti- body), Benralizumab (anti-CD125 antibody), Bertilimumab (anti-CC L11 (eotaxin-1) antibody), besilesomab (also known as Scintimun, anti-CEA-related antibody) Antibody), bevacizumab (also known as Avastin, anti-VEGF antibody), biciromab (also known as Fi briScint, anti-fibrin II beta chain antibody), bivatuzumab (anti-CD44v6 antibody), brina Blinatumomab (also known as BiTE, anti-CD19 antibody), brentuximab (Br entuximab) (cAC10, anti-CD30 TNFRSF8 antibody), briakinumab (Briak inumab) (anti-IL-12, IL-23 antibody), canakinumab (also known as Ilaris, anti-IL L-1 antibody), cantuzumab (also known as C242, anti-CanAg antibody), capromab (Capr omab), catumaxomab (also known as removab, anti-EpCAM, anti-CD3 antibody), CC49( Anti-TAG-72 antibody), Cedelizumab (anti-CD4 antibody), Certolizumab Bupegol (also known as CIMZIA, an anti-TNF-α antibody), cetuximab (also known as Erbitux, I MC-C225, anti-EGFR antibody), sitatuzumab (anti-EpCAM antibody), cixtumba Cixutumumab (anti-IGF-1 antibody), clenoliximab (anti-CD4 antibody), cliva Clivatuzumab (anti-MUC1 antibody), conatumumab (anti-TR AIL-R2 antibody), CR6261 (anti-influenza A hemagglutinin antibody), Dasein Dacetuzumab (anti-CD40 antibody), daclizumab (also known as Zenapax, anti-CD25) C (IL-2 receptor α chain) antibody), daratumumab (anti-CD38 (cyclin anti-RANKL (ADP-ribose hydrolase) antibody), denosumab (also known as Prolia, anti-RANKL antibody), detumomab (anti-B-lymphoma cell antibody), dorlimomab, druxizumab (Dorl ixizumab), Ecromeximab (anti-GD3 ganglioside antibody), lisumab (also known as Soliris, anti-C5 antibody), edovacomab (anti-endotoxin antibody), Dorecolomab (also known as Panorex, MAb17-A1, anti-EpCAM antibody), Efalizumab Raptiva (anti-LFA-1 (CD11a) antibody), Efungumab (also known as Mycograb, anti-Hsp90 antibody), Elotuzumab (anti-SLAMF7 antibody), Elsilimomab (anti-IL-6 antibody), Enlimomab pegol (anti ICAM-1 (CD54) antibody), Epitumomab (anti-episialin antibody) , epratuzumab (anti-CD22 antibody), erlizumab (anti-ITGB2 (C D18) antibody), Ertumaxomab (also known as Rexomun, anti-HER2 / ne u, CD3 antibody), etaracizumab (also known as Abegrin, anti-integrin αvβ3), Sibilumab (anti-hepatitis B surface antigen antibody (HBs antibody)), Fanolesomab b) (also known as NeutroSpec, anti-CD15 antibody), faralimomab antibody (anti-I Interferon receptor antibody), farletuzumab (anti-folate receptor 1 antibody) ), Felvizumab (antibody against respiratory syncytial virus), Fezakinumab ( zakinumab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor Antibody), Fontolizumab (anti-IFN-γ antibody), Foravirumab ( Foravirumab (anti-rabies virus glycoprotein antibody), Fresolimumab ) (anti-TGF-β antibody), Galiximab (anti-CD80 antibody), Gantenol Gantenerumab (anti-β-amyloid antibody), Gavilimomab (anti-CD 147 (basigin) antibody), gemtuzumab (anti-CD33 antibody), girentuximab (Girentuximab) tuximab (anti-carbonic anhydrase 9 antibody), Glembatumumab (also known as CR01 1, anti-GPNMB antibody), golimumab (also known as Simponi, anti-TNF-α antibody), golimumab Gomiliximab (anti-CD23C (IgE receptor) antibody), Ibalizumab umab (anti-CD4 antibody), ibritumomab (anti-CD20 antibody), igobo Igovomab (also known as Indimacis-125, anti-CA-125 antibody), imciromab omab) (also known as Myoscint, an anti-cardiac myosin antibody), infliximab (also known as Remicade, Anti-TNF-α antibody), Intetumumab (anti-CD51 antibody), Innolimoma Inolimomab (anti-CD25 (IL-2 receptor α chain) antibody), Inotuzumab ab) (anti-CD22 antibody), ipilimumab (anti-CD152 antibody), iratumumab ab) (anti-CD30 (TNFRSF8) antibody), Keliximab (anti-CD4 antibody) body), labetuzumab (also known as CEA-Cide, anti-CEA antibody), lebrikizumab ) (anti-IL-13 antibody), Lemalesomab (anti-NCA-90 (granulocyte antigen) ) antibody), lerdelimumab (anti-TGFβ-2 antibody), lexatumumab ( Lexatumumab (anti-TRAIL-R2 antibody), Libivirumab (anti-hepatitis B surface antigen antibody), Lintuzumab (anti-CD33 antibody), Lucatumumab tumumab (anti-CD40 antibody), lumiliximab (anti-CD23 (IgE level) Sceptor antibody), mapatumumab (anti-TRAIL-R1 antibody), maslimomab ab) (anti-T cell receptor antibody), matuzumab (anti-EGFR antibody), mepoliz Mab (also known as Bosatria, anti-IL-5 antibody), Metelimumab (anti-TGFβ -1 antibody), Milatuzumab (anti-CD74 antibody), Minretumomab tumomab (anti-TAG-72 antibody), Mitumomab (also known as BEC-2, anti-gas Glycoside antibody (GD3), Morolimumab (anti-rhesus factor antibody) , Motavizumab (also known as Numax, an anti-RS virus antibody), muromonab (Mu romonab)-CD3 (also known as Orthoclone OKT3, anti-CD3 antibody), nacolomab ) (anti-C242 antibody), naptumomab (anti-5T4 antibody), natalizumab ( Alias: Tysabri, anti-integrin α4 antibody, nebacumab (anti-endothelin Necitumumab (anti-EGFR antibody), Nerelimomab omab) (anti-TNF-α antibody), nimotuzumab (also known as Theracim, Theraloc, anti-EGFR anti- Nofetumomab, ocrelizumab (anti-CD20 antibody), odulizumab Momab (also known as Afolimomab, anti-LFA-1 (CD11a) antibody), Ofatumumab (also known as :Arzerra, anti-CD20 antibody), Olaratumab (anti-PDGF-Rα antibody) , Omalizumab (also known as Xolair, anti-IgE Fc region antibody), Opolz Oportuzumab (anti-EpCAM antibody), Oregovomab (also known as Ova Rex, anti-CA-125 antibody), otelixizumab (anti-CD3 antibody), Pagibaximab (anti-LTA antibody), Palivizumab (also known as Synagis, Abbosy) nagis, anti-RS virus antibody), panitumumab (also known as Vectibix, ABX-EGF, anti-E GFR antibody), panobacumab (anti-pseudomonas aeruginosa antibody), pascolizumab lizumab) (anti-IL-4 antibody), pemtumomab (also known as: Theragyn, anti-MU C1 antibody), pertuzumab (also known as Omnitarg, 2C4, anti-HER2 / neu antibody), pec Pexelizumab (anti-C5 antibody), Pintumomab (anti-adenocarcinoma antigen) antibody), Priliximab (anti-CD4 antibody), Pritumumab (anti-vimentin antibody), PRO140 (anti-CCR5 antibody), racotumomab (racotumomab ) (Also known as: 1E10, Anti-(N-glycolylneuraminic acid (NeuGc, NGNA) - Cancer Glycoside (GM3) antibody), Rafivirumab (anti-rabies virus glycoprotein) Ramucirumab (anti-VEGFR2 antibody), Ranibizumab ( Alias: Lucentis, anti-VEGF-A antibody, Raxibacumab (anti-anthrax toxin) Antibody (protective antigen antibody), Regavirumab (anti-CMV glycoprotein B antibody), Reslizumab (anti-IL-5 antibody), rilotumumab (anti-H GF antibody), rituximab (also known as MabThera, Rituxanmab, anti-CD20 antibody), Robatum Robatumumab (anti-IGF-1 receptor antibody), Rontalizumab ( Anti-IFN-α antibody), Rovelizumab (also known as LeukArrest, anti-CD11, CD18 antibody), Ruplizumab (also known as Antova, anti-CD154 (CD40 antibody) L antibody), satumomab (anti-TAG-72 antibody), sevirumab ) (anti-CMV antibody), sibrotuzumab (anti-FAP antibody), sifalimumab ) (anti-IFN-α antibody), siltuximab (anti-IL-6 antibody), Sipliz Mab (anti-CD2 antibody), (Smart) MI95 (anti-CD33 antibody), solanezumab (sola nezumab (anti-β-amyloid antibody), sonepcizumab (anti-sphingomyelinase) Anti-episialin-1-phosphate antibody), Sontuzumab (anti-episialin antibody), Stam Stamulumab (anti-myostatin antibody), sulesomab (also known as Leuko Scan, (anti-NCA-90 (granulocyte antigen) antibody))), Tacatuzumab (anti-NCA-90 (granulocyte antigen) antibody))) α-fetoprotein antibody), tadocizumab (anti-integrin αIIb β3 antibody), talizumab (anti-IgE antibody), tanezumab (anti-NGF antibody) , taplitumomab (anti-CD19 antibody), tefibazumab ) (also known as Aurexis, anti-clumping factor A antibody), telimomab, tenatum Tenatumomab (anti-tenascin-C antibody), Teneliximab (anti-CD 40 antibody), Teplizumab (anti-CD3 antibody), TGN1412 (anti-CD2 antibody) 8 antibody), ticilimumab (also known as tremelimumab, anti-CTLA-4 antibody), tigatuzumab (Tigatuzumab) (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody), Tocilizumab (also known as Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor antibody) Toralizumab (anti-CD154 (CD40L) antibody), tositumomab (anti-CD 20 antibody), trastuzumab (also known as Herceptin, anti-HER2 / neu antibody), tremelli Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab-cell molleukin zumab celmoleukin) (anti-EpCAM antibody), tuvirumab (anti-hepatitis B antibody) ), Urtoxazumab (anti-Escherichia coli antibody) , Ustekinumab (also known as Stelara, anti-IL-12, IL-23 antibody), Vapaliximab (anti-AOC3 (VAP-1) antibody), vedolizumab olizumab), (anti-integrin α4β7 antibody), veltuzumab (anti-CD20 antibody), Vepalimomab (anti-AOC3 (VAP-1) antibody), visilizumab (also known as Nuv ion, anti-CD3 antibody), Vitaxin (anti-angiogenic integrin avb3 antibody), Boroshiki Volociximab (anti-integrin α5β1), Votumumab (also known as HumaSPECT, anti-tumor antigen CTAA16.88 antibody), Zalutumumab (also known as HuMax-EGFr, (anti-EGFR antibody), zanolimumab (also known as HuMax-CD4, anti-CD4 antibody), ziralimumab Ziralimumab (anti-CD147 (basic immunoglobulin) antibody), zolimomab ) (anti-CD5 antibody), etanercept (registered trademark "Enbrel"), alefacept (Alefacept) ept) (registered trademark "Amevive"), abatacept (registered trademark "Orencia"), rilonacept (Rilonacept) (Arcalyst), 14F7 [anti-IRP-2 (iron regulatory protein 2) antibody], 14G2a (Anti-Ganglioside GD from Nat. Cancer Inst.) for Melanoma and Solid Tumors 2 antibody), J591 (anti-P from Weill Cornell Medical School for treating prostate cancer), SMA antibody, ), 225.28S [anti-HMW-MAA (high molecular weight melanoma associated antibody) for melanoma (antigen) antibody, Sorin Radiofarmaci SRL (Milan, Italy)], COL-1 (Nat. Cancer Institute, anti-CEACAM3 antibody for colorectal cancer and gastric cancer, CGM1), CYT -356 (registered trademark "Oncoltad"), prostate cancer; HNK20 (RS virus from Ora Vax Inc.); for non-Hodgkin's lymphoma), ImmuRAIT (from IMMUNOMEDICS for non-Hodgkin's lymphoma), Lym-1 (anti-HLA-DR10 antibody, from Peregrine Pharm for tumors), MAK-1 95F [Anti-TNF (tumor necrosis factor; TN F) from Abbott / Knoll for sepsis and toxic shock] FA, TNF-α; TNFSF2) antibody], MEDI-500 [also known as: T10B9, MedImmune anti-CD3 antibody for graft-versus-host disease from Inc, TRαβ (T-cell receptor α / β),]; RING SCAN [Anti-TAG from Neoprobe Corp. for breast, colon, and colorectal cancer] 72 (tumor-associated glycoprotein 72) antibody)], Avicidin (anti-EPCAM (epithelial cell Anti-TACSTD1 (tumor-associated calcium signal transducer and activator of calcium ion transporter) antibody -1) antibody, anti-GA733-2 (gastrointestinal tumor-associated protein 2) antibody, anti-EGP-2 (epithelial Glycoprotein 2) antibodies; anti-KSA antibodies; KS1 / 4 antigen; M4S; tumor antigen 17-1A; CD32 from NeoRx Corp. for colon cancer, ovarian cancer, prostate cancer, and non-Hodgkin's lymphoma 6. LYMPHOCIDE (IMMUNOMEDICS, NJ), Smart ID10 (Protein Design) Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), Anti-V EGF antibody (Genentech, CA); CEAcide (Immunomedics, NJ), IMC -1C11 (ImClone Systems, NJ), and cetuximab (ImClone, NJ) However, it is not limited to these.

[0118] Other antibodies as cell binding molecules / ligands include, but are not limited to, the following antigens: : Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various CA125 (ovarian), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (cancer) Lewis Y (carcinoma), Lewis X (carcinoma), α-fetoprotein (carcinoma), CA24 2 (colon rectum), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), Prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), CD2 (Hodgkin's disease, NH L 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 erythematosus), CD3 0 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma) ), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma , sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma and erythroblastoma) CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer), whip CD221 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer) cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM 3 (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 body, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hematopoietic tumors, solid tumors ), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head NHL (nose, cervix, colon cancer, NHL prostate cancer, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2 ; lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian GD2 ganglioside (cancer), G-28G (cell surface antigen glycolipid, melanoma), GD 3 Idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HE R2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL , B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor receptor factor-1 receptor, solid tumors, blood cancers), IL-2 receptor (interleukin-2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6-dependent tumors), integrins (αVβ for various cancers) 3, α5β1, α6β4, αIIβ3, α5β5, αVβ5), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE-4 (carcinoma), anti-transferase Phosphoreceptor (carcinoma), p97 (melanoma), MS4A1 (transmembrane 4-domain family A Member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast Cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), C EA (colon), gp100 (melanoma), MART1 (melanoma), MPG (melanoma), MS 4A1 (transmembrane 4-domain family A member 1, small cell lung cancer, NHL), nucleo Phosphorus, neuro-oncogene product (carcinoma), P21 (carcinoma), anti-(N-glycolylneuraminic acid paratope (breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian cancer, sperm prostate cancer), PSMA (prostate cancer), PSA (prostate), ROBO4, TAG72 (tumor-associated Glycoprotein 72, leukemia (AML, gastric cancer, colorectal cancer, ovarian cancer), T cell transmembrane protein Protein (cancer), Tie (CD202b), TNFRSF10B (tumor necrosis factor receptor serotonin receptor 10B) necrosis factor receptor supramolecular family member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor supramolecular family member 10B, cancer), Parr family member 13B, multiple myeloma, NHL, other cancers, RA and SLE), T PBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (TNF-related apoptosis Ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106 , melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers) Other tumor-associated antigens recognized by antibodies have already been reported (Gerber, et al, mAbs 1:3, 247-253 (2009); Novellino et al, cancer immunol immunother. 5 4 (3), 187-207 (2005) Franke et al, cancer biother radiopharm. 2000, 15,459 -76).

[0119] 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 binding to lanocytes. The cell binding agent may be any one of the following antigens or receptors: Any drug / molecule can be used: CD3, CD4, CD5, CD6, CD7, CD8 , CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, C D15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53, CD5 4, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, C D62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83, CD8 6, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD9 8, CD100, CD103, CD105, CD106, CD109, CD117, CD 120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD15 1, CD147, CD152, CD154, CD156, CD158, CD163, CD 166, CD168, CD174, CD180, CD184, CDw186, CD194 , CD195, CD200, CD200a, CD200b, CD209, CD221, C D227, CD235a, CD240, CD262, CD271, CD274, CD27 6(B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activation inhibitor 1) or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase enzyme 1, AFP, AKAP-4, ALK, α integrin, αvβ6, aminopeptidase ZeN, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, Annexin A1, anthrax toxin protective antigen, anti-transferrin receptor, AOC3( VAP-1), B7-H3, anthrax, BAFF (B-cell activating factor), B-lymphomatosis Cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate Carbonic anhydrase 9 (CAIX, carbonic anhydrase 9), CAL LA, CanAg, canine IL31, carbonic anhydrase IX, cardiac myosin, CCL11(C- C motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD194), C CR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEAC AM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK 2R), CLDN18 (claudin-18), clumping factor A, CRIPTO, F CSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2 , granulocyte-macrophage colony-stimulating factor (GM-CSF), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184 ), CXC chemokine receptor type 4, cADP-ribose hydrolase, Cyclin B1 , CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B, Dabiga Tran, DLL4 (Delta-like ligand 4), DPP4 (Dipeptidyl peptidase 4), DR5 (death receptor 5), Escherichia coli Shiga toxin type 2, ED-B, EGFL7 (protein 7-containing EGF-like domain), EGFR, EGFRII, EGFRvIII, Endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion molecule EphA2, episialin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS2ETS fusion gene), E. coli, ETV6-AML, FAP (fibrosis) Blast activation protein α), FCGR1, α-fetoprotein, fibrin II, β chain, fibronectin ectodomain B, FOLR (folate receptor), folate receptor α, folate receptor Dolorase, Fos-related antigen 1, F protein of respiratory syncytial virus, Frizzled receptor , fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 Diotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, G MCSF receptor α chain, growth differentiation factor 8, GP100, GPNMB (transmembrane protein NM B), GUCY2C (guanylate cyclase 2C, guanylate cyclase C (GC-C) , intestinal guanylate cyclase, guanylate cyclase-C receptor, heat-stable enterotoxin receptor (hSTAR), heat shock proteins, hemagglutinin, hepatitis B surface antigen, B Hepatitis virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu , HER3 (ERBB-3), IgG4, HGF / SF (stem cell growth factor / cell scattering factor) ), HHGFR, HIV-1, histone complex, HLA-DA (human leukocyte antigen), HL A-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, Human cell scatter factor receptor kinase, HPV E6 / E7, Hsp90, hTERT, IC AM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin -like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2 receptor) , IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9 , IL-10, L-12, IL-13, IL-17, IL-17A, IL-20, IL- 22, IL-23, IL-31RA, ILGF2 (insulin-like growth factor 2), integrin Phosphorus (α4, α IIIb β3, αvβ3, α4β7, α5β1, α6β4, α7β7, α IIβ3, α5β5, αvβ5), interferon-γ-inducible protein, ITAGA2, ITGB2, KIR2D, LCK, Le, legumain, Lewis-Y antigen, LFA-1 (Li lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LI V1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MA GE-2, MAGE-3, MAGEA1, MAGEA3, MAGEA4, MART1, M CP-1, MIF (macrophage 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 cell surface ithelial mucin (PEM)), MUC1-KLH, MUC16 (CA12 5), MCP1 (monocyte chemotactic protein 1), MelanA / MART1, ML-IAP , MPG, MS4A1 (transmembrane 4 domain subfamily A), MYCN, myelin-related Glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen) , Nectin-4 (ASG-22ME), NGF, neuroapoptosis-regulating protein Ze1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-B R-1, NY-ESO-1, OX-40, Oxidized low-density lipoprotein (OxLDL), O Y-TES1, P21, non-mutant p53, P97, Page4, PAP, anti-(N-glycosyltransferase) Cholinuric acid (PAX3, PAX5, PCSK9, PDCD1(P D-1, programmed cell death protein 1, CD279), PDGF-Rα, (blood cell Plate-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLAP-like testis Alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate cotransporter, P MEL17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (phosphatidylinositol phosphate) Dyserin), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus glycoprotein Protein, RHD (Rh polypeptide 1 (RhPI), CD240), Rhesus factor (Rhesus factor), RANKL, PhoC, Ras mutant, RG55, R OBO4, respiratory syncytial virus, RON, sarcoma metastasis breakpoint, SART3, scleros SLAMF7 (SLAM family member 7), selectin P, SDC1 (synthesis) Decane 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate somatostatin, sperm protein 17, SSX2, STEAP1 (prostate 1, 6 Transmembrane epithelial antigen), STEAP2, STn, TAG-22 (tumor-associated glycoprotein 72) , survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor epithelial marker 1) ), TENB2, tenascin C (TN-C), TGF-α, TGF-β (transforming 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 apoptosis) Death-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), major Tumor-specific glycosylation of MUC1, a related calcium signal transducer 2, WEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase, VCA M-1(CD106), VEGF, VEGF-A, VEGF-2(CD309), VEG FR-1, VEGFR2, or vimentin, WT1, XAGE1, or any insulin A cell expressing a growth factor receptor, or any epidermal growth factor receptor.

[0120] In another specific embodiment, the cell-binding ligand-drug conjugates of the present invention are The method is used to treat cancers, including but not limited to adrenal gland cancer. Interstitial cancer, anal cancer, bladder cancer, brain tumors (adult, brainstem glioma, children, cerebellar astrocytoma, cerebral astrocytoma) tumors, ependymomas, medulloblastomas, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and optic nerve tumors 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), lip and oral Cavity cancer, liver cancer, lung cancer (non-small cell, small cell), lymphoma (AIDS-related, central nervous system, skin T Cells, Hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, Merkel cell carcinoma, and of unknown primary origin Metastatic squamous neck cancer, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes group, 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 sinuses and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasmacytoma, prostate cancer, Rhabdomyosarcoma, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter (transitional cell), salivary gland cancer, Cezari - syndrome, skin cancer, skin cancer (dermatoid T-cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft Tissue sarcoma, stomach cancer, testicular cancer, thymoma (malignant), thyroid cancer, urethral cancer, uterine cancer (sarcoma), children abnormal cancers of the vagina, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0121] In another specific embodiment, the cell-binding molecule-drug conjugate of the present invention via a bridge linker comprises: The composition and method can be used to treat or prevent autoimmune diseases. Epidemic diseases include autoimmune gastric achlorhydria, chronic active hepatitis, acute disseminated encephalomyelitis, and acute hemorrhagic leukemia. Encephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, rigidus Spondylitis, anti-GMB / TBM nephritis, antiphospholipid syndrome, antisynthetase syndrome, joint inflammation, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune Cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune phosphorus Pancreatic cancer, autoimmune peripheral nervous system disease, autoimmune pancreatitis, multiple autoimmune endocrine disorders Disorders type I, II, III, autoimmune progestational dermatitis, autoimmune thrombocytopenic purpura Plaque disease, autoimmune uveitis, Barlow's disease / Barlow concentric sclerosis, Behçet's disease, B Berger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, catarrh Suleman's disease, Chagas' disease, chronic fatigue and immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy Disorders, chronic recurrent multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergic granulomatosis Tumorous vasculitis, cicatricial pemphigoid, celiac disease, Cogan's syndrome, cold agglutinin disease, complement components C2 deficiency, cephalic arteritis, CREST syndrome, Crohn's disease (idiopathic inflammatory bowel disease), ng syndrome, cutaneous leukocytoclastic vasculitis, malignant atrophic papulopathy, painful steatosis, dermatitis herpetiformis, Dermatomyositis, type 1 diabetes, diffuse cutaneous scleroderma, myocardial infarction, discoid lupus erythematosus, eczema, Endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa acquisita, erythema nodosum, idiopathic Gender-specific mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, fibromyalgia fibromyositis, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis , Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroid inflammation, hemolytic anemia, allergic purpura, herpes gestationis, hidradenitis suppurativa, Hughes syndrome (anticoagulant) Phospholipid syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis Idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Ber ger's disease), inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, irritable bowel syndrome , juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymph node syndrome, Lambert-I Peterson's myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease ( LAD), Lou Gehrig's disease (amyotrophic lateral sclerosis), lupus-like hepatitis, lupus erythematosus, bradykinin Cow's syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed combination Histological diseases, scleroderma, Mucha-Jakob disease, Muckle-Wells syndrome, multiple myeloma, multiple Neuropathic sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neurogenic Muscle, ocular cicatricial pemphigoid, opsoclonus-myoclonus syndrome, thyroiditis, thyroid Intractable rheumatism, Panda syndrome (childhood autoimmune neuropsychiatric disorder complicated by streptococcal infection), tumors Cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry-Romberg syndrome, Parsonage-Jones syndrome Zia syndrome, pars plana inflammation, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, PO EMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis Primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, Pure red cell aplastic anemia, Rasmussen's encephalitis, Raynaud's disease, relapsing polychondritis, Reiter's disease syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Glenn's syndrome, spondyloarthropathy, sticky blood syndrome, Still's disease, stiff-man syndrome, Subacute bacterial endocarditis, Susac syndrome, acute febrile neutrophilic dermatosis, Sydenham chorea, sympathetic nervous system Ophthalmitis, Takayasu's arteritis, temporal arteritis (giant cell arteritis), Tolosa-Hunt syndrome, transverse spinal cord injury Myelitis, ulcerative colitis (idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, blood Vasculitis, vitiligo, Wegener's granulomatosis, Wilson's syndrome, Westcott-Aldrich disease This includes, but is not limited to, the following:

[0122] In another specific embodiment, the cross-linkers of the present invention are used to treat or prevent autoimmune diseases. The binding molecules used for conjugation include anti-elastin antibody; Abys anti-epithelial cell antibody; Antibody; anti-basement membrane type IV collagen protein antibody; antinuclear antibody; anti-double-stranded DNA antibody, anti-single Double-stranded DNA antibody, anticardiolipin antibody IgM, IgG; anti-celiac antibody; anti-phospholipid Antibodies IgK, IgG; anti-SM antibody; anti-mitochondrial antibody; thyroid antibody; microparticle antibody, T Cellular antibodies; thyroglobulin antibodies, anti-scleroderma-70 antibodies (AntiSCL-70); anti-joint antibodies -Antibody (Anti-Jo), anti-U1RNP antibody (Anti-U1RNP); anti-La / SS B antibody; anti-SSA antibody; anti-SSB antibody; anti-parietal cell antibody; anti-histone antibody; anti-RNP antibody; C-ANCA; P-ANCA; anti-centromere antibody; anti-fibrin antibody, anti-GBM antibody, Anti-ganglioside antibody; anti-desmosomal glycoprotein 3 core antibody (anti-Desmo gein3); anti-p62 antibody; anti-sp100 antibody; anti-mitochondrial (M2) antibody; Liu Mat factor antibody; anti-MCV antibody; anti-topoisomerase antibody; antineutrophil cytoplasmic antibody (cANCA) These include, but are not limited to, antibodies.

[0123] 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).

[0124] 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.

[0125] The cell-binding molecule-drug conjugates prepared by the crosslinkers of the present invention are useful for the treatment of infectious diseases. The infectious diseases include Acinetobacter infection, actinomycosis, African flu, and the like. Sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), amebiasis , Anaplasma, Anthrax, Bacillus anthracis, Bacillus tuberculosis infection, Argentine hemorrhagic fever, Ascariasis, Aspergillus infection, astrovirus infection, babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis , Bacteroides infection, Balantidiosis, Baileyi roundworm infection, BK virus infection , black sand hair, Blastocys hominis infection, Blastomyces, Bolivian hemorrhagic fever, Borrelia Infectious diseases, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, Cholderia infection, Buruli ulcer, Calicivirus infection (Norovirus, Sapovirus) ), Campylobacter infection, Candida infection (candidiasis, thrush), cat scrofula Lacchia, cellulitis, Chagas disease (American trypanosomiasis), chancroid, chickenpox, Kinuhara body, pneumococcal infection, huaoran, pigmented mycosis, liver fluke disease, Clostridium difficile infection Infection, coccidioidomycosis, Colorado tick fever, colds (acute viral nasopharyngitis, acute rhinitis) ), Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever, cryptococcosis, cryptococcosis Tosporidium, cutaneous larval migration, Cyclospora infection, cysticercosis, cytomegalovirus infection Infection, dengue fever, diamoebiasis, diphtheria, diphyllobothriasis, dracunculiasis, Ebola hemorrhagic fever, Hydatid disease, ehrlichiosis, pinworms (pinworm infection), enterococcal infection, enterovirus infection , typhus, erythema infectiosum (fifth disease), acute rash in children, trematode fluke disease, thrombocytopenia, fatal thrombocytopenia Insomnia, filariasis, food poisoning caused by Clostridium perfringens, non-parasitic amoeba infection, Fusobacterium infection, gas gangrene (Clostridial myonecrosis), diotrichosis jaundice, Gerstmann-Straussler-Scheinker syndrome, Giardiasis, glanders, Gnathostomiasis, gonorrhea, granuloma groin (donovanosis), group A streptococcal infection, group B streptococcal infection Infectious diseases, Haemophilus influenzae infection, hand, foot and mouth disease (HFMD), Hantavirus pulmonary syndrome, Helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever with renal syndrome, hepatitis A, hepatitis B, C Hepatitis, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human varicella Human HIV infection, human ehrlichiosis Evans, human granulocytic anaplasmosis, human methamphetamine Human papillomavirus infection, human monocytic aerosol infection, human parasite infection Influenza virus infection, microtaeniasis, influenza, isosporosis, Kawasaki disease, Mononucleosis, Mycobacterium kamylosis, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease Neillosis (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis Lyme disease (Lyme borrelia), lymphatic filariasis (elephantiasis), lymphocytic choriocarcinoma Meningitis, malaria, Marburg hemorrhagic fever, measles, melioidosis (Whitmore's disease), meningitis, myelitis Meningococcal disease, metagonism, microsporidiosis, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasma pneumonia, mycetoma, myiasis, neonatal conjunctivitis (neonatal ophthalmitis) Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiosis, oncolytic filariasis (blinding filariasis), paracoccidioidomycosis (South American blastomycosis), Insect lice, Pasteurellosis, Head lice (head lice), Body lice (body lice) , pubic lice (Crab ice), pelvic inflammatory disease, whooping cough ing cough), plague, pneumococcal infection, carinii pneumonia, pneumonia, polio, Prevotella Infectious diseases, PAME, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, Respiratory syncytial virus infection, rhinovirus infection, rickettsial infection, rickettsia, rif Tovalley fever, Rocky Mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS ( Severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, diarrhea (dysentery), shingles (Herpes zoster) pes zoster), smallpox, sporotrichum, staphylococcal food poisoning, staphylococcal infection , nematodes, syphilis, tapeworms, tetanus (trismus), Barber's itch ), tinea manubriata, pityriasis nigricans, tinea pedis, tinea unguium, tinea versicolor, toxocariasis (ocular larva migrans) , toxocariasis (visceral larva migrans), toxoplasmosis, trichinosis, trichomoniasis, chestnut Pathobiosis (whipworm infection), pulmonary tuberculosis, tularemia, urea-degrading Mycoplasma infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, rhizobium spp. Diseases including, but not limited to, Yersinia pseudotuberculosis, yersiniosis, yellow fever, and zygomycosis .

[0126] More preferably, the cell-binding molecule is an antibody against a pathogenic strain described herein, The pathogenic strains include Acinetobacter baumannii, Actiomyces israelii, and Acinetobacter pneumoniae. Actinomyces odontolyticus s), Propionibacterium propionicus, Trypanosoma brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma spp., Bacillus anthracis, Methylshemolites Arcanobacterium haemolyticum, Junin virus Aspergillus, Ascaris, Astroviridae, Babesia, Bacillus cereus, Malaria Tipul bacteria, Bacteroides spp., Colonic pouch ciliates, Bailey roundworms, Nematode spp., BK Virus, Piedraiahortae, Blastocyst Diphtheria hominis, Dermatitis gracilis, Makupo virus, Borrelia spp., Clostridium botulinum, Sabia , Brucella spp., typically Burkholderia cepacia and other Burkholderia species, Mycobacterium Caliciviridae family, Campylobacter, commonly known as Caliciviridae Candida albicans and other Candida species, Bartonella henselae ella henselae, group A streptococci and staphylococci, Trypanosoma cruzi , Haemophilus chancroid, Varicella-zoster virus (VZV), Chlamydia trachomatis, Chlamydia A. pneumoniae, Vibrio cholerae, Fonseca pedrosoi, Clonorchiasis, Clostridium difficile, Coccidioides immitis, Coccidioides posadaci, Corola Tick ​​fever virus, rhinovirus, coronavirus, Creutzfeldt-Jakob disease Lyon, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Crimean-Congo hemorrhagic fever virus ... Ptosporidium, cat hookworm, co-parasites, Cyclospora, Taenia solium, Cytomegalovirus dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) - flavivirus Viruses, Dimorphonucleate amoeba, Corynebacterium diphtheriae, Diphyllobothria, Dracunculia spp., Guinea worm (D racunculusmedinensis), Ebola virus, Echinococcus spp., - Rickettsia spp., pinworms, Enterococcus spp., Enterovirus spp., Rickettsia typhi , Parvovirus B19, Human Herpesvirus 6, Human Herpesvirus 7, Hypertrophy Flukes, Liver Fluke and Liver Fluke Giant, FFI Prion, Filariae, Clostridium perfringens, Fuso Bacterium, Clostridium perfringens, other Clostridium species, Geotrichum candidum, G SS prion, Giardia lamblia, Burkholderia Burkholderia mallei, Gnathostoma nematode, Gnathostoma strigiforme, Neisseria gonorrhoeae, Bordetella granulomatosis, Streptococcus pyogenes, Streptococcus pyogenes agalactiae, Haemophilus influenzae, enteric viruses, most Coxsackieviruses A Enterovirus type 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O 158:H7, Bunyaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus Hepatitis D virus, Hepatitis E virus, Herpes simplex virus type 1, Herpes simplex Virus type 2, Histoplasma capsulatum, hookworm, American hookworm, Influenza Lactobacillus casei, Boca human virus, Ehrlichia ewingii ngii), Anaplasma phagocytophilum, human metapneumovirus, Yale Richter chaffeensis, human papillomavirus, human parainfluenza virus , dwarf tapeworm, diminutive tapeworm, Epstein-Barr virus, Orthomyxoviridae, Iso Isospora belli, Kingell a kingae), Klebsiella pneumoniae, Klebsiella ozena, Klebsiella rhinosierrero Klebsiellarhinoscleromotis, Klebsiella purpurea Lassa fever virus, Legionella pneumophila, Legionella pneumophila, Mycobacterium leprosum, Mycobacterium lepromatosis, and Mycobacterium lepromatosis erium lepromatosis), Leptospira, Listeria, Borreliosis and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus Rus (LCMV), Plasmodium genus, Marble Virus, measles virus, Burkholderia pseudomallei lei), Neisseria meningitidis, Fluke yokogawai, Microsporidia, Molluscum contagiosum virus (MCV), mumps Viruses, Rickettsia typhi, Mycoplasma pneumoniae, various bacteria (Actinomyces Flea mycetoma) and fungi (mycetoma), parasitic fly larvae of the Diptera order, Chlamydia trachomatis Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia species, Onchocerca volvulus, Blastomyces brasiliensis, Paragonimus and other Paragonimus species, Pasteurella spp. Head lice, body lice, Phthiruspubis, 100-day Cough bacillus, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis cysticercosis, Poliovirus, Prevotella spp., Naegleria ameba, JC virus, Chlamydia psittacosis, Coxiella burnetii , rabies virus, bead-chain E. coli and rat bite fever spirochetes, respiratory RS virus, Rhinosporidium sayberi, rhinovirus, rickettsia, rickettsiada 2. Rift Valley fever virus, Rocky Mountain spotted fever rickettsia, rotavirus, rubella virus Salmonella, atypical pneumonia coronavirus, scabies mites, Schistosoma, Shigella, Varicella Herpes virus, Variola major or minor, Sporothrix schenckii, Staphylococcus spp., Yellow fever Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Syphilis spirochete, Cestodes spp., Clostridium tetani, Ringworm, Lycophyton tonsurans, Ringworm, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, Trichophyton rubrum, Holtea werneckii, Ringworm, Malassezia, Roundworm and cat roundworm, toxoplasmosis, trichinella, vaginal trichomonas, whipworm, tuberculosis, Tula hotof Bacteria urinary tract infections, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Cholera Lactobacillus casei, Guanarito virus, West Nile virus, Trichosporonium spp., Yersinia pseudotuberculosis, Yersinia enteritidis A, Yellow fever virus, Mucorales (mucormycosis) and insect mesh mold (Entomophthora infection) , Pseudomonas aeruginosa, Campylobacter fetus (Vibrio), Aeromonas bacteria, Edwardsiella spp. .tarda, Yersinia pestis, Shigella dysenteriae, Shigella sonnei, Salmonella typhimurium, Treponema Pertenue, Treponema carateneum, Fensenburgdorferi, Borrelia b Lugdorferi, Leptospira hemorrhagic jaundice, Pneumocystis carinii, Brucella abortus, porcine Brucella abortus, Malta fever, Mycoplasma spp., Rickettsia typhi, Rickettsia tsutsuga Chlamydia, pathogenic fungi (Aspergillus fumigatus, Candida albicans) protozoa (Entamoeba histolytica, Trichomonas vaginalis, Trichomonas chinensis); Monas, Trypanosoma gambiens, Trypanosoma rhodesiense, Leishmania donovani Leishmania, Leishmania tropicalis, Leishmania brasiliensis, Pneumocystis carinii pneumonia, San Plasmodium vivax, Plasmodium falciparum, malignant malaria); or helminths (Schistosoma japonicum, These include, but are not limited to, Schistosoma mansoni, Schistosoma haematobium and hookworms.

[0127] 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.

[0128] According to a further object, the present invention provides a method for the preparation of a conjugate of the present invention via a crosslinked conjugate and a pharmaceutical It also relates to pharmaceutical compositions for treating cancer and autoimmune diseases, together with an acceptable carrier. The methods for treating cancer and autoimmune diseases include in vitro, Can be performed in vivo or ex vivo An example of an in vitro therapy is to transfect all cells except the desired mutant that do not express the target antigen. cell culture treatment to kill mutants expressing undesired antigens Examples of ex vivo therapies include hematopoietic stem cell transplantation prior to performing a hematopoietic stem cell transplant (HSCT). Treating (HSCs) and returning them to the same patient to kill diseased or malignant cells For example, the extraction of cancer cells from bone marrow prior to autologous transplantation in the treatment of cancer and autoimmune diseases. prior to transplantation to remove lymphocytes or cells or to prevent graft-versus-host disease Clinical extractions to remove T cells and other lymphoid cells from allogeneic bone marrow or tissue are also available. In vivo treatment can be performed by obtaining bone marrow cells from the patient or other individual. After that, the serum containing the conjugate of the present invention was added to the serum so that the concentration range was 1 pM to 0.1 mM. Incubate in a suitable medium at 37°C for 30 minutes to approximately 48 hours. The amount of serum (mg / mL) can be easily determined by an experienced clinician. The cells are washed with culture medium and then returned to the body by known methods, such as intravenous injection. If the patient is receiving other treatments (e.g., ablative chemotherapy or total body irradiation) during the course of the treatment After processing, the bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0129] For in vivo clinical applications, the conjugate drug of the present invention may be administered in the form of a solution or in sterile water for injection. It is provided in the form of a lyophilized solid that can be redissolved in water. An example of a method is as follows: 1 weekly intravenous bolus of the conjugate for 8 to 20 weeks. The bolus dose is dissolved in 50 to 500 mL of saline, and the saline is then injected into human blood. Serum albumin can be added (e.g., 0.5-5 ml of concentrated human serum albumin) The drug dosage is approximately 50 μg to 20 mg / kg body weight per week. Intravenous injection (injection dose ranges from 10 μg to 200 mg / kg per injection) for 4 to 20 weeks After treatment is completed, patients can receive a second course of treatment. The exact treatment method, including the agent, diluent, dosage, and duration of treatment, will be determined by an experienced surgeon. It is possible.

[0130] 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 (viruses). (including viruses, bacteria or parasites).

[0131] Several factors affect the amount of conjugated drug required for the 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.

[0132] Generally, the conjugates of the present invention via the linker are used at a concentration of 0.1 to 10% w / v. It may be a formulation for parenteral administration dissolved in a physiological buffer so as to contain the compound. Typical dosage ranges are 1 μg / kg body weight to 0.1 g / kg body weight per day, preferably The recommended dose range is 0.01 mg / kg to 20 mg / kg of body weight per day, or The preferred drug dosage depends, for example, on the type and stage of progression of the disease or disorder. the overall health of the individual patient, the relative biological activity of the selected drug, and the compound Dosage form, mode of administration (intravenous, intramuscular, or other), and dosage of the drug in the selected mode of administration Pharmacokinetic properties, as well as the rate of administration (single injection or continuous infusion) and schedule of administration (constant The dose depends appropriately on variables such as the frequency of administration over time.

[0133] 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.

[0134] Drugs / Cytotoxic Agents Drugs that can be linked 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 molecular weight of the drug may be, for example, 100 to 1800, more preferably 120 to 1400. Organic, inorganic or organometallic compounds are widely used. For a better definition of small molecule drugs, Reference is made to WO05058367A2 and U.S. Pat. No. 4,956,303, as well as other documents. The drugs may be any of the known drugs listed above, which are incorporated by reference in their entirety. This includes substances and potential drugs.

[0135] Known drugs include, but are not limited to:

[0136] 1) Chemotherapeutic agents: a) Alkylating agents: e.g., nitrogen mustard: chlorambucil Sil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, iho Sufamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobromide Ronitol, melphalan, pipobroman, nobembrine, fenesterine, prednisolone sucralose, thiotepa, trofosfamide, uracil mustard; CC-1065 (Adzere Synthetic analogues of cin, carzelesin and bizelesin; duocarmycin (synthetic analogs, including KW-2189 and CBI-TMI; benzodiazepine dimers (e.g., For example, pyrrolobenzodiazepines (PBDs) or tomaymycin, indolinobenzodiazepines dimers of benzodiazepines, imidazobenzothiazepines, or oxazolidinobenzodiazepines Nitrosourea compounds: (carmustine, lomustine, chlorozotocin, fotemus) cin, nimustine, ranimustine); alkyl sulfonates (busulfan, treosulfan) phan, improsulfan and piposulfan); triazene (dacarbazine); platinum Contains compounds: (carboplatin, cisplatin, oxaliplatin); benzodopa, carboplatin Aziridines such as boquone, metuledopa and uredopa; ethyleneimines, and alto methylamine, triethylenemelamine, triethylenephosphoramide and triethylenethiophosphatide b) plant alkaloids: e.g., vinca alkaloids; Idols: (vincristine, vinblastine, vindesine, vinorelbine, navelbine) Taxoids: (paclitaxel, docetaxel); and their analogs, maytansine steroids (DM1, DM2, DM3, DM4, maytansine, ansamitocin) and These analogs, cryptophycins (particularly cryptophycin 1 and cryptophycin 8), ); Epothilones, eluterobins, discodermolide, bryostatins, dolomides Statins, auristatins, tubulysins, cephalostatins; pancratic statins; sarcodictyins; spongistatins; c) DNA topoisomerase inhibitors: e.g. For example, epipodophyllins (9-aminocamptothecin, camptothecin, chrysanthecin, Thor, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxant ron, novantrone, retinoic acid (retinols), teniposide, topotecan, 9-ni Trocamptothecin (RFS 2000); Mitomycins: (Mitomycin C) d) Antimetabolites: for example, {[antifolates: dihydrofolate reductase inhibitors: (methotrexate) Rexate, trimetrexate, denopterin, pteropterin, aminopterin (4 -aminopteroic acid), or other folic acid analogs); IMP dehydrogenase inhibitors ( mycophenolic acid, tiazofurin, ribavirin, EICAR); ribonucleotide reductase Pyrimidine inhibitors (hydroxyurea, deferoxamine)]; [Pyrimidine analogs: uracils analogs (ancitabine, azacitidine, 6-azauridine, capecitabine (Xeloda), Lumofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5- Fluorouracil, floxuridine, raltitrexed (Tomudex); Cytosine Purine analogs: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (Arabine Zathioprine, Fludarabine, Mercaptopurine, Thiamiprine, Thioguanine)]; e) Hormone therapy agents: for example, {receptor antagonists: [anti-estrogens LHRH agonists: (megestrol, raloxifene, tamoxifen) (goserelin, leuprolide acetate); Antiandrogens: (bicalutamide, flutamide, Calsterone, dromostanolone propionate, epithiostanol, goserelin, thiaprolide, mepitiostane, nilutamide, testolactone, trilostane, and other Androgen inhibitors)]; Retinoids / deltoids: [Vitamin D3 analogs: (CB1093 , EB1089, KH1060, cholecalciferol, ergocalciferol); light Radiation therapy agents: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy- Hypocrellin A; Cytokines: (Interferon α, Interferon γ, Tumor necrosis factor (TNF, TNF domain-containing human protein)]}; f) kinase inhibitors: For example, BIBW2992 (anti-EGFR / Erb2), imatinib, gefitinib, and pegasus Putanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib Nib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubu Litinib, ponatinib (AP24534), bafetinib (INNO-406), bosuti Nib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib , CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab g) Antibiotics: e.g., trastuzumab, ranibizumab, panitumumab, ispinesib; For example, enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1 , δ1, α1 and β1, e.g., J.Med.Chem., 39(11), 2103-2 117(1996),Angew Chem Intl.Ed.Engl.33:183 -186 (1994); dynemicin A and deoxydynemicin esperamicin, kedarcidin, C-1027, maduropeptin, and neocardi Nostatin chromophores and related chromoprotein enediyne antibiotic chromophores, Aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin Cactinomycin, carabic in), carminomycin, carzinophilin; chromomycins, dactinomycin, Daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxol Bicine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidine Nodoxorubicin and deoxydoxorubicin, epirubicin, idarubicin, marcescens thromycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins , peplomycin, potfilomycin, puromycin, querramycin, rodolubicin Syn, streptonigrin, streptozocin, tubercidin, ubenimex, Zinosta f) Other categories: e.g., polyketides (acetogenins), In particular, bullatacin and bullatacinone; gemcitabine, epoxomicins (e.g., calfi bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostasis , Zyblestat, PLX4032, STA-9090, Stimvax muvax), allovectin-7, Zygeba, Provenge, Elvoy, isoprenylation inhibitors anti-inflammatory drugs (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-furan), phenylpyridine ions), cell cycle inhibitors (e.g., staurosporine), actinomycin antimycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g., , bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epinephrine, Rubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., Vera Pamil), Ca 2+ ATP inhibitors (thapsigargin, etc.), histone deacetylase inhibitors agents (vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB 939, resminostat, gibinostat, AR-42, CUDC-101, sulfonamide Laphan, trichostatin A); thapsigargin, celecoxib, glitazones, epigastric locatechin gallate, disulfiram, salinosporamide A, antiadrenal drugs such as aminoglycoside Lutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; Aminolevulinic acid; arabinoside, Bestravsil; bisantrene; edatrexate; Defofamine, demecolcine, diazicon, elfornithine (DFMO), eritin acetate Putinium, etocluzid, gallium nitrate, gacitosine, hydroxyurea; ibandrone lute, lentinan; lonidamine; mitoguazone; mopidamol; nitraelin; pentos Tatin; Fenamet; Pirarubicin; Podophyllic acid; 2-ethylhydrazide; Proca Rubadin; PSK (registered trademark); Razoxane; Rhizoxin; Sizofiran; Spirogerma TRIAZICONE;2,2',2''-TRICHLOROTRIETHYLAMINO ACID;TRIAZICONE;2,2',2''-TRICHLOROTRIETHYLAMINO ACID trichothecenes (especially T2 toxin, verrucarin A, roridin A, and anguidine); urethane, siRNA, antisense drugs, and nucleolytic enzymes.

[0137] 2) Anti-autoimmune disease drugs: cyclosporine, cyclosporine A, aminocaproic acid, aza Thiopurine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, steroids (e.g., hormones, betamethasone, budesonide, flunisolide, fluticasone) propionate, hydrocortisone, dexamethasone, fluocorticoid, danazol ol, triamcinolone acetonide, beclomethasone dipropionate), dehydroisoamin Drosterone, etanercept, hydroxychloroquine, infliximab, meloxicam methotrexate, mofetil, mycophenylate, prednisone, sirolimus, including but not limited to clolimus.

[0138] 3) Anti-infectives: a) Aminoglycosides: Amikacin, Astromicin, Gentamycin Syn (netilmicin, sisomicin, isepamicin), hygromycin, kanamycin Cin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobrama Isin), neomycin (neomycin B, paromomycin, ribostamycin), lumycin, spectinomycin, streptomycin, tobramycin, verdamycin b) Amphenicols: azidamphenicol, chloramphenicol, florphenicol c) Ansamycins: geldanamycin, herbimycin d) Carbapenems: biapenem, doripenem, ertapenem, imipenem / silapenem Statins, meropenem, panipenem; e) Cephems: carbacephem (loracarbef) , cephacetrile, cefaclor, cephradine, cefadroxil, cephalonium, Farolidine, cephalothin or cephalosporin, cephalexin, cephaloglycin , cefamandole, cephapirin, cefatrizine, cefazaflur, cefazedone, Fazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminoc cefoxitin, cefprozil, cephalosporins, ceftezole, cefuroxime, Fixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, Cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefo Thiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome , cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, ceftera ceftibuten, ceftiolene, ceftizoxime, ceftazidime, ceftriaxone, Cefuroxime, cefazolin furan, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephems (flomoxef, latamoxef); f) glycopeptides : Bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin, cubicin; g) glycylcyclines: e.g. , tigecycline; h) β-lactamase inhibitors: penams (sulbactam, tazobactam ), clavams (clavulanic acid); i) lincosamides: clindamycin, lincomycin j) Lipopeptides: Daptomycin, A54145, calcium-dependent antibiotics ( CDA);k) Macrolides: azithromycin, cethromycin, clarithromycin dirithromycin, erythromycin, flurithromycin, josamycin, keto Lyde (telithromycin, ethethromycin), midecamycin, miocamycin, Leandomycin, rifamycin (rifampicin, rifampin, rifabutin, Rifapentine), rokitamycin, roxithromycin, spectinomycin, spiramycin mycin, tacrolimus (FK506), troleandomycin, telithromycin; l) Monobactams: Aztreonam, Tigemonam; M) Oxazolidinones: Linezoli N) Penicillins: Amoxicillin, Ampicillin (Pivampicillin, Hetacillin, Bacampicillin, methampicillin, talampicillin), azidocillin, azlocillin, Penicillin, benzathine penicillin, phenoxybenzathine penicillin, clometocillin, Procaine penicillin, carbenicillin (calindacillin), cloxacillin, diclofenac Saccharin, cephalosporins, flucloxacillin, mecillinam (pivmecillinam), Dulocillin, methicillin, nafcillin, oxacillin sodium, feneticillin, penicillin Cillin, pheneticillin, penicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) polypeptides: bacitracin, polymyxin E, polymyxin B Myxin B; p) Quinolones: Alatrofloxacin, Balofloxacin, Ciprofloxacin Sasin, clinafloxacin, danofloxacin, difloxacin, enoxacin, en Lofloxacin, ofloxacin, garenoxacin, gatifloxacin, gemifloxacin Grepafloxacin, Kano trovafloxacin, levofloxacin, lomefloxacin Sacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, gre Pafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin q) Streptozotocins: Pristinamycin, Quinu Pristine / dalfopristine; r) sulfonamides: mafenide, prontosil, Sulfacetamide, sulfamethoxazole, sulfanilamide, sulfasalazine, Lupafurazol, trimethoprim, trimethoprim-sulfamethoxazole (Cotri Moxazole; s) steroidal antibacterial agents: e.g., fusidic acid; t) tetracycline Class: doxycycline, chlortetracycline, clomocycline, demeclocycline cycline, lymecycline, meclocycline, methacycline, minocycline, oxytet Lacycline, penimepicycline, rolitetracycline, tetracycline, glycine Lucyclines (e.g., tigecycline); u) Other types of antibiotics: annonacin, Arsphenamine, bacitracin, DADAL / AR inhibitors (cycloserine), dictyostatin, discodermolide, eleutherobin, epo Tiron, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isopropyl alcohol Niazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e.g., fosfomycin), nitrofurantoin, paclitaxel, platensis mycin, pyrazinamide, quinupristin / dalfopristin, rifampin (rifampin) These include, but are not limited to, tazobactam tinidazole, and uvaricin.

[0139] 4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, vicriviroc b) Ink, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab); Tegrase inhibitors: raltegravir, elvitegravir, globoidin A; c) maturation inhibitors Antineoplastic agents: Bevirimat, Vivicon; d) Neuraminidase inhibitors: Oseltamivir, Zanthamivir Namivir, Peramivir; e) Nucleosides and Nucleotides: Abacavir, Acyclovir , adefovir, amdoxovir, apricitabine, brivudin, cidofovir, Kleb DDI, dexeruvucitabine, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'- Fluoro-substituted 2',3'-deoxynucleoside analogs (e.g., 3'-fluoro-2' ,3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanidine Anosine (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine ( 3TC), L-nucleosides (e.g., β-L-thymidine, β-L-2'-deoxycysteine, djin), penciclovir, rasibir, 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, efavirdine Virenz, foscarnet (phosphoryl formate), imiquimod, interferon alpha, Robinson Lidl, rhodenosine, methisazone, nevirapine, NOV-205, peginterferon alpha , podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), Lomantadine; g) Protease inhibitors: Amprenavir, Atazanavir, Boceprevir , darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, prednisolone Conaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir;h )Other types of antiviral drugs: Abzyme, Arbidol, Calanolide A, Seragenin, Cyanovirin-N, DAPY, epigallocatechin gallate (EGCG), foscarnet , Griffithsin, taribavirin (viramidine), hydroxycarbamide, KP-146 1, Pleconaril, portmanteau inhibitors, ribavirin, seliciclib.

[0140] 5) Drugs used for conjugation via the bridge linkers of the present invention 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 , 125 I, 131 I, 133 Xe, 177 Lu, 211 At, and 213 Bi mentioned Radiolabeled antibodies are very useful in receptor-targeted imaging experiments, or For example, the invention of antibody-drug conjugates (Wu et al. (2005) Nature Biotechnology 23(9):1137 -1146), which can be used for direct relative targeting therapy, cell-binding molecules, e.g. The antibody may be conjugated, chelated, or otherwise complexed with a cross-linker of the present invention, as described above. This labeling technique is described in the Current Protocol. s in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York k, NY, Pubs. (1991). Chelating agents capable of forming complex metal complexes DOTA, DOTP, DOTMA, DTPA, and TETA (Macrocyclics, Dallas, TX) , Tex.).

[0141] 6) Pharmaceutically acceptable salts, acids, or derivatives of any of the above drugs.

[0142] In another embodiment, the drugs of formula (II) and (IV) bind to cell-binding molecules and target cells. Chromophore molecules that can be conjugated to detect, monitor, or study interactions Chromophore molecules can emit light such as UV light, fluorescent light, IR light, near-IR light, and visible light. A compound that has the ability to absorb certain types of light; chromophore molecules are found in xanthophores, erythrophores, including the classes or subclasses of iridophores, leucophores, melanophores, cereuphores, and fluorophores; The phosphors are fluorescent chemicals that re-emit light in light; Class or subclass of luminescent molecules; Class or subclass of luminescent molecules subclass; a class or subclass of luciferin compounds.

[0143] 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.

[0144] 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).

[0145] 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.

[0146] 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+ )), SNARF(pH6 / 9), Allophycocyanin (APC), AmCyan1 (tetramer, Clontech), AsRed2 ( Tetramer, Clontech), Azami Green (monomer, MBL), azurite, B-phycoe Lithrin (BPE), Cerulean, CyPet, DsRed monomer (Clontech), DsR ed2 (“RFP”, Clontech), EBFP, EBFP2, ECFP, EGFP (weak dimeric 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, K usabira Orange (monomer, MBL), mCFP, mCherry, mCit rine, Midoriishi Cyan (dimer, MBL), mKate (TagFP6 35, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOran ge, mPlum, mRaspberry, mRFP1 (monomer, Tsien Lab) , mStrawberry, mTFP1, mTurquoise2, P3 (Phycobilizo Peridinin Chlorophyll (PerCP), R-Philips Coerythrin (RPE), T-Sapphire, TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP ( dimer, Evrogen), tdTomato (tandem dimer), Topaz, TurboFP 602 (dimer, Evrogen), TurboFP635 (dimer, Evrogen), TurboG FP (dimer, Evrogen), TurboRFP (dimer, Evrogen), TurboYFP6 35 (dimer, Evrogen), Venus, native GFP, YPet, Zs Green 1 (tetramer) Zs Yellow 1 (tetramer, Clontech).

[0147] In another embodiment, the drugs in formula (II) and (IV) when administered to a mammal It is a polyalkylene glycol used to extend the half-life of cell-binding molecules in Polyalkylene glycols include, but are not limited to, poly(ethylene glycol) Poly(ethylene oxide), poly(propylene glycol), and ethylene oxide and propylene Particularly preferred is PEG, and even more particularly preferred is a copolymer with PEG and PEG-10. The hydroxyPEG is a monofunctionally activated hydroxyPEG (e.g., hydroxyPEG-monoca Carboxylic acid, hydroxyPEG-monoaldehyde, hydroxyPEG-monoamine, hydroxy HydroxyPEG-monohydrazide, HydroxyPEG-monocarbazate, HydroxyPEG -monoiodoacetamide, hydroxyPEG-monomaleimide, hydroxyPEG-monoiodoacetamide Orthopyridyl disulfide, hydroxyPEG-monoxime, hydroxyPEG- Monophenyl carbonate, hydroxy PEG-monophenyl glyoxal, hydroxy HydroxyPEG-monothiazolidine-2-thione, HydroxyPEG-monothioester, HydroxyPEG-monothiazolidine-2-thione HydroxyPEG-monothiol, hydroxyPEG-monotriazine, and hydroxyPEG G - Single terminal activated hydroxyl containing reactive ester of monovinyl sulfone PEG.

[0148] In certain embodiments, the polyalkylene glycol has a molecular weight of from about 10 Daltons to about 200 kDa. , preferably having a molecular weight of about 88 Da to about 40 kDa; The molecular weight of the two more preferred branches is about 88 Da to about 40 kDa. In one particular embodiment, the polyalkylene glycol is poly (ethylene) glycol, a molecule of about 10 kDa, about 20 kDa, or about 40 kDa In certain embodiments, the PEG is PEG 10 kDa (linear or branched), PE PEG 20kDa (linear or branched), or PEG 40kDa (linear or branched). Many US patents describe linear or branched "non-antigenic" PEG polymers and their derivatives or The preparation of conjugates has been disclosed, for example, in U.S. Patents 5,428,128; 5,621,039; 5,622,988. 6; 5,643,575; 5,728,560; 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; 5,9 69,040; 5,981,709; 6,011,042; 6,042,822; 6,113,906; 6,127,355; 6,132,713; 6,177, 087, and 6,180,095. Conjugation of antibodies to polyalkylene glycols via this bridge linker An example of a body structure is PG01, as shown below.

[0149] [ka]

[0150] where mAb is an antibody; n is 1 to 30; R' and R'' are independently H or C. H3. m3 and m4 are independently 0 to 5000. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). R4 is OH, H, or R1 or R3 as defined in formula (I).

[0151] In yet another embodiment, a compound suitable for conjugation to a cell-binding molecule via a bridge linker of the present application is Preferred cytotoxic agents are tubulysins, maytansinoids, taxanoids (taxanoids), compounds), CC-1065 analogs, daunorubicin and doxorubicin compounds, benzodiazepines Zepine dimers (e.g., pyrrolobenzodiazepines (PBDs), tomaymycin, anthraquinones, mycin, indolinobenzodiazepines, imidazobenzothiadiazepines, or oxazolidinone Dimers of zolidinobenzodiazepines, calicheamicins and enediyne antibiotics , actinomycin, azaserine, bleomycin, epirubicin, tamoxifen , idarubicin, dolastatins, auristatins (e.g., monomethyl auristatin Auristatin E, MMAE, MMAF, Auristatin PYE, Auristatin TP, Auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP), duocalmycin amines, thiotepa, vincristines, hemiasterins, nazumamides ides), microginins, radiosumins sumins, alterobactins, microsclero Dermicins (microsclerodermins), theonellamides (theon ellamides, esperamicins, PNU-15 9682, and their analogs and derivatives.

[0152] 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. Fries father, 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.). with cell-binding molecules Preferred structures of tubulysins for use in the treatment of cancer are described in PCT / IB2012 / 053554 is described in.

[0153] An example of the structure of an antibody-tubulysin analog conjugate via this bridge linker is shown below. These include T01, T02, T03, T04, T05, T06, and T07 below.

[0154] [ka] TIFF2026041725000033.tif177169

[0155] where mAb is an antibody. Z3 and Z'3 are independently H, OP(O)(OM1)( OM2), OCH2OP(O)(OM1)(OM2), OSO3M1, R1, or O-dimer Glycosides (glucosides, galactosides, mannosides, glucuronosides, alosides, fructosides) M1 and M2 are independently selected from the group consisting of NH-glycoside, S-glycoside, and CH2-glycoside. The elements are H, Na, K, Ca, Mg, NH4, and NR1R2R3. n is 1 to 30. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). That is correct.

[0156] Calicheamicin and related enzymes are preferred in the cell-binding molecule-drug conjugates of the present application. Diyne antibiotics are described in the following literature: Nicolaou, KC et al, Science 1 992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. 1993, 90, 5881-5888), US Pate nt Nos. 4,970,198; 5,053,394; 5,108,912; 5,264,586; 5,384,412; 5,606,040; 5,712, 374; 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 5,877,296; 6,015,562 6,124,310; 8,153,768. Conjugation of antibody-calicheamicin analogs via this bridge linker An example of a body structure is C01 as follows:

[0157] [ka]

[0158] In the formula, mAb is an antibody, and n is 1 to 30. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). That is correct.

[0159] 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 M01. It can be obtained.

[0160] [ka]

[0161] In the formula, mAb is an antibody, and n is 1 to 30. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). That is correct.

[0162] Paclitaxel (Taxol) is preferred for conjugation via the bridge linkers of this patent. Taxanes, cytotoxic natural products including docetaxel (Taxane), and their semisynthetic derivatives The tails, as well as their analogues, are described in K. C. Nicolaou et al. l., J. Am. Chem. Soc. 117, 2409-2420, (1995); Ojima et al, J. Med. Chem. 39:38 89-3896 (1996); 40:267-278 (1997); 45, 5620-5623 (2002); Ojima et al., Pro c. Natl. Acad. Sci., 96:4256-4261 (1999; Kim et al., Bull. Korean Chem. Soc., 2 0, 1389-1390 (1999); Miller, et al. J. Med. Chem., 47, 4802-4805(2004); United States Patent Numbers: 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.

[0163] An example of the structure of an antibody-taxane conjugate via a bridge linker is Tx01 below: Tx02, and Tx03.

[0164] [ka]

[0165] In the formula, mAb is an antibody, and n is 1 to 30. [ka] X1, X2, R1, and R2 are the same as defined in formulas (I) and (II). do.

[0166] CC-1065 analogs and Duocarmycin analogs are also Also preferred for use in conjugation with the bridge linkers of the present invention. CC-1065 analogs and Examples of duocarmycin analogues, as well as 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 via bridge linkers Examples of the structures of the antibody-CC-1065 analog conjugates are CC01 and CC0 2, and CC03.

[0167] [ka]

[0168] In the formula, mAb is an antibody, n is 1 to 30, Z4 and Z'4 are independently H, P O(O)(OM1)(OM2), CH2PO(OM1)(OM2), SO3M1, CH3 N(CH2CH2)2NC(O)-, O(CH2CH2)2NC(O)-, or glycoside X3 and X'3 are independently O, NH, NHC(O), OC(O), C(O)O, R1, or absent. [ka] X1, X2, R1, R2, M1, and M2 are as defined in formulas (I) and (II). It is the same as:

[0169] Daunorubicin / doxorubicin analogs are also suitable for conjugation via the bridge linkers of this patent. Its preferred structure and synthesis method are exemplified below: Hurwitz, E., et al. ., Cancer Res. 35, 1175-1181 (1975). Yang, HM, and Reisfeld, RA, Proc. N atl. Acad. Sci. 85, 1189-1193 (1988); Pietersz, CA, E., et al., E., et al. ," Cancer Res. 48, 926-9311 (1988); Trouet, et al., 79, 626-629 (1982); Z. B rich et al., J. Controlled Release, 19, 245-258 (1992); Chen et al., Syn. Com m., 33, 2377-2390, 2003; King et al., Bioconj. Chem., 10, 279-288, 1999; King et al. al., J. Med. Chem., 45, 4336-4343, 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, 1305-1312, 1995; Scott et al., Bioorg. Med.l Chem. Lett. 6, 1491 -1496; 1996; Watanabe et al., Tokai J. Experimental Clin. Med. 15, 327-334, 1990 ; Zhou et al., J. Am. Chem. Soc. 126, 15656-7, 2004; WO 01 / 38318; US Patent No. 5, 106,951; 5,122,368; 5,146,064; 5,177,016; 5,208,323; 5,824,805; 6,146,658; 6,214 ,345; 7569358; 7,803,903; 8,084,586; 8,053,205. Antibody-CC-1 via cross-linking Examples of the conjugate structure of 065 analogues are Da01, Da02, Da03, and Da04 is an example.

[0170] [ka]

[0171] In the formula, mAb is an antibody, n is 1 to 30, and X3 and X'3 are independently H, O, or , NH, NHC(O), NHC(O)NH, C(O), R1, or OC(O). [ka] X1, X2, R1, and R2 are the same as defined in formulas (I) and (II). do.

[0172] Auristatins and dolastatins ins) are preferred for conjugation via the bridge linkers of the present invention. 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, p pp. 921-932 (2004); U.S. Patent Applications 11134826, 20060074008, 2006022925. U.S. Patent No. 4414205, 4753894, 4764368, 4816444, 4879278, 4943628, 4978744, 5122368, 5165923, 5169774,5286637, 5410024, 5521284, 5530097, 5554725, 5585089, 5599902, 5629197, 5635483, 5654399, 5663149, 5665860, 5708146, 5714586, 5741892, 5767236, 5767237 , 5780588, 5821337, 5840699, 5965537, 6004934, 6033876, 6034065, 6048720, 605429 7, 6054561, 6124431, 6143721, 6162930, 6214345, 6239104, 6323315, 6342219, 63422 21, 6407213, 6569834, 6620911, 6639055, 6884869, 6913748, 7090843, 7091186, 7097 840, 7098305, 7098308, 7498298, 7375078, 7462352, 7553816, 7659241, 7662387, 774 5394, 7754681, 7829531, 7837980, 7837995, 7902338, 7964566, 7964567, 7851437, 79 94135. An example of the structure of an antibody-auristatin conjugate via a bridge linker is shown below. Examples of such alloys include Au01, Au02, Au03, Au04, and Au05.

[0173] [ka]

[0174] In the formula, mAb is an antibody; n is 1 to 30; X3 and X'3 are independently CH2 , O, NH, NHC(O), NHC(O)NH, C(O), OC(O), R1, or absent X4 and X'4 are independently CH2, C(O), C(O)NH, C(O)N( R1), R1, NHR1, NR1, C(O)R1, or C(O)O. Z3 and Z' 3 independently represents H, R1, OP(O)(OM1)(OM2), NHR1, OCH2OP( OM1) (OM2), OSO3M1, or O-glycosides (glucosides, galactosides, mannitol glycosides, glucuronosides, alosides, fructosides, etc.), NH-glycosides, S-glycosides, or M1 and M2 are independently H, Na, K, Ca, Mg, NH4 , NR1R2R3. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). That is correct.

[0175] 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, and PB11.

[0176] [ka] TIFF2026041725000048.tif251156TIFF2026041725000049.tif159169

[0177] In the formula, mAb is an antibody; n is 1 to 30; X3 and X'3 are independently CH2 , O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(N R3), R1, NHR1, NR1, C(O)R1, or absent. X4 and X'4 are Independently, CH2, C(O), C(O)NH, C(O)N(R1), R1, NHR1, N R1, C(O)R1, or C(O)O. M1 and M2 are independently H, Na, K, C a, Mg, NH4, NR1R2R3. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). In addition, R1 and / or R2 may be absent.

[0178] In yet another embodiment, two or more different cytotoxic agents are delivered to cells via a bridge linker of the invention. Preferably, two or more different cytotoxic agents are conjugated to a tubulin-binding molecule. maytansinoids, maytansinoids, taxanoids (taxanes), CC-1065 analogs, Daunorubicin and doxorubicin compounds, benzodiazepine dimers (e.g., pyrrolobe Benzodiazepines (PBDs), tomaymycin, anthramycin, indolinobenzodiazepines azepines, imidazobenzothiadiazepines, or oxazolidinobenzodiazepines dimer), calicheamicin and enediyne antibiotics, actinomycin, azaserine , bleomycins, epirubicin, tamoxifen, idarubicin, dolastatins , auristatins (e.g., monomethyl auristatin E, MMAE, MMAF, auristatin Auristatin PYE, Auristatin TP, Auristatin 2-AQ, 6-AQ, EB(A EB), and EFP (AEFP), duocarmycins, thiotepa, vincristine Hemiasterins, nazumamides, microginins (m icroginins, radiosumins, alterobactins alterobactins, microscl erodermins, theonellamides, Espera Esperamicins, PNU-159682, and their analogues and derivatives thereof. An example of the structure of a conjugate containing different cytotoxic agents is shown below: Z01, Z02, Z03, Z04 , Z05, Z06, Z07, Z08, Z09, Z10, Z11, Z12, Z13, Z14 , Z15, Z16, Z17, and Z18.

[0179] [ka] TIFF2026041725000052.tif242164TIFF2026041725000053.tif255162TIFF2026041725000054.tif186164

[0180] In the formula, mAb is an antibody; n is 1 to 30; X3 and X'3 are independently CH2 , O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(N R3), R1, NHR1, NR1, C(O)R1, or absent. X4 and X'4 are Independently, H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1 , NHR1, NR1, C(O)R1, or C(O)O. M1 and M2 are independently H , Na, K, Ca, Mg, NH4, NR1R2R3. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). In addition, R1 and / or R2 may be absent.

[0181] In yet another embodiment, the cell-bound ligand or receptor may be linked via a bridge linker of the present patent. These conjugated cell-binding ligands or receptors can be conjugated to cell-binding molecules. In particular, antibody-receptor conjugates serve as targeted conductors / decoders for delivering the conjugate to malignant cells. not only act as directors, but also to modulate or co-stimulate the desired immune response, or signal In immunotherapy, cell-bound ligands or receptors can be used to alter neural signaling pathways. The receptor is TCR (T cell receptor) T cells, or CAR (chimeric antigen receptor) T cells, or Preferably, the antibody is conjugated to the B cell receptor (BCR) or to a cytotoxic cell. Ligands or receptors include, but are not limited to, folic acid derivatives (folate receptor, ovarian cancer and and other malignancies) (Low, PS et al 2008, Acc. Ch em. Res. 41, 120-129); glutamic acid urea derivatives (prostate-specific membrane antigen, prostate cancer cells) (Hillier, SM et al, 2009, Cancer Res. 69, 6932-6940) Somatostatin (growth hormone-inhibiting hormone (GHIH), somatotropin release inhibitor) somatotropin-releasing inhibitor (SRIF), also known as somatotropin-releasing hormone (SRIF) and its Their derivatives, such as octreotide (Sandostatin) and lanreotide (Somatulin) ) (especially neuroendocrine tumors, GH-producing pituitary adenomas, paragangliomas, dysfunctional pituitary adenomas, brown chromocytoma) (Ginj, M., et al, 2006, Proc. Natl. Acad. Sci. USA 103, 16436-1644 1); specific aromatic sequences specific for carbonic anhydrase IX (a marker for hypoxia and renal cell carcinoma) Sulfonamide (Neri, D., et al, Nat. Rev. Drug Discov. 2011, 10, 767-777); brown Pituitary adenylate cyclase-activating peptide (PAC) for carcinoma and paraganglioma tumors AP) (PAC1); for cancers of the lung, stomach, colon, rectum, breast, prostate, pancreatic duct, liver, and bladder Vasoactive intestinal peptide (VIP / PACAP) (VPAC1, VCAP2); Cholecystokinin (CCK) for cell lung cancer, medullary thyroid cancer, astrocytoma, and ovarian cancer (CCK1 (formerly CCK-A) and CCK2; renal cell carcinoma, breast cancer, lung cancer, gastric cancer, and pre- Bombesin (Pyr- for prostate cancer and neuroblastoma) Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-H is-Leu-Met-NH2) / gastrin-releasing peptide GRP receptor subtypes (BB1, 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 Neurotensin (NTR1, NTR2, NTR3) for pancreatic, and colon cancer; Substance P (NK1 receptor) for glial tumors; neuropeptide Y (Y1) for breast cancer -Y6; RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), receptors on the tumor surface (in Dimeric and multimeric cyclic RGD peptides (e.g., cRGDfV) that recognize tegrin ( Laakkonen P, Vuorinen K. 2010, Integr Biol (Camb). 2(7-8): 326-337; Chen K, Chen X. 2011, Theranostics. 1:189-200; Garanger E, et al, Anti-Cancer Agents Med Chem. 7 (5): 552-558; Kerr, JS et al, Anticancer Research, 19(2A), 959-9 68; Thumshirn, G, et al, 2003 Chem. Eur. J. 9, 2717-2725), and TAASGV RSMH and LTLRWVGLMS (chondroitin sulfate proteoglycan NG2 receptor ) and F3 peptide (a 31 amino acid peptide that binds to cell surface-expressed nucleolin receptors) )(Zitzmann, S., 2002 Cancer Res., 62, 18, pp. 5139-5143, Temminga, K., 2005, D rug Resistance Updates, 8, 381-402; P. Laakkonen and K. Vuorinen, 2010 Integrat ive Biol, 2(7-8), 326-337; MA Burg, 1999 Cancer Res., 59(12), 2869-2874; K. Porkka, et al 2002, Proc. Nat. Acad. Sci. USA 99(11), 7444-9) Cell-penetrating peptides (CPPs) (Nakase I, et al, 2012, J. Control Re lease. 159(2),181-188); peptide hormones, e.g., testosterone production, as well It acts by targeting follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, and Gonadotropin-releasing hormone (GnRH) agonists, such as buserelin (Pyr-His-Trp-Se r-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-NHEt), gonadorelin (Pyr-His-Trp-Ser-Tyr-Gly-Le u-Arg-Pro-Gly-NH2), goserelin (Pyr-His-Trp-Ser-Tyr-D-Ser(OtBu)-Leu-Arg-Pro-A zGly-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 (Py r-His-Trp-Ser-Tyr-2Nal-Leu-Arg-Pro-Gly-NH2), Triptorelin (Pyr-His-Trp-Ser-Ty rD-Trp-Leu-Arg-Pro-Gly-NH2), nafarelin, deslorelin, abarelix (Ac-D- 2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Ser-(N-Me)Tyr-D-Asn-Leu-isopropylLys-P ro-DAla-NH2), Cetrorelix (Ac-D-2Nal-D-4-chloroPhe-D-3-(3-pyridyl)Ala-Se r-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2), degarelix (Ac-D-2Nal-D-4-chloroPhe-D-3 -(3-pyridyl)Ala-Ser-4-aminoPhe(L-hydroorotyl)-D-4-aminoPhe(carba-moyl)-Leu -isopropylLys-Pro-D-Ala-NH2), and ganirelix (Ac-D-2Nal-D-4-chloroPhe-D-3- (3-pyridyl)Ala-Ser-Tyr-D-(N9, N10-diethyl)-homoArg-Leu-(N9, N10-diethyl)-h omoArg-Pro-D-Ala-NH2)(Thundimadathil, J., J. Amino Acids, 2012, 967347, doi:10 .1155 / 2012 / 967347; Boccon-Gibod, L.; et al, 2011, Therapeutic Advances in Urolo gy 3 (3): 127-140; Debruyne, F., 2006, Future Oncology, 2(6), 677-696); and for example, small molecules (imiquimod, guanidine and adenosine analogues) to lipopolysaccharides. (LPS), nucleic acids (CpG DNA, poly I;C) and lipopeptides (Pam3CSK4 )(Kasturi, SP, et al, 2011, Nature 470, 543-547; Lane, T., 2001, JR Soc Med. 94, 316; Hotz, C., and Bourquin, C., 2012, Oncoimmunology 1, 227-228; Dud (K, AZ, et al, 2007, Clin. Cancer Res. 13, 7119-7125) Toll-like receptors (TLRs), C-type lectins, and No d-like receptors (NLRs) (Fukata, M., et al, 2009, Semin. Immunol. 21, 242-2 53; Maisonneuve, C., et al, 2014, Proc. Natl. Acad. Sci. USA 111, 1-6; Bot os, I., et al, 2011, Structure 19, 447-459; Means, TK, et al, 2000, Life Sci. 68, 241-258) and other pattern recognition receptors (PRRs).

[0182] Cell-binding ligands or receptors are synthesized using Ig-based and non-Ig-based protein scaffolds (sc Ig-based scaffolds can be, but are not limited to, nanobodies (V HH (a derivative of camelid Ig) (Muyldermans S., 2013 Annu Rev Biochem. 82, 775-797; domain antibodies (dAb, derivatives of VH or VL domains) (Ho lt, L. J, et al, 2003, Trends Biotechnol. 21, 484-490); (BiTE, bispecific dimer) (Baeuerle, P. A, et al, 2009, Curr. Opin. Mol . 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 (Derivatives of lipocalins) (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 B iophys 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 (10th FN3 (fibronectin)) (Koide, A, et al., 1998 J. Mol. Biol., 284(4):114 1-1151; Batori V, 2002, Protein Eng. 15(12): 1015-1020; Tolcher, A. W, 2011, Clin. Cancer Res. 17(2):363-371; Hackel, B. J, 2010, Protein Eng. Des. Sel. 2 3(4):211-219; designed ankyrin repeat proteins (DARPins) (An Derivatives of cullin repeat (AR) proteins (Boersma, YL, et al, 2011 Curr Opin Biotechnol. 22(6): 849-857), such as DARPinC9, DARPinEc4, and DARPinE69_LZ3_E01(Winkler J, et al, 2009 Mol Cancer Ther. 8(9 ), 2674-2683; Patricia MK. M., et al, Clin Cancer Res. 2011;17(1):100-110; B oersma Y. L, et al, 2011 J. Biol. Chem. 286(48),41273-41285); Avimer (Av imer) (domain A / low-density lipoprotein (LDL) receptor) (Boersma Y. L, 20 11 J. Biol. Chem. 286(48): 41273-41285; Silverman J, et al, 2005 Nat. Biotechn ol., 23(12):1556-1561).

[0183] An example of the structure of an antibody-cell binding ligand or receptor conjugate via a bridge linker is shown below: As follows: LB01 (PMSA ligand conjugate), LB02 (folate receptor conjugate), L B03 (somatostatin receptor conjugate), LB04 (octreotide, somatostatin analogs) somatostatin analog receptor conjugate), LB05 (lanreotide, somatostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07 (CAIX receptor conjugate), LB08 (yellow Somatogenic hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 ( Luteinizing hormone-releasing hormone (LH-RH) and GnRH ligand conjugate), LB10 (GnRH antagonist, abarelix conjugate), LB11 (cobalamin, VB12 analogue conjugate), LB12 (gastrin-releasing peptide receptor (GRPr), MBA conjugate ), LB13(α v β3 integrin receptor, cyclic RGD pentapeptide conjugate), LB 14 (hetero-bivalent peptide ligand of VEGF receptor conjugate), LB15 (Neuromed LB16 (G protein-coupled receptor, bombesin conjugate), and LB17 (Toll-like receptor, TLR2 conjugate).

[0184] [ka] TIFF2026041725000057.tif232169TIFF2026041725000058.tif215165TIFF2026041725000059.tif236167TIFF2026041725000060.tif49166

[0185] In the formula, mAb is an antibody; n is 1 to 30; X3 and X'3 are independently CH2 , O, NH, NHC(O), NHC(O)NH, C(O), OC(O), OC(O)(N R3), R1, NHR1, NR1, C(O)R1, or absent. X4 and X'4 are Independently, H, CH2, OH, O, C(O), C(O)NH, C(O)N(R1), R1 , NHR1, NR1, C(O)R1, or C(O)O. M1 and M2 are independently H , Na, K, Ca, Mg, NH4, NR1R2R3. m3 and m4 are 0 to 5000 is. [ka] X1, X2, R1, R2, and R3 are as defined in formulas (I) and (II). In addition, R1 and / or R2 may be absent.

[0186] The drugs / cytotoxic agents used for conjugation via the bridge linkers of the present invention are It may be any analogue and / or derivative of the indicated drug / cytotoxic agent. Those skilled in the art will readily appreciate that the specificity and / or activity of the starting compound may be maintained under conditions that preserve the specificity and / or activity of the starting compound. It will be readily understood that each drug / cytotoxic agent described herein can be modified. Those skilled in the art will recognize that many of these compounds may be substituted for the drugs / cytotoxic agents described herein. It is also understood that the drugs / cytotoxic agents of the present invention may be analogs of the above compounds. and derivatives.

[0187] All references cited in the specification and examples that follow are expressly incorporated by reference in their entirety. It can be enjoyed. [Example]

[0188] 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 from Thailand, USA, unless otherwise specified. American College of Microbiology and Cell Culture Collection (ATCC), German Collection of Microbial Cell Cultures (DSMZ) ), or maintained in culture medium based on the conditions specified by the Shanghai Institute of Cell Culture, Chinese Academy of Sciences. Unless otherwise stated, all cell culture reagents were provided by Invitrogen. All anhydrous reagents were commercially available and stored in nitrogen-filled sealed bottles. Chemicals and solvents were purchased according to the highest standards and used without further purification. Separation was performed using a Varain PreStar HPLC. NMR spectra The chemical shifts (Δ ) is in ppm, tetramethylsilane is used as the standard at 0.00, and the unit of the coupling constant (J) is Mass spectrometry data was obtained using a Waters Acquity UPLC separator and Waters Xevo QTOF mass spectrometer equipped with an Acquity TUV detector Retrieved from.

[0189] Example 1: tert-butyl 3-(2-(2-hydroxyethoxy)ethoxy)propanoate Lu (84) [ka]

[0190] Add 80 mg (0.0025 mol) of sodium hydroxide to 350 ml of anhydrous THF with stirring. Add sodium and triethylene glycol (83) (150.1 g, 1.41 mol) After the metallic sodium was completely dissolved, tert-butyl acrylate (24 ml, 0. 33 mol) was added. The solution was stirred at room temperature for 20 hours and then neutralized with 8 mL of 1.0 M HCl. The solvent was removed under reduced pressure and the residue was suspended in brine (250 ml) and ethyl acetate (3× The combined organic layers were extracted with brine (100 ml) and then water (100 ml). ), dried over sodium sulfate, and the solvent was removed. The resulting colorless oil was Drying gave 60.27 g of product (84) (78% yield). 1 H NMR: 1.4 1(s,9H),2.49(t,2H,J=6.4Hz),3.59-3.72(m,1 0H);ESI MS m / z- C 11 H21 O5(MH), calculated value 233.15, Actual value: 233.40.

[0191] Example 2: 3-(2-(2-(tosyloxy)ethoxy)ethoxy)propanoic acid tert -Butyl (85) [ka]

[0192] A solution of compound (84) (10.0 g, 42.70 mmol) in dichloromethane (50.0 ml) was treated with pyridine (20.0 ml). A solution of 5.81 mmol) in dichloromethane (50 ml) was added dropwise through a funnel over 30 minutes. After 5 hours, TLC analysis revealed the reaction was complete. The pyridine hydrochloride formed was filtered off and the solvent was removed. The residue was purified by elution with 20% ethyl acetate in hexane containing ethyl acetate, and 1 0.39 g of compound (85) was obtained (yield 76%). 1 H NMR: 1.40 (s, 9H ),3.23(s,3H),2.45(t,2H,J=6.4Hz),3.54-3.7 0 (m, 10H); ESI MS m / z + C 12 H 25 O7S(M+H), calculated value 31 3.10, actual value 313.30.

[0193] Example 3: tert-Butyl 3-(2-(2-azidoethoxy)ethoxy)propanoate (86) [ka]

[0194] In 50 mL of DMA, add 3-(2-(2-(mesyloxy)ethoxy)ethoxy)ethoxy (c) tert-Butyl propanoate (85) (4.0 g, 12.81 mmol) and azide Sodium (0.90 g, 13.84 mmol) was added with stirring. After 4 hours, TLC analysis revealed the reaction was complete. The reaction solution was cooled to room temperature and quenched with water (25 mL). The aqueous layer was separated and The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and Concentrate under reduced pressure and eluate on silica gel with pure ethyl acetate in 15% ethyl acetate in hexane. The mixture was purified by elution with hexane to give 2.88 g of compound (86) (yield 76%). 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,8H).ESI MS m / z+C 11 H 22 N3O7(M+H), calculated value 260.13, found value 260 .20.

[0195] Example 4: 3-(2-(2-azidoethoxy)ethoxy)propanoic acid (87) [ka]

[0196] Azide compound (86) (2.51 g, 9.68 mmol) was dissolved in 1,4-dioxane (30 The mixture was stirred for 35 min and then dissolved in EtO (30 The mixture was diluted with methanol (5% to 10 mL) and toluene (30 mL) and removed under vacuum. %) and a mixture of 1% formic acid in methylene chloride as the eluent. The product was purified on silica gel to give the title compound (87) (1.63 g, 83% yield). SI MS m / z-C7H 12 N3O4(MH), calculated value 202.06, measured value 20 2.30.

[0197] Example 5: 2,5-Dioxopyrrolidin-1-yl 3-(2-(2-azidoethoxy) Ethoxy)propanoate (88). [ka]

[0198] To compound (87) (1.60 g, 7.87 mmol) in 30 mL of dichloromethane, Add NHS (1.08 g, 9.39 mmol) and EDC (3.60 g, 18. 75 mmol) was added. After 8 hours, TLC analysis revealed the reaction was complete. The reaction mixture was evaporated and a mixture of ethyl acetate (5%-10%) in methylene chloride was added. Purification on silica gel using as the eluent gave the title compound (88). (1.93g, yield 82%). ESI MS m / z+C 11 H 17 N4O6(M+H) ,Calculated value 301.11,Measured value 301.20.

[0199] Example 6: (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3 S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamide) Pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3 -(3-(2-(2-azidoethoxy)ethoxy)propanamido)-4-hydroxybenzoate (phenyl)-2-methylpentanoic acid (94) [ka]

[0200] Mix DMA (10 mL) and NaH2PO4 buffer (5 mL, 1.0 M, pH 7.5) (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S )-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamide)pe thiazole-4-carboxamido)-5-(3-methylpentyl)thiazole-4-carboxamido Amino-4-hydroxyphenyl)-2-methylpentanoic acid (93) (Huang Y. et al, Med Chem. #44, 249 th ACS National Meeting, Denver, CO, Mar. 22-26, 2015; WO20140 09774) (100 mg, 0.131 mmol) to compound (88) (80.0 mg, 0. 266 mmol) was added in four portions over 2 hours. The mixture was stirred overnight, concentrated, and -18 Preparative chromatography (3.0 x 25 cm, 25 ml / min) with 80% water / methyl The product was purified by elution with 20% ethanol, 10% water, and 90% methanol for 45 minutes. The title compound was obtained (101.5 mg, 82% yield). LC-MS (ESI) m / z calculation Value C 45 H 70 N9O 11 S[M+H] + :944.48, actual value 944.70.

[0201] Example 7: (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3 S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamide) Pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5-(3 -(3-(2-(2-aminoethoxy)ethoxy)propanamido)-4-hydroxybenzoate (phenyl)-2-methylpentanoic acid (95) [ka]

[0202] Compound (94) (10 mL) in 0.1% HCl in methanol (25 mL) in a hydrogenation vessel 0.0 mg, 0.106 mmol) and Pd / C (25 mg, 10% Pd, 50% wet) After evacuating the air from the vessel, hydrogen gas was introduced at 35 psi. The mixture was shaken for 2 hours, filtered through Celite, concentrated, and purified by C-18 preparative HPLC (3.0×25 cm, 25 ml / min) 85% water / 15% methanol to 10% water / 90% methanol The mixture was purified by elution with hexane for 45 minutes to give the title compound (101.5 mg, yield 8 2%). LC-MS(ESI) m / z calculation value C 45 H 72 N7O 11 S[M+H] + :9 18.49, actual value 918.60.

[0203] Example 8: 4-(benzyloxy)-3-methoxybenzoic acid [ka]

[0204] Ethanolic acid of 4-hydroxy-3-methoxybenzoic acid (50.0 g, 297.5 mmol) In a mixture of ethanol (350 mL) and NaOH solution (2.0 M, 350 mL), BnBr (140.0 g, 823.5 mmol) was added. The mixture was stirred at 65° C. for 8 hours and concentrated. Coevaporate with water (2 x 400 mL) to approximately 400 mL and adjust to pH 3.0 with 6 M HCl. The solid was filtered, crystallized with EtOH, dried under vacuum at 45° C., and the title compound was obtained. A compound was obtained (63.6g, yield 83%). ESI MS m / z+ 281.2(M+N a).

[0205] Example 9: 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid [ka]

[0206] 4-(benzyloxy)-3-methoxybenzoic acid (63.5 g, 246.0 mmol) To a mixed solution of CH2Cl2 (400 mL) and HOAc (100 mL), Smoke, 25.0 mL, 528.5 mmol) was added. The mixture was stirred for 6 h, concentrated, and Crystallization from HCl and drying under vacuum at 40°C gave the title compound (63.3 g, yield rate 85%). ESI MS m / z+ 326.1(M+Na).

[0207] Example 10: (2S,4R)-4-Hydroxypyrrolidine-2-carboxylic acid methyl ester Hydrochloride [ka]

[0208] Dried trans-4-hydroxy-L-proline (15.0 g, 114.3 mmol) Thionyl chloride (17 mL, 231 mmol) was dissolved in methanol (250 mL) at 0-4°C. The resulting mixture was stirred at room temperature overnight, concentrated, and rehydrated with EtOH / hexane. Crystallization afforded the title compound (18.0 g, 87% yield). ESI MS m / z+ 168.2(M+Na).

[0209] Example 11: (2S,4R)-1-tert-butyl 2-methyl 4-hydroxypyrrolidone Lysine-1,2-dicarboxylate [ka]

[0210] trans-4-hydroxy-L-proline methyl ester (18.0 g, 107.0 m mol) in MeOH (150 mL) and sodium bicarbonate solution (2.0 M, 350 mL) To the mixed solution, (BOC)2O (30.0 g, 137.6 mmol) was added in three portions for 4 hours. After stirring for an additional 4 h, the reaction was concentrated to approximately 350 mL and The combined organic layers were washed with brine (100 mL), dried (M gSO4), filtered, concentrated, and purified by SiO2 chromatography (1:1 hexane / EtOA Purification by ESI MS c) gave the title compound (22.54 g, 86% yield). m / z+ 268.2 (M+Na).

[0211] Example 12: (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine-1, 2-dicarboxylate [ka]

[0212] The title compound was prepared according to the method described in Franco Manfre et al. J. Org. Chem. 1992, 57, 2060-2065. The synthesis was carried out by the Ess-Martin oxidation. The Swern oxidation procedure is as follows: (COCl)2 (13.0 mL, 74.38 mmol) in CHCl2 (35 The solution was cooled to -78°C and dry DMSO (26.0 mL) was added. Stir at 8°C for 15 minutes, then add (2S,4R)-1-tert-butyl 2-methyl 4 -hydroxypyrrolidine-1,2-dicarboxylate (8.0 g, 32.63 mmol) A solution of 100 mL of triethyl methyl ether in CH2Cl2 was added. After stirring at -78°C for 2 hours, Dimethylamine (50 mL, 180.3 mmol) was added dropwise and the solution was allowed to warm to room temperature. The solution was diluted with NaH2PO4 (400 mL, 1.0 M) and separated. The aqueous layer was (2 x 60 mL). The organic layers were combined, dried over MgSO4, filtered, and concentrated. , purified by SiO2 chromatography (7:3 hexane / EtOAc) to give the title compound. The compound was obtained (6.73g, yield 85%). ESI MS m / z+266.2(M+N a).

[0213] Example 13: (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1 ,2-dicarboxylate [ka]

[0214] At 0°C, methyltriphenylphosphonium bromide (19.62 g, 55.11 mmol) To a THF solution (150 mL) of 1.2g of potassium t-butoxide (6.20 g, 55.30 After stirring at 0°C for 2 hours, the obtained The yellow ylide suspension was treated with (S)-1-tert-butyl 2-methyl 4-oxopyrrolidine A solution of 4-methyl-1,2-dicarboxylate (6.70 g, 27.55 mmol) in THF 0 mL) was added. After stirring at room temperature for 1 h, the reaction mixture was concentrated and Dilute with 1 L of water, wash with H2O (150 mL) and brine (150 mL), and add MgSO4 The solids were dried and purified by flash chromatography on SiO2 (9:1 hexane / EtOAc). The title compound was obtained by filtration and purified using a HPLC with HPLC (5.77 g, 87% yield). EIMS m / z +264 (M+Na).

[0215] Example 14: (S)-Methyl 4-methylenepyrrolidine-2-carboxylate [ka]

[0216] At 4°C, (S)-1-tert-butyl 2-methyl 4-methylenepyrrolidine-1, 2-dicarboxylate (5.70 g, 23.63 mmol) in 40 ml of EtOAc To the resulting solution was added HCl (10 mL, 12 M). The mixture was stirred for 1 hour and toluene (5 0 mL), concentrated, and crystallized with EtOH / hexane to give the title compound as the HCl salt. (3.85 g, 92% yield). EIMS m / z +142.2 (M+H).

[0217] Example 15: (S)-Methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrilo (benzoyl)-4-methylenepyrrolidine-2-carboxylate [ka]

[0218] A catalytic amount of DMF (30 μl) was added to 4-(benzyloxy)-5- Methoxy-2-nitrobenzoic acid (2.70 g, 8.91 mmol) and oxalyl chloride ( 2.0 mL, 22.50 mmol) and the resulting mixture was stirred at room temperature (RT) for 2 h. Excess CH2Cl2 and oxalyl chloride were removed on a rotary evaporator. The acyl chloride was resuspended in fresh CH2Cl2 (70 mL) and stirred for 4 h at 0 °C under an argon atmosphere. - methylene-L-proline methyl ester HCl salt (1.58 g, 8.91 mmol), A solution of Et3N (6 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirring was continued for 8 h. After removing CH2Cl2 and Et3N, the residue was diluted with H2O and EtOAc (70 / 70 mL). The aqueous layer was further extracted with EtOAc (2 x 60 mL). The combined organic layers The residue was washed with brine (40 mL), dried (MgSO4), and concentrated. by purification by chromatography (silica gel, 2:8 hexane / EtOAc) , (S)-methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl (4-methylpyrrolidine-2-carboxylate) was obtained (2.88 g, yield 76. 1%). EIMS m / z +449.1([M] + +Na).

[0219] Example 16: (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl) (yl)-4-methylenepyrrolidine-2-carbaldehyde [ka]

[0220] Vigorous stirring of (S)-methyl 1-(4-benzyloxy)-5-methoxy-2- Nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate (2.80 g, 6 A solution of 0.57 mmol of HCl in anhydrous CHCl (60 mL) was added to -7 DIBAL-H (10 mL of 1 M CH2Cl2 solution) was added dropwise at 8 °C. After stirring for 90 min, the reaction was diluted with 2 mL of methanol followed by the addition of 5% HCl (10 mL). The excess reagent was decomposed. The resulting mixture was warmed to 0° C. The layers were separated and the aqueous layer was further diluted with CH The combined organic layers were washed with brine and dried (M gSO4), concentrated, and purified by flash chromatography (silica gel, 95:5 CHCl The residue was purified with 5-(4-benzyloxy)-5-methoxybenzoate (MeOH) to give (S)-1-(4-benzyloxy)-5-methoxybenzoate. (2-nitrobenzoyl)-4-methylenepyrrolidine-2-carbaldehyde was obtained. 0.19g, 84% yield. EIMS m / z +419.1 ([M] + +Na).

[0221] Example 17: (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3- Dihydro-1H-benzo[e]pyrrolo[1,2-a]azepin-5(11aH)-one [ka]

[0222] (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4 -methylenepyrrolidine-2-carbaldehyde (2.18 g, 5.50 mmol) and Na A solution of 2SO4 (8.0 g, 45.97 mmol) in THF (60 mL) and HO (40 m The mixture was stirred at room temperature for 20 hours. The solvent was removed under high vacuum. The residue was dissolved in MeOH (60 mL) and HCl (6 M) was added dropwise until the pH reached 2. The mixture was stirred at room temperature for 1 hour. The reaction was worked up by removing most of the MeOH. The EtOAc solution was then diluted with 100 mL of saturated aqueous NaHCO3. The residue was washed with HCl and brine, dried (MgSO4), and concentrated. The compound was purified by chromatography (silica gel, 97:3 CHCl3 / MeOH) to give (S)-8-( benzyloxy)-7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e ]pyrrolo[1,2-a]azepin-5(11aH)-one (1.52 g, 80%) EIMS m / z +372.11([M] + +Na).

[0223] Example 18: (S)-8-Hydroxy-7-methoxy-2-methylene-2,3-dihydro -1H-Benz[e]pyrrolo[1,2-a]azepin-5(11aH)-one [ka]

[0224] (S)-8-(benzyloxy)-7-methoxy-2-methylene-2,3-dihydro- 1H-Benz[e]pyrrolo[1,2-a]azepin-5(11aH)-one (1.50g , 4.32 mmol) in CH2Cl2 (70 mL) at 0 °C in CH2SO3H (25 The mixture was stirred at 0°C for 10 minutes, then at room temperature for 2 hours, and The solution was diluted and neutralized to pH 4 with cold 1.0 N NaHCO3 and filtered. The aqueous layer was diluted with CHCl The organic layers were combined, dried over Na2SO4, filtered, and evaporated. The mixture was extracted with CH3OH / CH2Cl2 (1:15) and purified by SiO2 chromatography. Purification by filtration gave 811 mg (73% yield) of the title product. EIMS m / z +28 1.1([M] + +Na).

[0225] Example 19: (11aS,11a'S)-8,8'-(pentane-1,5-diylbis( Oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e] Pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one) (97) [ka]

[0226] Stirring suspension of Cs2CO3 (0.761 g, 2.33 mmol) in butanone (8 mL) The solution was added with (S)-8-hydroxy-7-methoxy-2-methylene-2,3-dihydro-1 H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one ( 401 mg, 1.55 mmol) and 1,5-diiodopentane (240 mg, 0.74 0 mmol) was added. The mixture was stirred at room temperature overnight, concentrated, and Purification by SiO2 chromatography eluting with HCl (1:10) gave 337 mg (yield: The title product was obtained in a 78% yield. EIMS m / z +607.2 ([M] + +Na).

[0227] Example 20: (S)-7-Methoxy-8-((5-(((S)-7-methoxy-2-methyl 5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e ]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy) -2-Methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4 ]diazepin-5(11aH)-one (98) [ka]

[0228] (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy)) Bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1 ,2-a][1,4]diazepin-5(11aH)-one) (150 mg, 0.256 m mol) in anhydrous dichloromethane (1 mL) and absolute ethanol (1.5 mL), A solution of sodium borohydride in methoxyethyl ether (85 μl, 0.5 M, 0. After 5 min, the ice bath was removed and the mixture was stirred at room temperature for 3 h, then Cool to 0 °C, quench with saturated ammonium chloride, dilute with dichloromethane, and The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered through Celite, and After concentration, the residue was purified by reverse phase HPLC (C18 column, acetonitrile / water). The corresponding fractions were extracted with dichloromethane and concentrated to give the title compound (98), (S)-7- Methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3 ,5,10,11,11a-Hexahydro-1H-benzo[e]pyrrolo[1,2-a][ 1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methylene-2,3- Dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5(11a H)-one) (64.7 mg, 43%), MS m / z +609.2 (M+Na), 62 5.3 (M+K), 627.2 (M+Na+H2O); fully reduced compound, (11aS, 1 1a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy) 2-methylene-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diazepin-5(10aH)-one) (16.5 mg, 11.1 %), MS m / z+611.2(M+Na),627.2(M+K),629.2(M +Na+H2O); and unreacted starting material (10.2 mg, 6.8%), MS m / z +6 Obtained 07.2 (M+Na), 625.2 (M+Na+H2O).

[0229] Example 21: (S)-8-((5-(((S)-10-(3-(2-(2-azidoethoxy) ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo-2,3,5 ,10,11,11a-Hexahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]Diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene -2,3-Dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine- 5(11aH)-On(99) [ka]

[0230] Compound (98) (60.0 mg, 0.102 mmol) in dichloromethane (5 mL) A mixture of 100 mg of EDC and compound (88) (40.5 mg, 0.134 mmol) was added. The mixture was stirred at room temperature overnight, concentrated, and Si Purification on an O2 column eluted with EtOAc / CH2Cl2 (1:6) provided the target The title product (99) was obtained. ESI MS m / z+ C 40 H 50 N7O9(M+H), Calculated value: 772.36, measured value: 772.30.

[0231] Example 22: (S)-8-((5-(((S)-10-(3-(2-(2-aminoethoxy) ethoxy)propanoyl)-7-methoxy-2-methylene-5-oxo-2,3,5 ,10,11,11a-Hexahydro-1H-benzo[e]pyrrolo[1,2-a][1, 4]Diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methylene -2,3-Dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine- 5(11aH)-On(100) [ka]

[0232] Mix THF (5 mL) and NaH2PO4 buffer (50 mM, pH 5.0, 1 mL) Compound (99) (60 mg, 0.078 mmol) in the mixture was added to PPh3 (70 mg, 0.2 67 mmol) was added. The mixture was stirred at room temperature overnight, concentrated, and purified by C-18 chromatography. The product was purified by eluting with water / CH3CN (90% water to 35% water in 35 min). ), and after drying under high vacuum pump, 45.1 mg of the title product (100) was obtained (yield 7 9%). ESI MS m / z+ C 40 H 52 N5O9 (M+H), calculated value 746.3 7. Actual value: 746.50.

[0233] Example 23: (S)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate Ruboxylate. [ka]

[0234] Boc-L-proline (10.0 g, 46.4 mmol) dissolved in 50 mL of THF The mixture was cooled to 0° C. and a solution of BH 3 in THF (1.0 M, 46.4 mL) was added carefully. The mixture was stirred at 0° C. for 1.5 hours, then poured into ice water and extracted with ethyl acetate. Washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give a white solid The title compound (8.50 g, yield 91%) was obtained as a solid. 1 H NMR (500 MHz, CDCl3) δ 3.94 (dd, J = 4.9, 2.7 Hz, 2H), 3.60 (ddd, J = 18.7, 11.9, 9.3 Hz, 2H), 3.4 9 - 3.37 (m, 1H), 3.34 - 3.23 (m, 1H), 2.06 - 1.91 (m, 1H), 1.89 - 1.69 ( m, 2H), 1.65 - 1.51 (m, 1H), 1.49 - 1.40 (m, 9H).

[0235] Example 24: (S)-tert-butyl 2-formylpyrrolidine-1-carboxylate to [ka]

[0236] (S)-tert-Butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate To a solution of 13.0 g (64.6 mmol) of thiamin in 90 mL of dimethyl sulfoxide, Triethylamine (40 mL) was added and stirring was continued for 15 minutes. The mixture was cooled on an ice bath and Sulfotrioxide-pyridine complex (35.98 g, 226 mmol) was added over 40 min. The reaction was allowed to warm to room temperature and stirred for 2.5 hours. Ice (250 g) was added. The mixture was then extracted with dichloromethane (150 mL x 3). The organic phase was diluted with 50% citric acid. solution (150 mL), water (150 mL), saturated sodium bicarbonate solution (150 mL), and Wash with brine (150 mL) and dry over anhydrous Na2SO4. Remove the solvent in vacuo. This gave the title compound (10.4 g, 81% yield) as a dense oil. This was used without further purification. 1 H NMR (500 MHz, CDCl3) δ 9.45 (s, 1H), 4.04 (s, 1H), 3.53 (dd, J = 14.4, 8.0 Hz, 2H), 2.00 - 1.82 (m, 4H), 1.44 (d, J = 22.6 Hz, 9H).

[0237] Example 25: (4R,5S)-4-methyl-5-phenyl-3-propionyl oxazolidinyl Jin-2-On [ka]

[0238] 4-Methyl-5-phenyloxazolidin-2-one (8.0 g, 45.17 mmol) A solution of 100 mL of n-butyllithium in THF was heated at -78°C under a nitrogen atmosphere. A solution of 21.6 mL of sucrose (2.2 M, 47.43 mmol) was added. The solution was then cooled to -78 °C. Hold for 1 hour, then slowly add propionyl chloride (4.4 mL, 50.59 mmol). The reaction mixture was warmed to -50°C and stirred for 2 hours, then added to a saturated solution of ammonium chloride. The reaction mixture was quenched by the addition of acetic acid (100 mL). The organic solvent was removed under reduced pressure, and the resulting solution was diluted with acetic acid. The organic layer was extracted with ethyl acetate (3 x 100 mL). Washed with 100 mL of HCl (1 L) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (20% ethyl acetate / hexane) to give the title compound. The mixture was obtained as a dense oil (10.5 g, 98% yield). 1 H NMR (500 MHz, CDCl 3) δ 7.45 - 7.34 (m, 3H), 7.30 (d, J = 7.0 Hz, 2H), 5.67 (d, J = 7.3 Hz, 1H), 4.82 - 4.70 (m, 1H), 2.97 (dd, J = 19.0, 7.4 Hz, 2H), 1.19 (t, J = 7. 4 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H).

[0239] Example 26: (S)-tert-butyl 2-((1R,2R)-1-hydroxy-2- Methyl-3-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidine -3-yl)-3-oxopropyl)pyrrolidine-1-carboxylate [ka]

[0240] (4R,5S)-4-methyl-5-phenyl-3-propanediol in dichloromethane (60 mL) A solution of lopionyloxazolidin-2-one (9.40 g, 40.4 mmol) was added at 0°C. EtN (6.45 mL, 46.64 mmol) was added, followed by 1 M dibutylboron A solution of triflate in dichloromethane (42 mL, 42 mmol) was added. The mixture was heated to 0°C. The mixture was stirred at rt for 45 min, cooled to -70°C, and then (S)-tert-butyl 2-formylpyrrolidone was added. Dichloromethane solution of zinzyl-1-carboxylate (4.58 g, 22.97 mmol) (40 mL) was added slowly over 30 min. The reaction was stirred at -70 °C for 2 h and at 0 °C for 2 h. Stir for 1 hour and 15 minutes at room temperature, then quench with phosphate buffer (pH 7, 38 mL). MeOH-30% H2O2 (2:1, 100 mL) was added at 10°C or below, and the mixture was stirred for 20 minutes. After stirring for 1 min, water (100 mL) was added and the mixture was concentrated under reduced pressure. 0 mL) was added and the mixture was extracted with ethyl acetate (3 x 100 mL). HSO4 (100 mL), sodium bicarbonate solution (100 mL), and saline (100 m The residue was washed with HCl, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The title compound was purified by chromatography (10% to 50% ethyl acetate / hexane) to give a white solid. Obtained as a solid (7.10 g, 71% yield). 1 H NMR (500 MHz, CDCl3) δ 7.39 (dt , J = 23.4, 7.1 Hz, 3H), 7.30 (d, J = 7.5 Hz, 2H), 5.67 (d, J = 7.1 Hz, 1H) , 4.84 - 4.67 (m, 1H), 4.08 - 3.93 (m, 3H), 3.92 - 3.84 (m, 1H), 3.50 (d, J = 9.0 Hz, 1H), 3.24 (d, J = 6.7 Hz, 1H), 2.15 (s, 1H), 1.89 (dd, J = 22 .4, 14.8 Hz, 3H), 1.48 (d, J = 21.5 Hz, 9H), 1.33 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H).

[0241] Example 27: (S)-tert-butyl 2-((1R,2R)-1-methoxy-2-methyl) 3-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidine- 3-yl)-3-oxopropyl)pyrrolidine-1-carboxylate [ka]

[0242] (S)-tert-butyl 2-((1R,2R)-1-hydroxy-2-methyl-3 -((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidin-3-yl )-3-oxopropyl)pyrrolidine-1-carboxylate (5.1 g, 11.9 mm A mixture of 1000 ml of HCl (1000 ml) and molecular sieves (4 Å, 5 g) was added to anhydrous dichloromethane under a nitrogen atmosphere. Ethane (30 mL) was added. The mixture was stirred at room temperature for 20 minutes and cooled to 0°C. thiamin sponge (6.62 g, 30.9 mmol), followed by trimethyloxonium tetrahydrofuran Fluoroborate (4.40 g, 29.7 mmol) was added. The mixture was stirred at 0° C. for 2 hours and at room temperature. The reaction mixture was stirred for 48 hours, filtered, and the filtrate was concentrated and purified by column chromatography. (20-70% ethyl acetate / hexanes) to give the title compound as a colorless solid ( 1.80g, 35% yield. 1 H NMR (500 MHz, CDCl3) δ 7.46 - 7.27 (m, 5H), 5. 65 (s, 1H), 4.69 (s, 1H), 3.92 (s, 1H), 3.83 (s, 1H), 3.48 (s, 3H), 3. 17 (s, 2H), 2.02 - 1.68 (m, 5H), 1.48 (d, J = 22.3 Hz, 9H), 1.32 (t, J = 6.0 Hz, 3H), 0.91 - 0.84 (m, 3H).

[0243] Example 28: (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyridinone Roridin-2-yl)-3-methoxy-2-methylpropanoic acid [ka]

[0244] (S)-tert-Butyl 2-( (1R,2R)-1-Methoxy-2-methyl-3-((4R,5S)-4-methyl-2- Oxo-5-phenyloxazolidin-3-yl)-3-oxopropyl)pyrrolidine- 1-carboxylate (1.80 g, 4.03 mmol) was added to 30% H2O2 (1.44 A solution of LiOH (0.27 mL, 14.4 mmol) was added over 5 min at 0 °C, followed by g, 6.45 mmol) aqueous solution (5 mL) was added. After stirring at 0°C for 3 hours, 1N sulfur dioxide was added. Sodium carbonate (15.7 mL) was added and the mixture was allowed to warm to room temperature and stirred overnight. The aqueous phase was removed in vacuo and washed with dichloromethane (3 x 50 mL) to obtain the oxazolidinone co-reductant. The auxiliary was removed. The aqueous phase was acidified to pH 3 with 1N HCl and diluted with ethyl acetate (3 x 50 mL). The organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and Concentration under reduced pressure gave the title compound as a colorless oil (1.15 g, 98% yield). 1 H NMR (500 MHz, CDCl3) δ 3.99 - 3.74 (m, 2H), 3.44 (d, J = 2.6 Hz, 3H), 3. 23 (s, 1H), 2.60 - 2.45 (m, 1H), 1.92 (tt, J = 56.0, 31.5 Hz, 3H), 1.79 - 1.69 (m, 1H), 1.58 - 1.39 (m, 9H), 1.30 - 1.24 (m, 3H).

[0245] Example 29: (4S,5S)-4-((tert-butoxycarbonyl)amino)-5- Ethyl methyl-3-oxoheptanoate [ka]

[0246] (2S,3S)-2-((tert-butoxycarbonyl)amino)-3-methylpentene To an ice-cold solution of 1,1' benzoic acid (4.55 g, 19.67 mmol) in THF (20 mL), -Carbonyldiimidazole (3.51 g, 21.63 mmol) was added. Gas evolution After the reaction had ceased, the resulting mixture was stirred at room temperature for 3.5 hours. A solution of propylmagnesium in THF (123 mmol, 30 mL) was added to a pre-cooled (0°C) ) Add ethyl hydrogen malonate (6.60 g, 49.2 mmol) to a solution and keep the internal temperature below 5°C. The mixture was stirred at room temperature for 1.5 hours. The magnesium enolate solution was cooled on an ice-water bath, followed by the imidazolide solution. The resulting mixture was stirred at 0° C. for 30 minutes, and then added slowly over 1 hour at 0° C. The mixture was then stirred at room temperature for 64 hours. 10% aqueous citric acid solution (5 mL) was added to the reaction mixture. The mixture was quenched by adding 10% aqueous citric acid (110 mL) to pH 3. The mixture was extracted with ethyl acetate (150 mL × 3). The organic extract was diluted with water (50 mL), Saturated aqueous sodium bicarbonate solution (50 mL) and saturated aqueous sodium chloride solution (50 mL ), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using ethanol (1:4) to give The title compound was obtained (5.50 g, 93% yield). 1 H NMR (500 MHz, CDCl3) δ 5.04 ( d, J = 7.8 Hz, 1H), 4.20 (p, J = 7.0 Hz, 3H), 3.52 (t, J = 10.7 Hz, 2H), 1. 96 (d, J = 3.7 Hz, 1H), 1.69 (s, 2H), 1.44 (s, 9H), 1.28 (dd, J = 7.1, 2. 9 Hz, 3H), 0.98 (t, J = 6.9 Hz, 3H), 0.92 - 0.86 (m, 3H).

[0247] Example 30: (3R,4S,5S)-4-((tert-butoxycarbonyl)amino) Ethyl 3-hydroxy-5-methylheptanoate [ka]

[0248] (4S,5S)-4-((tert-butoxycarbonyl)amino)-5-methyl-3 -Ethyl oxoheptanoate (5.90 g, 19.38 mmol) in ethanol (6 m L), sodium borohydride (3.77 g, 99.2 mmol) was added in one portion at -60 °C. The reaction mixture was stirred at -55°C or below for 5.5 hours, and then 10% aqueous citric acid ( The resulting solution was further acidified to pH 2 with 10% aqueous citric acid. The organic extract was diluted with saturated sodium chloride solution. The mixture was washed with aqueous ethanol (100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by chromatography (10-50% ethyl acetate / hexanes) to give pure dichloromethane. Stereomeric (3R,4S,5S)-4-((tert-butoxycarbonyl)amino) Ethyl 3-hydroxy-5-methylheptanoate (2.20 g, 37% yield) and (3R ,4S,5S)-4-((tert-butoxycarbonyl)amino)-3-hydroxy- Ethyl 5-methylheptanoate and (3S,4S,5S)-4-((tert-butoxycarbonyl) Mixture of ethyl 3-hydroxy-5-methylheptanoate (2.0g) , 34% yield, approximately 9:1 ratio) was obtained. 1 H NMR (500 MHz, CDCl3) δ 4.41 (d, J = 9 .3 Hz, 1H), 4.17 (tt, J = 7.1, 3.6 Hz, 2H), 4.00 (t, J = 6.9 Hz, 1H), 3.55 (dd, J = 11.7, 9.3 Hz, 1H), 2.56 - 2.51 (m, 2H), 2.44 (dd, J = 16.4, 9.0 Hz , 1H), 1.79 (d, J = 3.8 Hz, 1H), 1.60 - 1.53 (m, 1H), 1.43 (s, 9H), 1.27 (dd, J = 9.3, 5.0 Hz, 3H), 1.03 - 0.91 (m, 7H).

[0249] Example 31: (3R,4S,5S)-4-((tert-butoxycarbonyl)amino) -3-hydroxy-5-methylheptanoic acid [ka]

[0250] Compound (3R,4S,5S)-4-((tert-butyl)-2-(2-methyl-2-propanol)-4-one in ethanol (22 mL) Ethyl (dicarbonyl)amino)-3-hydroxy-5-methyl-heptanoate (2.20g) , 7.20 mmol) was added to a 1N aqueous solution of sodium hydroxide (7.57 mL, 7.57 mmol). l) was added. The mixture was stirred at 0°C for 30 minutes and then at room temperature for 2 hours. The resulting solution The mixture was acidified to pH 4 with 1N aqueous hydrochloric acid, and then extracted with ethyl acetate (50 mL x 3). The organic extract was diluted with 1N aqueous potassium hydrogen sulfate (50 mL) and saturated aqueous sodium chloride (10 mL). (50 mL), dried over Na2SO4, and concentrated under reduced pressure to give the title compound (1 0.90g, 95% yield. 1 H NMR (500 MHz, CDCl3) δ 4.50 (d, J = 8.7 Hz, 1H), 4.07 (d, J = 5.5 Hz, 1H), 3.59 (d, J = 8.3 Hz, 1H), 2.56 - 2.45 (m, 2H), 1.76 - 1.65 (m, 1H), 1.56 (d, J = 7.1 Hz, 1H), 1.45 (s, 9H), 1.26 (t, J = 7.1 Hz, 3H), 0.93 (dd, J = 14.4, 7.1 Hz, 6H).

[0251] Example 32: (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl )Amino)-3-methoxy-5-methylheptanoic acid [ka]

[0252] (3R,4S,5S)-4-((tert-butoxycarbonyl) (1.90 g, 6.9 mmol)amino)-3-hydroxy-5-methylheptanoic acid Sodium hydride (60% oil suspension, 1.93 g, 48.3 mmol) was added to the mixture at 0°C. After stirring for 1 hour, methyl iodide (6.6 mL, 103.5 mmol) was added. After stirring at 0°C for 40 hours, saturated aqueous sodium bicarbonate solution (50 mL) was added and The mixture was washed with diethyl ether (50 mL × 2) and water (100 mL) was added. The aqueous layer was acidified to pH 3 with 1N aqueous potassium hydrogen sulfate solution, and then ethyl acetate (50 mL × The combined organic extracts were washed with 5% aqueous sodium thiosulfate (50 mL) and Wash with saturated aqueous sodium chloride (50 mL), dry over Na2SO4, and concentrate under reduced pressure. The title compound was obtained (1.00 g, yield 48%). 1 H NMR (500 MHz, CDCl3) δ 3.95 (d, J = 75.4 Hz, 2H), 3.42 (d, J = 4.4 Hz, 3H), 2.71 (s, 3H), 2.62 (s, 1H) ), 2.56 - 2.47 (m, 2H), 1.79 (s, 1H), 1.47 (s, 1H), 1.45 (d, J = 3.3 Hz, 9H), 1.13 - 1.05 (m, 1H), 0.96 (d, J = 6.7 Hz, 3H), 0.89 (td, J = 7.2, 2. 5 Hz, 3H).

[0253] Example 33: General procedure for removal of the Boc functionality with trifluoroacetic acid To the N-Boc amino acid (1.0 mmol) in methylene chloride (2.5 mL), triflate Oroacetic acid (1.0 mL) was added. After stirring at room temperature for 1 to 3 hours, the reaction mixture was concentrated under reduced pressure. Co-evaporation with toluene gave the deprotected product, which was further purified. It was used without

[0254] Example 34: (S)-tert-butyl 2-((1R,2R)-1-methoxy-3-( ((S)-1-Methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2 -methyl-3-oxopropyl)pyrrolidine-1-carboxylate [ka]

[0255] (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidine- 2-yl)-3-methoxy-2-methylpropanoic acid (100 mg, 0.347 mmol) and L-phenylalanine methyl ester hydrochloride (107.8 mg, 0.500 mmol ) in DMF (5 mL) at 0 °C. 51 mmol), followed by EtN (131 μL, 0.94 mmol). The mixture was stirred at 0° C. for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (8 Dilute with 1N potassium hydrogen sulfate aqueous solution (40 mL), water (40 mL), saturated charcoal Wash with aqueous sodium hydrogen carbonate solution (40 mL) and saturated aqueous sodium chloride solution (40 mL). The residue was purified by column chromatography (15 ~75% ethyl acetate / hexanes) to give the title compound as a white solid (130 mg, yield 83%). 1 H NMR (500 MHz, CDCl3) δ 7.28 (dd, J = 7.9, 6.5 Hz, 2H ), 7.23 (t, J = 7.3 Hz, 1H), 7.16 (s, 2H), 4.81 (s, 1H), 3.98 - 3.56 (m, 5H), 3.50 (s, 1H), 3.37 (d, J = 2.9 Hz, 3H), 3.17 (dd, J = 13.9, 5.4 Hz, 2H), 3.04 (dd, J = 14.0, 7.7 Hz, 1H), 2.34 (s, 1H), 1.81 - 1.69 (m, 2H), 1.65 (s, 3H), 1.51 - 1.40 (m, 9H), 1.16 (d, J = 7.0 Hz, 3H).

[0256] Example 35: (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4 S,5S)-4-((tert-butoxycarbonyl)-(methyl)amino)-3-meth (5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylprop propanamido)-3-phenylpropanoate [ka]

[0257] (S)-tert-butyl 2-((1R,2R)-1-methoxy-3-(((S)- 1-Methoxy-1-oxo-3-phenylpropan-2-yl)amino)-2-methyl- Deprotection product from 3-oxopropyl)pyrrolidine-1-carboxylate (0.29 mmol) and (3R,4S,5S)-4-((tert-butoxycarbonyl)(methyl (amino)-3-methoxy-5-methylheptanoic acid (96.6 mg, 0.318 mmol) To a DMF solution (5 mL) of 1,4-diethyl cyanophosphonate (58 μL, 0.3 μL) was added at 0°C. 47 mmol), followed by EtN (109 μL, 0.78 mmol). The mixture was stirred at 0° C. for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (8 Dilute with 1N potassium hydrogen sulfate aqueous solution (40 mL), water (40 mL), saturated charcoal Wash with aqueous sodium hydrogen carbonate solution (40 mL) and saturated aqueous sodium chloride solution (40 mL). The residue was purified by column chromatography ( Purification with 15-75% ethyl acetate / hexane gave the title compound (150 mg, 81% yield) ) was obtained as a white solid. LC-MS (ESI) m / z C 34 H 55 N3O8[M+H ] + : Calculated value 634.40, measured value 634.40.

[0258] Example 36: (S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4 S,5S)-4-((S)-2-((tert-butoxycarbonyl)amino)-N,3 -dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2- yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate [ka]

[0259] (S)-Methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S) -4-((tert-butoxycarbonyl)-(methyl)amino)-3-methoxy-5- Methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamine (do)-3-phenylpropanoate (0.118 mmol) and Boc-Val-OH( A solution of 51.8 mg (0.236 mmol) of bromotris( Dimethylamino)-phosphonium hexafluorophosphate (BroP, 70.1 mg , 0.184 mmol), followed by diisopropylethylamine (70 μL, 0.425 m The mixture was protected from light and stirred at 0°C for 30 minutes, then at room temperature for 2 days. The mixture was diluted with ethyl acetate (80 mL), 1N aqueous potassium hydrogen sulfate solution (40 mL), Water (40 mL), saturated aqueous sodium bicarbonate solution (40 mL), and saturated sodium chloride solution The residue was washed with aqueous solution (40 mL), dried over Na2SO4, and concentrated in vacuo. The title compound was purified by column chromatography (20-100% ethyl acetate / hexane). The product (67 mg, 77% yield) was obtained as a white solid. LC-MS (ESI) m / z C3 9H 64 N4O9[M+H] + : Calculated value 733.47, actual value 733.46.

[0260] Example 37: Preparation of compound Boc-N-Me-Val-OH [ka]

[0261] Boc-L-Val-OH (2.00 g, 9.2 mmol) and methyl iodide (5.7 A solution of 4 mL of 4-chloro-4-benzotriazole (92 mmol) in anhydrous THF (40 mL) was added to sodium hydride (3 The reaction mixture was stirred at 0° C. for 1.5 hours and then at room temperature. The reaction was quenched with ice water (50 mL). Water (100 mL) was added. After the addition of ethyl acetate (50 mL × 3), the reaction mixture was washed with ethyl acetate and the aqueous solution was acidified to pH 3. The combined organic phase was washed with Na2SO4 and then extracted with ethyl acetate (50 mL x 3). After drying and concentration, Boc-N-Me-Val-OH (2.00 g, 94% yield) was obtained as white solids. Obtained as a coloured solid. 1 H NMR (500 MHz, CDCl3) δ 4.10 (d, J = 10.0 Hz, 1H), 2.87 (s, 3H), 2.37 - 2.13 (m, 1H), 1.44 (d, J = 26.7 Hz, 9H), 1.02 (d, J = 6. 5 Hz, 3H), 0.90 (t, J = 8.6 Hz, 3H).

[0262] Example 38: (S)-Methyl 2-((2R,3R)-3-((S)-1-((6S,9S ,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-1 3-Methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxo (5,8,11-triazapentadecan-15-yl)pyrrolidin-2-yl)-3- Methoxy-2-methylpropanamido)-3-phenylpropanoate [ka]

[0263] (S)-Methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S) -4-((S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethyl Butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3 -Methoxy-2-methylpropanamido)-3-phenylpropanoate (0.091m mol) and Boc-N-Me-Val-OH (127 mg, 0.548 mmol) To the MF solution (5 mL), diethyl cyanophosphonate (18.2 μL, 0.114 mmol), followed by 4-methylmorpholine (59 μL, 0.548 mmol) The reaction mixture was stirred at 0°C for 2 hours, then warmed to room temperature and stirred overnight. Dilute with ethyl acetate (80 mL), 1N aqueous potassium hydrogen sulfate solution (40 mL), water (40 mL ), saturated aqueous sodium bicarbonate solution (40 mL), and saturated aqueous sodium chloride solution (40 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography. The product was purified by chromatography (20-100% ethyl acetate / hexanes) to give the title compound (30m g, 39% yield) as a white solid. LC-MS (ESI) m / z C 45 H75 N 5O 10 [M+H] + : Calculated value 846.55, measured value 846.56.

[0264] Example 39: (S)-2-((2R,3R)-3-((S)-1-((6S,9S,12 S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methyl Toxo-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5 ,8,11-Triazapentadecan-15-oyl)pyrrolidin-2-yl)-3-meth Oxy-2-methylpropanamido)-3-phenylpropanoic acid [ka]

[0265] (S)-Methyl 2-((2R,3R)-3-((S)-1-((6S,9S,12S ,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-meth Oxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5, 8,11-Triazapentadecan-15-oyl)pyrrolidin-2-yl)-3-methoxy Di-2-methylpropanamido)-3-phenylpropanoate (30 mg, 0.035 To a THF solution (1.0 mL) of 1.0 mmol, a 1.0 M LiOH aqueous solution (0.8 mL) was added. The mixture was stirred at room temperature for 35 min, neutralized to pH 6 with 0.5 M H3PO4, and concentrated. Then, a SiO2 column was used to separate CH3OH / CH2Cl2 / HOAc (1:10:0.01 ) to obtain the title compound (25.0 mg, 85% yield). ESI) m / z calculated value C 44 H 74 N5O 10 [M+H]+ :832.54, Actual value:8 32.60.

[0266] Example 40: (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S )-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino )Butanamido)butanamido)-3-methoxy-5-methylheptanoyl)-pyrrolidine (2-phenyl-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (101) [ka]

[0267] (S)- in a mixture of HCl (conc. 0.3 mL) and 1,4-dioxane (0.9 mL) 2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12- ((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-2,2,5, 11-Tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triaza Pentadecane-15-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropional (25 mg, 0.030 mmol)-3-phenylpropanoic acid at room temperature for 3 The mixture was stirred for 5 min and diluted with EtOH (1.0 mL) and toluene (1.0 mL). , concentrated, and re-evaporated with EtOH / toluene (2:1) to give the title compound as a white solid This was used in the next step without further purification. -MS(ESI)m / z+calculated value C 39 H 66 N5O8[M+H] + :732.48,Real Measurement: 732.60.

[0268] Example 41: (2S)-2-((2R,3R)-3-((2S)-1-((11S,14 S,17S)-1-Azido-17-((R)-sec-butyl)-11,14-diisopropyl propyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6 -Dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl (phenyl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid [ka]

[0269] DMA (0.8 mL) and NaH2PO4 buffer (0.7 mL, 1.0 M, pH 7.5 To the crude compound (101) (22 mg, 0.033 mmol) in a mixture of ) (18.0 mg, 0.060 mmol) was added in four portions over 2 hours. The mixture was stirred overnight, concentrated, and purified by SiO2 column with CH3OH / CH2Cl2 / HOAc (1 :8:0.01) to give the title compound (22.5 mg, yield 82%). LC-MS(ESI)m / z+calculated value C 46 H 77 N8O 11 [M+H] + :917.56, Actual value:917.60.

[0270] Example 42: (2S)-2-((2R,3R)-3-((2S)-1-((11S,14 S,17S)-1-amino-17-((R)-sec-butyl)-11,14-diisopropyl propyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6 -Dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl (2-methyl-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (103) [ka]

[0271] Compound (102) (22.0 mg, 0.02 mL) in methanol (5 mL) in a hydrogenation vessel To the solution (4 mmol), Pd / C (5 mg, 10% Pd, 50% wet) was added. After evacuating the gas, 25 psi of H2 was introduced. The mixture was shaken for 4 hours and filtered through Celite. The mixture was filtered and concentrated to give the crude title compound (~20 mg, yield ~92%). This was used in the next step without further purification. ESI MS m / z+C 46 H 79 NO 11 (M+H ), calculated value: 891.57, measured value: 891.60.

[0272] Example 43: 2,3-Dibromosuccinic anhydride (70) [ka]

[0273] 2,3-Dibromosuccinic acid (10.00 g, 36 mL) in dry CHCl (100 mL) Phosphorus pentoxide (12.21 g, 85.84 mmol) was added to the solution at 0°C. The mixture was stirred at 0°C for 2 hours, then at room temperature for 5 hours, filtered through a short SiO2 column, and The column was rinsed with tOAc / CH2Cl2 (1:6). The filtered solutions were combined and evaporated. The residue was solidified with EtOAc / hexane to give the title compound (6.63 g, 71% yield). ESI MS m / z + C4H2Br2O3 (M+H), calculated: 256.85, found :256.70.

[0274] Example 44: 2,3-Dibromo-4-((2-(2-(3-((S)-7-methoxy-8 -((5-(((S)-7-Methoxy-2-methylene-5-oxo-2,3,5,11a -Tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8- yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a -Tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10 (5H)-yl)-3-oxopropoxy)ethoxy)ethyl)amino)-4-oxobutanol Tannic acid (124) [ka]

[0275] Compound in a mixture of DCM (4 mL) and DIPEA (12 uL, 0.069 mmol) The compound (100) (40.0 mg, 0.068 mmol) was treated with 2,3-dibromoanhydrochloride at 0°C. Cinic acid (38.0 mg, 0.148 mmol) was added. The mixture was stirred at 0° C. for 2 hours, then The mixture was stirred at room temperature for 5 hours, concentrated, and purified by column chromatography using CHOH / CHC Purification by elution with I2 / HOAc (1:6:0.01) gave the title compound. (56.5 mg, 83% yield). LC-MS (ESI) m / z calculated: C 44 H 53 B r2N5O 12 [M+H] + :1002.21, Actual value:1002.40,1004.4 0(M+2+H).

[0276] Example 45: 2,5-Dioxopyrrolidin-1-yl 2,3-dibromo-4-((2- (2-(3-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl ethylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl 5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diazepine-10(5H)-yl)-3-oxopropoxy) (2-Oxy)ethyl)amino)-4-oxobutanoate (125) [ka]

[0277] Compound 125 (55.0 mg, 0.054 mmol) in CHCl (3 mL) NHS (10.0 mg, 0.086 mmol) and EDC (30.5 mg, 0.15 mmol) were added. The mixture was stirred at room temperature overnight, concentrated, and purified by SiO2 column. Purification by elution with tOAc / CH2Cl2 (1:5) gave the title compound (50.5 mg, yield 85%). LC-MS(ESI)m / z calculation value:C 48 H 56 Br 2N6O 14 [M+H] + :1099.22, Actual value:1099.40,1101.40 (M+2+H), 1119.50 (M+2+H+H2O).

[0278] Example 46: (2S)-2-((2R,3R)-3-((2S)-1-((13S,26 S,29S,32S)-12,13-dibromo-32-((R)-sec-butyl)-2 6,29-diisopropyl-33-methoxy-1-((S)-7-methoxy-8-((5 -(((S)-7-Methoxy-2-methylene-5-oxo-2,3,5,11a-tetramethyl- Hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)o (oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetra Hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H) -yl)-25,31-dimethyl-1,11,14,24,27,30-hexaoxo- 4,7,18,21-Tetraoxa-10,15,25,28,31-pentaazapenta Triacontan-35-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropional propanamido-3-phenylpropanoic acid (126) [ka]

[0279] DMA (1mL) and NaH2PO4 buffer (0.6mL, 0.15M, pH7.5) Compound (103) (~20 mg, 0.022 mmol) in a mixture of (30.0 mg, 0.027 mmol) was added. The mixture was stirred for 7 hours, concentrated, and C- 18 Chromatography (φ2.0 cm × 25 cm) eluted with water / CH3CN (10 ml / min, 90% water to 15% water for 50 minutes) and purified by high vacuum pump. After drying, the title compound (126) was obtained (26.1 mg, 63% yield). S m / z+C 90 H 130 Br2N 11 O 22 (M+H), calculated value: 1874.77, 1874.50.

[0280] Example 47: Conjugation of compound (126) with antibody for compound (127) [ka]

[0281] 100 ml of 10 mg / ml Herceptin at pH 6.0-8.0 in a 2.0 mL mixture 0.70-2.0 mL of PBS buffer with M NaH2PO4, pH 6.5-7.5 , TCEP (28 μl, 20 mM in water), and compound (126) (14 μl, 2 in DMA). The mixture was incubated at room temperature for 4 to 16 hours, and then DHAA (135 μl, 50 mM) was added at RT. After continuous incubation overnight at room temperature, , and a G-25 column was used to separate the eluate in 100 mM NaH2PO4, 50 mM NaCl buffer (pH 6. The mixture was purified by elution with 13.1 to 15.0 ml of buffer (1 mL). 6.5 mg to 17.7 mg of conjugate compound (127) was obtained (yield: 82% to 88%). The drug / antibody ratio (DAR) (combination of PBD dimer and MMAF per antibody) was UP The LC-Qtof mass spectrum determined the value to be 3.85. Tosoh Bioscience, Tskgel G3000SW, 7.8mm ID x 30cm, 0.5 ml / min, 100 min), they were 96-99% monomeric, S A single band was detected on the DS-PAGE gel.

[0282] Example 48: (4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S ,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamide Pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-5- (3-(12,13-dibromo-24-((S)-7-methoxy-8-((5-(((S )-7-Methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1 H-Benz[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pen (ethyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1 H-Benzo[e]pyrrolo[1,2-a][1,4]diazepin-10(5H)-yl)- 11,14,24-trioxo-4,7,18,21-tetraoxa-10,15-diazo Tetracosanamide)-4-hydroxyphenyl)-2-methylpentanoic acid (128) [ka]

[0283] DMA (1mL) and NaH2PO4 buffer (0.6mL, 0.15M, pH7.5) Compound (95) (20 mg, 0.021 mmol) in a mixture of The mixture was stirred for 8 hours, concentrated, and the C-18 The mixture was eluted with water / CH3CN using a chromatography column (φ2.0 cm × 25 cm). The mixture was purified (10 ml / min, 90% water to 20% water for 50 minutes) and dried with a high vacuum pump. After filtration, the title compound (128) was obtained (26.6 mg, 64% yield). ESI MS m / z+C 89 H 123 Br2N 12 O 22 S(M+H), calculated value: 1901.69, actual value: Measurement: 1901.90.

[0284] Example 49: Conjugation of Compound 128 with an Antibody for Compound (129) [ka]

[0285] 100 ml of 10 mg / ml Herceptin at pH 6.0-8.0 in a 2.0 mL mixture 0.70-2.0 mL of PBS buffer with M NaH2PO4, pH 6.5-7.5 , TCEP (28 μl, 20 mM in water), and compound (128) (14 μl, 2 in DMA). The mixture was incubated at room temperature for 4 to 16 hours, and then DHAA (135 μl, 50 mM) was added at RT. After continuous incubation overnight at room temperature, , and a G-25 column was used to separate the eluate in 100 mM NaH2PO4, 50 mM NaCl buffer (pH 6. The mixture was purified by elution with 13.2 to 15.1 ml of buffer (0 to 7.5 ml). 6.4 mg to 17.6 mg of conjugate compound (129) was obtained (yield: 82% to 88%). The drug / antibody ratio (DAR) (PBD dimer and tubulysin analog conjugate per antibody) is The HCl concentration was determined by UPLC-Qtof mass spectrometry to be 3.9. (Tosoh Bioscience, Tskgel G3000SW, 7.8mm ID x 30cm , 0.5 ml / min, 100 min), they are 96-99% monomer, A single band was observed on the SDS-PAGE gel.

[0286] Example 50: Bis(2,5-dioxopyrrolidin-1-yl) 2,3-dibromosuccine (9) [ka]

[0287] 2,3-Dibromosuccinic acid (5.0 g, 18.25 mmol), N-hydroxysuccinic acid NHS (5.01 g, 43.56 mmol), and EDC (12.02 g, A solution of 62.60 mmol) in dichloromethane (100 mL) was stirred overnight, concentrated, and Si Purification on an O2 column eluted with EtOAc / CH2Cl2 (1:6) provided the target The title compound was obtained (6.74 g, 79% yield). LC-MS (ESI) m / z calculated value C 12 H 11 Br2N2O8[M+H] + :468.88,[M+H+2] + :470.88, Actual measurements: 468.70, 470.70

[0288] Example 51: (R,R,S,S,R,4R,4'R)-5,5'-(((12,13-di Bromo-11,14-dioxo-4,7,18,21-tetraoxa-10,15-diazo Tetracosane-1,24-dioyl)bis(azanediyl)bis(4-hydroxy- 3,1-phenylene))-bis(4-(2-((1R,3R)-1-acetoxy-3-( (2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carbohydrate (Thiazole-4-carboxamide)pentanamido)-4-methylpentyl)thiazole-4-carboxamide -2-methylpentanoic acid) (141) [ka]

[0289] Compound (95) (40 mg, 0.042 mmol) in DMA (1 mL) and NaHPO 4. Bis(2,5-dioxopyrrolidine) in buffer (0.6 mL, 0.15 M, pH 7.5) -1-yl) 2,3-dibromosuccinate (9) (18.0 mg, 0.038 mmol The mixture was stirred for 8 hours, concentrated, and purified by C-18 chromatography (φ2.0c The column was purified by eluting with water / CH3CN on a column (10 ml / min, 90% water / 10% CH3CN to 20% water / 90% CH3CN for 50 minutes, high vacuum pump After drying at 40°C, the title compound (141) was obtained (38.5 mg, 49% yield). MS m / z+C 94 H 143 Br2N 14 O 24 S2(M+H), calculated value: 2073. 81, Actual value: 2073.60.

[0290] Example 52: Conjugation of compound (141) with an antibody for compound (142) [ka]

[0291] 100 ml of 10 mg / ml Herceptin at pH 6.0-8.0 in a 2.0 mL mixture 0.70-2.0 mL of PBS buffer with M NaH2PO4, pH 6.5-7.5 , TCEP (28 μl, 20 mM in water), and compound (141) (14 μl, 2 in DMA). The mixture was incubated at room temperature for 4 to 16 hours, and then DHAA (135 μl, 50 mM) was added at RT. After continuous incubation overnight at room temperature, , and a G-25 column was used to separate the eluate in 100 mM NaH2PO4, 50 mM NaCl buffer (pH 6. The mixture was purified by elution with 13.1 to 15.2 ml of buffer (0 to 7.5 ml). The yield of conjugate compound (92) ranged from 6.4 mg to 17.6 mg. The drug / antibody ratio (DAR) was The HCl concentration was determined by UPLC-Qtof mass spectrometry to be 3.9. (Tosoh Bioscience, Tskgel G3000SW, 7.8mm ID x 30cm , 0.5 ml / min, 100 min), they are 95-99% monomer, A single band was observed on the SDS-PAGE gel.

[0292] Example 53: In vitro cell differentiation of conjugates 127, 129, and 142 compared to T-DM1 Toxicity evaluation Cell lines used for cytotoxicity assays were the human leukemia cell line HL-60 and the human gastric cancer cell line NCI-N78, human invasive ductal carcinoma cell line BT-474, and human ovarian cancer cell line SKOV 3 was used. For HL-60, NCI-N87, and BT-47 cells, The cells were cultured in RPMI-1640 containing 10% FBS. To perform the assay, cells (180 μl, 6000 cells) was added to each well of a 96-well plate and incubated at 37°C in 5% CO The cells were then incubated at 2°C for 24 hours. The cells were treated with various concentrations of test compounds (20 μl) in the control wells. Plates containing cells and medium but lacking test compounds were incubated at 37°C and 5% CO2 for 120 min. MTT (5 mg / ml) was added to the wells (20 μl) and the plate was incubated for 1 hour. The plates were incubated at 37°C for 1.5 hours. After that, the medium was carefully removed and DMS After shaking for 15 minutes, the sample was analyzed using a 620 nm reference filter. The absorbance was measured at 490 nm and 570 nm. The percent inhibition was calculated according to the following formula: Injury rate suppression % = [1 - (analytical value - blank) / (control - blank)] x 100

[0293] Cytotoxicity results: [Table 1]

[0294] The specificity of conjugate (127) for N87 cells was greater than 889 (IC 50 >8 / IC5 0 = 0.009), and in SK-OV-3 cells it was over 800. The specificity of the complex (129) is over 666 (IC 50 >8 / IC 50 =0.012), In SK-OV-3 cells, the specificity of the conjugate (142) in N87 cells was Over 155 (IC 50 >15 / IC 50 =0.097) and 18 in SK-OV-3 cells. The specificity of the conjugate T-DM1 for N87 cells was ≥ 57 (IC 50 >15 / IC 50 =0.263), and in SK-OV-3 cells it was over 80.

[0295] The three new conjugates (127), (129), and (142) were synthesized from commercially available conjugates T-DM1. It was much more powerful than 1.

[0296] Example 54: Antitumor activity in vivo The in vivo efficacy of T-DM1 and conjugates 127, 129, and 142 was evaluated using human immunization assays. The efficacy and safety of the compound was evaluated in a human gastric cancer N-87 cell line tumor xenograft model. N-87 carcinoma cells (5 × 10 cells) were added to 30 mice in 0.1 mL of serum-free medium. 6 Thin The tumors were inoculated subcutaneously into the area under the right shoulder. The tumors were allowed to grow for 8 days, reaching a size of 133 mm. 3 Average size of The animals were then randomly divided into 5 groups (6 animals per group). Group 1 One group of mice served as a control and was treated with phosphate-buffered saline vehicle. At a dose of 3 mg / kg administered intravenously, conjugates (127), (129), and (14 The three dimensions of the tumor were measured every 4 days, and tumor volume was calculated using the formula: tumor volume = 0.05; Tumor volume was calculated using 1 / 2 (length x width x height). The body weight of the animals was also measured at the same time. Mice were sacrificed if they met one of the following criteria: (1) lost more than 20% of their pretreatment weight; Weight loss, (2) 1500mm 3 (3) To reach food and water; Mice were considered to have tumors if they were too lethargic, or (4) skin necrosis. Tumors were not palpable. It was determined that there was no tumor.

[0297] The results are plotted in Figure 16. All four conjugates did not cause a decrease in animal weight. The control animals were then placed at 1500 mm on day 37. 3 were sacrificed due to tumor volumes exceeding They were too lethargic. 6 / 6 animals in the compound (127) and (129) groups In all cases, no tumors were measurable between days 13 and 60 (the end of the experiment). All 6 / 6 animals in the 42) group had no measurable tumors by day 21, and 2 / 6 animals Tumor growth (measurable) was observed on day 48. In contrast, T-DM1 at a dose of 3 mg / kg failed to eradicate the tumor and inhibited tumor growth for only 28 days.

Claims

1. Bridge linker compounds of formula (I): 【Chemistry 1】 During the ceremony, 【Chemistry 2】 represents any single bond; 【Transformation 3】 represents a single or double bond; 【Chemistry 4】 represents a single bond, U and U' are not both H; 【Transformation 5】 represents a double bond, either U or U' can be H, but at the same time do not have; a 2,3-disubstituted succinic acid group, or a 2-monosubstituted or 2,3-disubstituted fumaric acid group, or A component which may be a 2-monosubstituted or 2,3-disubstituted maleic acid 【Transformation 6】 can react with a pair of sulfur atoms of the cell-binding agent; is a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE) , L-glutathione (GSH) and tris(2-carboxyethyl)phosphine (TCE P), or / and β-mercaptoethanol (β-ME, 2-ME), a cell-binding agent a pair of thiols reduced from the interchain disulfide bond of U and U′ are the same or different leaving groups which may be substituted by thiol; Such leaving groups include, but are not limited to, halides (e.g., fluoride, chloride, etc.). methanesulfonyl (mesyl), p-toluenesulfonyl ( Tosyl), trifluoromethylsulfonyl (triflate), trifluoromethylsulfon nate, nitrophenol, N-hydroxysuccinimide (NHS), phenol; Nitrophenol; pentafluorophenol, tetrafluorophenol, difluoro Phenol, monofluorophenol, pentachlorophenol, imidazole, dichlorophenol chlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, 2-ethyl 5-phenylisoxazolium-3'-sulfonate, or for the Mitsunobu reaction It is an intermediate molecule produced by a condensation reagent. R １ and R ２ are the same or different and are absent, a linear alkyl having 1 to 6 carbon atoms, branched or cycloalkyl, straight-chain, branched or cycloalkenyl having 3 to 6 carbon atoms; alkynyl, ester, ether or amide having 1 to 6 carbon atoms, structural formula (OCH ２ CH ２ ) ｐ (p is an integer from 0 to about 1000) Formula (OCH ２ (CH ３ ) CH ２ ) ｐ (p is an integer of 0 to about 1000) oxy units, or combinations thereof; Additionally, R １ and R ２ are respectively, X １ or X ２ and Z １ or Z ２ Covalently bonded to is a chain of atoms selected from C, N, O, S, Si, and P, preferably 0 to 500 atom; R １ and R ２ The atoms used to form quinylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, Hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine amines, alkoxyamines, urethanes, amino acids, peptides, acyloxyamines, or All chemically related compounds that form hydroxamic acids, or combinations thereof They may also be joined by the method. X １ and X ２ is NH, N(R ３ ), O, S, or CH ２ independently selected from R ３ is H, straight chain alkyl having 1 to 6 carbon atoms, branched or cycloalkyl having 3 to 6 carbon atoms, straight chain , branched or cycloalkenyl or alkynyl, esters having 1 to 6 carbon atoms, ethers or amide, or the structural formula (OCH ２ CH ２ ) ｐ (p: 0 to about 1000) integer) polyethyleneoxy units, or combinations thereof; Z １ and Z ２ disulfides, thioethers, thioesters, peptides, hydrazones , ethers, esters, carbamates, carbonates, amines (secondary, tertiary or quaternary ), imine, cycloheteroalkane, heteroaromatic ring, alkoxide, or amide bond the same or different functional groups capable of reacting with a cytotoxic agent to form a Base Z １ and Z ２ is shown below: 【Transformation 7】 【change】 In the formula, X1 is F, Cl, Br, I, or Lv ３ and X2 is O, NH, N(R １ ), or CH ２ and R ５ and R ３ is H, R １ , aromatic ring, heteroaromatic ring, or one or more is a group in which several H atoms are independently １ , -halogen, -OR １ , -SR １ , -NR １ R ２ ,- NO ２ , -S(O)R １ , -S(O) ２ R １ or -COOR １ Aromatic groups substituted with And; Lv ３ is nitrophenol; N-hydroxysuccinimide (NHS); phenol dinitrophenol; pentafluorophenol; tetrafluorophenol; di Fluorophenol; Monofluorophenol; Pentachlorophenol; Triflate ; Imidazole; Dichlorophenol; Tetrachlorophenol; 1-Hydroxybenzo Triazole; Tosylate; Mesylate; 2-Ethyl-5-phenylisoxazolium- 3'-sulfonates, acid anhydrides formed with themselves or with other acid anhydrides (e.g., anhydrides acetic acid, formic anhydride); or for peptide coupling reactions, or for Mitsunobu reactions The leaving group is selected from the intermediates generated by the condensation reagents for the purpose of

2. A cell-binding agent-drug conjugate compound of formula (II): 【Transformation 8】 During the ceremony, Cb represents a cell-binding agent, preferably an antibody, and is bound to the antibody via a pair of sulfur atoms (thiols). Drug １ and Drug ２ the conjugable thiol atom is generally a dithiol Dithreitol (DTT), dithioerythritol (DTE), L-glutathione (GS H) and tris(2-carboxyethyl)phosphine (TCEP), and / or β-mer Captoethanol (β-ME, 2-ME) induces disulfide bond formation on cell-binding molecules can be produced from the reduction of "Drug １ " and "Drug ２ " is an alkyl, alkylene, alkenylene, alkylene ... Nylene, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, carbazide, alkoxyamine , urethane, amino acid, peptide, acyloxylamine, hydroxamic acid, disulfide amides, thioethers, thioesters, carbamates, carbonates, heterocycles, heteroalkoxy groups Cross-linked by alkyl, heteroaromatic, or alkoxyl bonds, or a combination thereof represents the same or a different cytotoxic agent linked to said cell-binding agent via a body; n is 1 to 30; 【Chemistry 9】 R １ , R ２ , X １ , and X ２ is the same as that described in claim 1 above.

3. Compounds of formula (III): 【Chemistry 10】 During the ceremony: 【Chemistry 11】 Cb, Z １ , Z ２ , n, R １ , R ２ , X １ , and X ２ has the same definition as in claims 1 and 2 do.

4. Compound of formula (IV): 【Chemistry 12】 During the ceremony: 【Chemistry 13】 U, U', Drug １ , Drug ２ , R １ , R ２ , X １ , and X ２ claims 1 and 2 and It's the same definition.

5. the 2,3-disubstituted succinic acid group, the 2-monosubstituted or 2,3-disubstituted fumaric acid group, or The 2-monosubstituted or 2,3-disubstituted maleic acid group can be synthesized as shown in the following scheme (Ia). 2,3-disubstituted succinic acid, 2-monosubstituted or 2,3-disubstituted fumaric acid, or 2 - Mono-substituted or 2,3-disubstituted maleic acid or its acid derivatives and by condensation with other components containing amines (1° or 2° amines), alcohols, or thiols 2. A bridge linker compound of formula (I) according to claim 1, synthesized by: 【Chemistry 14】 During the ceremony, X is NH, N(R ３ ), O, or S, １ or X ２ and ； R is R １ and / or R ２ and R １ , R ２ and R3 is as defined in claim 1 is the same as Lv １ and Lv ２ are the same or each independently OH; F; Cl; Br; I; Nit N-hydroxysuccinimide (NHS); phenol; dinitrophenol pentafluorophenol; tetrafluorophenol; difluorophenol; Monofluorophenol; Pentachlorophenol; Triflate; Imidazole; Dichlorophenol Tetrachlorophenol; 1-hydroxybenzotriazole; 2-ethyl-5-phenylisoxazolium-3'-sulfonate; mesylate; Formed by itself or with other anhydrides, such as acetic anhydride or formic anhydride or intermediate molecules for peptide coupling reactions, or for Mitsunobu reactions It is an intermediate produced by a condensation reagent for the reaction, for example, EDC (N -(3-dimethylaminopropyl)-N'-ethylcarbodiimide), DCC (dicyclo N,N'-diisopropylcarbodiimide (DIC), N -Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide and meso-p-toluene CMC, or CME-CDI, 1,1'-carbonyldiimidazole (CDI), TBTU (O-(benzotriazol-1-yl)-N,N,N',N'- Tetramethyluronium tetrafluoroborate), N,N,N',N'-tetramethyl —O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethylamino)phos Benzonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy) C) Tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl Cyanophosphonate (DEPC), chloro-N,N,N',N'-tetramethylformamide Midinium hexafluorophosphate, 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluoro Phosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H -[1,2,3]triazolo[4,5-b]pyridin-1-ium-3-oxide hexa Fluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium Hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluoro Fluorophosphate (PyCloP), fluoro-N,N,N',N'-bis(tetramethyl N,N,N',formamidinium hexafluorophosphate (BTFFH) N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluoro Phosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetra Methylthiuronium tetrafluoroborate (TPTU), S-(1-oxido-2-pi lysyl)-N,N,N',N'-tetramethylthiuronium tetrafluoroborate, O -[(ethoxycarbonyl)cyano-methylenamino]-N,N,N',N'-tetra Methyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy) C-(2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium Oxafluorophosphate (COMU), O-(benzotriazol-1-yl)-N, N,N',N'-bis(tetramethylene)uronium hexafluorophosphate (HB PyU), N-benzyl-N'-cyclohexylcarbodiimide (with polymer binding, Dipyrrolidino(N-succinimidyloxy)-carbenium hexafluoride Hexafluorophosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate Phate (PyCIU), 2-chloro-1,3-dimethylimidazolinium tetrafluoride Carborate (CIB), (benzotriazol-1-yloxy)dipiperidinocarbenyl Umbilical cord hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazole (N,N,N',N'-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)phosphonium hexafluorophosphate Propylphosphonic anhydride (BroP), propylphosphonic anhydride (PPACA, TsP®), 2- Morpholinoethyl isocyanide (MEI), N,N,N',N'-tetramethyl-O-( N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bro 1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxylated [N,N,N',N'-tetramethyluronium tetracarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetracarbonyl Trifluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazine N,N,-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate TSTU, O-(3,4-dihydro-4-oxo-1,2,3-benzotriazole N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'-(azodicarbonyl)dipiperidine (ADD), di-(4 -chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azo dicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD ), diethyl azodicarboxylate (DEAD).

6. In the formulas (II) and (IV) of claims 2 and 4, １ and Drug ２ However, or independently selected from: 1) Chemotherapeutic agents: a) Alkylating agents: Nitrogen mustards: Chlorambucil, Lornafadine, cyclophosphamide, dacarbazine, estramustine, ifosfamide mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobrontine le, melphalan, pipobroman, nobembine, fenesterine, prednimustine, Thiotepa, trofosfamide, uracil mustard; CC-1065 (adzelesin, ka including synthetic analogs of rzeresin and bizeresin; duocarmycins (synthetic analogs, KW-2189 and CBI-TMI); benzodiazepine dimers (pyrrolobenzoxazoles, Diazepine (PBD) or tomaymycin, indolinobenzodiazepines, imidazobe dimers of benzothiazepines or oxazolidinobenzodiazepines); nitrosourea Substances: (carmustine, lomustine, chlorozotocin, fotemustine, nimustine) , ranimustine); alkyl sulfonates (busulfan, treosulfan, impro sulfane and piposulfan); triazenes (dacarbazine); platinum-containing compounds: ( benzodopa, carboquone, methole Aziridines such as dopa and uredopa; ethyleneimines, as well as altretamine, triethylenediamine, Contains triethylene melamine, triethylene phosphoramide and triethylene thiophosphoramine b) Plant Alkaloids: Vinca Alkaloids: (Vincristine , vinblastine, vindesine, vinorelbine, navelbine); Taxoids: (Pakri Taxel, docetaxel); and their analogs, maytansinoids (DM1, DM2 , DM3, DM4, maytansine, ansamitocin) and their analogs, cryptofen cryptophycins (especially cryptophycin 1 and cryptophycin 8); epothilones, erythropoietins, Therobins, discodermolide, bryostatins, dolostatins, auristatins sarcodictin; sarcodictin; sarcodictin ; spongistatin; c) DNA topoisomerase inhibitors: [epipodophyllins: (9 - Aminocamptothecin, camptothecin, cristatol, daunomycin, etoposide , etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinols), teniposide, topotecan, 9-nitrocamptothecin (RFS 20 00); Mitomycins: (Mitomycin C); d) Antimetabolites: {[Antifolates: Dihydrofolate reductase inhibitors: (methotrexate, trimetrexate, denoptera) Phosphorus, pteropterin, aminopterin (4-aminopteroic acid), or other folic acids analogs); IMP dehydrogenase inhibitors (mycophenolic acid, tiazofurin, ribavirin ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine, cyclosporine, cyclosporine); Pyrimidine analogs: uracil analogs (ancitabine, azacitidine, 6-azacytidine) Sauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridin doxifluridine, enocitabine, 5-fluorouracil, floxuridine, ral Titrexed (Tomudex)); Cytosine analogues: (cytarabine, cytosine arabinoside) Purine analogues: (azathioprine, fludarabine, mercaptopropion) folic acid supplements such as folinic acid}; e) hormone therapy Drugs: {Receptor antagonists: [Anti-estrogens: (megestrol, raloxifene, tamoxifene LHRH agonists: (goserelin, leuprolide acetate); antiandrogens (bicalutamide, flutamide, calsterone, dromostanolone propionate, epinephrine) Thiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testora androgen inhibitors)]; retinoids / deltoids: [Bactone, trilostane, and other androgen inhibitors]; Tetanus D3 analogues: (CB1093, EB1089, KH1060, cholecalciferol Photodynamic therapy agents: (verteporfin, phthalocyanine Cytokines: (interferon, photosensitizer Pc4, demethoxy-hypocrelin A); Cytokines: (interferon Interferon α, interferon γ, tumor necrosis factor (TNF), TNF domain-containing human protein f) kinase inhibitors: BIBW2992 (anti-EGFR / Erb2), Imachi Nib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib Nib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (I NNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, Niparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, Bevacizumab, cetuximab, trastuzumab, ranibizumab, panitumumab, ispine g) Antibiotics: enediyne antibiotics (calicheamicins, especially calicheamicins) dynemicin γ1, δ1, α1, and β1; dynemicin including dynemicin A and deoxydynemicin Micin; Esperamicin, Kedarcidin, C-1027, Maduropeptin, and Neoca Ruzinostatin chromophore and related enediyne antibiotic chromophores. A, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin Mycins, cactinomycin, carabicin icin), carminomycin, carzinophilin; chromomycins, dactinomycin leucine, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin Sorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pi Lorino-doxorubicin and deoxydoxorubicin, epirubicin, idarubicin, malic acid Luceromycin, mitomycins, mycophenolic acid, nogalamycin, olivomycin peplomycin, potfilomycin, puromycin, querromycin, rhodomycin Rubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zino Statins, zorubicin; f) Other categories: Polyketides (acetogenins), especially Bullatacin and bullatacinone; gemcitabine, epoxomicins (carfilzomib), Bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, Xive Restat, PLX4032, STA-9090, Stimuvax , Allovectin-7, Zygeba, Provenge, Elvoy, isoprenylation inhibitors (Robas tatin), dopaminergic neurotoxin (1-methyl-4-phenylpyridine ion), cells Cycle inhibitors (staurosporine), actinomycins (actinomycin D, dactino bleomycin), bleomycins (e.g., bleomycin A2, bleomycin B2, bleomycin puromycin), anthracyclines (e.g., daunorubicin, doxorubicin (a) doriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxan Intron, MDR inhibitor (verapamil), Ca2+ ATP inhibitor (thapsigargin), steroid deacetylase inhibitors (vorinostat, romidepsin, panobinostat, Luproic acid, mocetinostat (MGCD0103), belinostat, PCI-2478 1. Entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); thapsigargin, celecoxib Sibu, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A , antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane; aceglatone; alveoli Dofosfamide glycoside; aminolevulinic acid; arabinoside, Bestravsil; Bisan Tren; Edatrexate; Defofamine, Demecolcine, Diazicon, Elforni DFMO, elliptinium acetate, etocluzid, gallium nitrate, gallic acid, Droxyurea; Ibandronate, Lentinan; Lonidamine; Mitoguazone; Mopidamol Nitraerin; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2 -Ethylhydrazide; Procarbazine; PSK®; Razoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2'' - trichlorotriethylamine; trichothecenes (especially T2 toxin, verrucarin A, Roridin A, and Anguidine); urethane, siRNA, antisense drugs, and nuclear Acid decomposition enzymes; 2) Anti-autoimmune disease drugs: cyclosporine, cyclosporine A, azathioprine, amino Caproic acid, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, Luticoids (hormonal drugs, betamethasone, budesonide, flunisolide, fluticasone protease inhibitors) Pionate ester, hydrocortisone, dexamethasone, fluocortodanazol, trimethoprim amcinolone acetonide, beclomethasone dipropionate), dehydroisoandrosterone ron, etanercept, hydroxychloroquine, infliximab, meloxicam, meth Trexate, mycophenolate mofetil, sirolimus, tacrolimus, prednisone ； 3) Anti-infectives: a) Aminoglycosides: Amikacin, Astromicin, Gentamycin Syn (netilmicin, sisomicin, isepamicin), hygromycin, kanamycin Cin (amikacin, arbekacin, aminodeoxykanamycin, dibekacin, tobrama isin), neomycin (neomycin B, paromomycin, ribostamycin), Lumycin, spectinomycin, streptomycin, tobramycin, verdamycin b) Amphenicols: azidamphenicol, chloramphenicol, florphenicol c) Ansamycins: geldanamycin, herbimycin d) Carbapenems: biapenem, doripenem, ertapenem, imipenem / silapenem Statins, meropenem, panipenem; e) Cephems: carbacephem (loracarbef) , cephacetrile, cefaclor, cephradine, cefadroxil, cephalonium, Farolidine, cephalothin or cephalosporin, cephalexin, cephaloglycin , cefamandole, cephapirin, cefatrizine, cefazaflur, cefazedone, Fazolin, cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminoc cefoxitin, cefprozil, cephalosporins, ceftezole, cefuroxime, Fixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, Cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefo Thiam, cefozopran, cephalexin, cefpimizole, cefpiramide, cefpirome , cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, ceftera ceftibuten, ceftiolene, ceftizoxime, ceftazidime, ceftriaxone, Cefuroxime, Cefazolin furan, Cephamycin (Cefoxitin, Cefotetan, cefmetazole), oxacephems (flomoxef, latamoxef); f) glycopeptides : Bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin, cubicin; g) glycylcyclines: chigesa Icrin; h) β-lactamase inhibitors: penams (sulbactam, tazobactam), clathrin; Bam (clavulanic acid); i) Lincosamides: clindamycin, lincomycin; j) Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDAs) k) Macrolides: azithromycin, cethromycin, clarithromycin, zirconium Thromycin, erythromycin, flurithromycin, josamycin, ketolide ( Telithromycin, Ethethromycin), Midecamycin, Miocamicin, Oleando mycin, rifamycin (rifampicin, rifampin, rifabutin, rifapentin) rokitamycin, roxithromycin, spectinomycin, spiramycin , tacrolimus (FK506), troleandomycin, telithromycin; l) mono Bactams: aztreonam, tigemonam; M) Oxazolidinones: linezolid; N) Penicillins: amoxicillin, ampicillin (pivanpicillin, hetacillin, bacampicillin) cillin, methampicillin, talampicillin), azidocillin, azlocillin, penicillin , benzathine penicillin, phenoxybenzathine penicillin, clometocillin, procaine penicillin, carbenicillin (calindacillin), cloxacillin, dicloxacillin , cephalosporins, flucloxacillin, mecillinam (pivmecillinam), mezlocillin methicillin, nafcillin, oxacillin sodium, phenethicillin, penicillin, Feneticillin, penicillin, piperacillin, propicillin, sulbenicillin, temocillin o) Polypeptides: bacitracin, polymyxin E, polymyxin B; p) quinolones: alatrofloxacin, balofloxacin, ciprofloxacin, Clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin ofloxacin, garenoxacin, gatifloxacin, gemifloxacin, gre Pafloxacin, kanotroafloxacin, levo Floxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin floxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, Trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, te Mafloxacin, tosufloxacin, trovafloxacin; q) streptozotocins: Pristinamycin, quinupristin / dalfopristin; r) sulfonamides: maf Enid, Prontosil, Sulfacetamide, Sulfamethoxazole, Sulfanilamido sulfasalazine, sulfafurazole, trimethoprim, trimethoprim-sul Famethoxazole (cotrimoxazole); s) steroidal antibacterial agents: e.g., Fusidi tetracyclines: doxycycline, chlortetracycline, chromosomal acid; Icrine, demeclocycline, lymecycline, meclocycline, methacycline, Minocycline, oxytetracycline, penimepicycline, rolitetracycline , tetracycline, glycylcycline (tigecycline); u) other types of antibiotics Quality: annonacin, arsphenamine, bactoprenol inhibitor (bacitracin), DA DAL / AR inhibitor (cycloserine), dictyostatin, discodermolide, Reuterobin, epothilone, ethambutol, etoposide, faropenem, fusidic acid, Furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolic acid methicone, NAM synthesis inhibitor (fosfomycin), nitrofurantoin, paclitaxel, Platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampin (rifampicin), tazobactamcinidazole, uvaricin; 4) Antiviral drugs: a) Entry / fusion inhibitors: aplaviroc, maraviroc, vicriviroc b) infliximab, gp41 (enfuvirtide), PRO140, CD4 (ibalizumab); c) maturation inhibitors: raltegravir, elvitegravir, globoidan A; d) neuraminidase inhibitors: oseltamivir, zolpidem namivir, peramivir; e) nucleosides and nucleotides: abacavir, acyclovir , adefovir, amdoxovir, apricitabine, brivudin, cidofovir, Kleb Din, 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 (FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC) , L-nucleosides (β-L-thymidine, β-L-2'-deoxycytidine), penicillin Lovir, Rasivir, Ribavirin, Stampidine, Stavudine (d4T), Talibavirin Viramidine, telbivudine, tenofovir, trifluridine, valacyclovir, f) non-nucleosides; Do: Amantadine, Ateviridine, Capravirine, Diarylpyrimidine (Etravirine , rilpivirine), delavirdine, docosanol, emivirine, efavirenz, fosca Lunet (phosphoryl formate), imiquimod, interferon alpha, loviride, rhodenosine , methisazone, nevirapine, NOV-205, peginterferon α, podophyllotoxin cin, rifampicin, rimantadine, resiquimod (R-848), tromantadine; g ) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, Suamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir vir, saquinavir, telaprevir (VX-950), tipranavir; H) antiviral drugs Other types: Abzyme, Arbidol, Calanolide A, Seragenin, Cyanovirin-N , DAPY, epigallocatechin gallate (EGCG), foscarnet, Griffithsin , taribavirin (viramidine), hydroxycarbamide, KP-1461, pleconaril , portmanteau inhibitors, ribavirin, seliciclib; 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、 １７７ Lu, ２１１ At, and ２１３ a radioisotope (radionuclide) selected from Bi; 6) Ability to absorb certain types of light such as UV light, fluorescent light, IR light, near-IR light, and visual light Chromophore molecules having: xanthophores, erythrophores, iridophores, leucophores, melanophores, cerelophores, and and a class or subclass of phosphors, said phosphors being fluorescent compounds that re-emit light with light. Chemical substances, visual light-sensitive molecules, light-emitting molecules, luminescent molecules, luciferin compounds Class or subclass, visible light transmitting molecule, light emitting molecule, luminescent molecule, luciferin compounds; for example, the following non-protein organic fluorophores: xanthene derivatives (fluorescein, thiazolidine, Oregon Green, Eosin, and Texas Red); cyanine derivatives (cyanine Carbocyanines, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, and merocyanines squalene derivatives and ring-substituted squaraines (Seta, SeTau, and Sq Naphthalene derivatives (including urea dyes); naphthalene derivatives (dansyl and prodan derivatives); coumarin derivatives Conductors: Oxadiazole derivatives (pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole); anthracene derivatives (DRAQ5, DRAQ7, and C anthraquinones including yTRAK Orange); pyrene derivatives (Cascade Blue etc.); oxazine derivatives (Nile Red, Nile Blue, Cresyl Violet, Oxazine Acridine derivatives (proflavine, acridine orange, acridine 170, etc.) Eraud et al.). Arylmethine derivatives (auramine, crystal violet, malachite) green). Tetrapyrrole derivatives (porphine, phthalocyanine, bilirubin); Any analogues and derivatives of the fluorescent compounds of: CF dyes (Biotium), DRAQ and CyT RAK probe (Bio-Status), BODIPY (Invitrogen), Alexa Fluor (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Att. o and Trancey (Sigma Aldrich), FluoProbes (Interchim), Abbe Abberior dyes (Abberior), DY and MegaStokes dyes (Dyomics), Sulf oCy dye (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau and Square dyes (SETA BioMedicals), Quasar and Cal Flour dyes (Biosearch Technologies), SureLight dyes (APC, RPEPerCP, F Cobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Ph yco-Biotech), allophycocyanin (APC), aminocoumarin, APC-Cy7 conjugate BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, Fluorescein, FluorX, Hydroxycoumarin, 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-NH S, Seta-680-NHS, Seta-780-NHS, Seta-APC-780 , Seta-PerCP-680, Seta-R-PE-670, SeTau380-N HS, SeTau405-maleimide, SeTau405-NHS, SeTau425- NHS, SeTau647-NHS, Texas Red, TRITC, TruRed, X- Rhodamine, 7-AAD (7-aminoactinomycin D, CG selective), acridine Range, chromomycin A3, CyTRAK Orange (Biostatus, red excitation dark), DA PI, DRAQ5, DRAQ7, ethidium bromide, Hoechst 33258, 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. Linking to the conjugates of the present invention for cell studies The fluorescent dye capable of this is selected from the following compounds or their derivatives: DCFH (2'7' Dichlorodihydrofluorescein, oxidized), DHR (dihydrorhodamine 123, acid (oxidized form, light catalyzed 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), Azami Green (monomer Body, MBL), azurite, B-phycoerythrin (BPE), cerulean, CyPet, DsRed monomer (Clontech), DsRed2 ("RFP", Clontech), EBFP, E BFP2, ECFP, EGFP (weak dimer, Clontech), Emerald (weak dimer, Invitrogen) ogen), EYFP (weak dimer, Clontech), GFP (S65A mutant), GFP (S65 C mutant), GFP (S65L mutant), GFP (S65T mutant), GFP (Y66F mutant), GFP (Y66H mutant), GFP (Y66W mutant), GFP ｕｖ , HcR ed1, J-Red, Katyusha, Kusabira Orange (monomer, MBL) , mCFP, mCherry, mCitrine, Midoriishi Cyan (dimeric mKate (TagFP635, monomer, Evrogen), mKeima-Red (monomer, MBL), mKO, mOrange, mPlum, mRaspberry, mR FP1 (monomer, Tsien Lab), mStrawberry, mTFP1, mTu rquoise2, P3 (phycobilisome complex), Peridinin Chlor ophyl (PerCP), R-phycoerythrin (RPE), T-Sapphire , TagCFP (dimer, Evrogen), TagGFP (dimer, Evrogen), TagRFP (dimer, Evrogen), TagYFP (dimer, Evrogen), tdTomato (tandem Dimer), Topaz, TurboFP602 (dimer, Evrogen), TurboFP63 5 (dimer, Evrogen), TurboGFP (dimer, Evrogen), TurboRFP (dimer Venus, natural GF P, YPet, Zs Green 1 (tetramer, Clontech), Zs Yellow 1 (tetramer, Clontech) ch). 7) Pharmaceutically acceptable salts, acids, or derivatives of any of the above drugs.

7. The aforementioned "Drug １ " and "Drug ２ " is a chromophore and is useful for detection, monitoring, or pre- The interaction of the cell-binding molecule and / or the conjugate with a target, in particular a target cell, and / or The compounds of formula (II) and (I) according to claims 2 and 4 can be used for functional studies. V).

8. The aforementioned "Drug １ " and "Drug ２ " is a cell-binding molecule that binds to a Polyalkylene glycols or polyalkylene glycols used to extend half-life The polyalkylene glycol can be a carboxylic acid analogue, and the polyalkylene glycol can be from about 10 daltons to about 200 daltons. Poly(ethylene glycol) (PEG), poly(propylene glycol) having a molecular weight of 100 kDa 2 and 4, which are copolymers of ethylene oxide, ethylene oxide or propylene oxide. Compounds of formula (II) and (IV) according to the formula (II) and (IV)

9. The aforementioned "Drug １ " and "Drug ２ " to deliver the conjugate to malignant cells, so that the conjugate can act as a targeting conductor / director, or They may also modulate or co-stimulate a desired immune response or alter signaling pathways. The ligand may be a cell-binding ligand or receptor, or a receptor analogue, such that the ligand can bind to the receptor. Compounds of formula (II) and (IV) according to claims 2 and 4.

10. The aforementioned "Drug １ " and "Drug ２ " is preferably a tubulysin, a calicheama Isins, auristatins, maytansinoids, CC-1065 analogs, daunorubicin cin and doxorubicin compounds, taxanoids (taxanes), cryptophycins, Epothilones, benzodiazepine dimers (e.g., pyrrolobenzodiazepines (PBDs), Tomaymycin, anthramycin, indolinobenzodiazepines, imidazobenzothiazolinone dimers of diazepines or oxazolidinobenzodiazepines), calicheamicins and enediyne antibiotics, actinomycin, azaserines, bleomycins, epi Rubicin, tamoxifen, idarubicin, dolastatins / auristatins (e.g. , monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin statin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEF P), duocarmycins, thiotepa, vincristines, hemiasterins, nas Nazumamides, microginins, la Radiosumins, Alterobactins ins), microsclerodermicins, Nelamides, esperamicins ns), siRNA, nucleases, and / or pharmaceutically acceptable derivatives of any of the above molecules.

5. The compound according to claim 2, wherein the compound is selected from the group consisting of salts, acids, and / or analogs and derivatives thereof. The conjugate compound described above.

11. The cell binding agent / molecule may be an antibody, a protein, a vitamin (e.g., folic acid), a peptide , polymer micelles, liposomes, lipoprotein-based drug carriers, nanoparticle drug carriers, dendrites and molecules coated with cell-binding ligands, or combinations thereof.

4. The conjugate compound according to claims 2 and 3.

12. The cell-binding molecules / agents of claims 2, 3, and 11 are antibodies, full-length antibodies (polyclonal antibodies), and the like. monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) single-chain antibodies, antibody fragments that bind to target cells, monoclonal antibodies, single-chain monoclonal antibodies monoclonal antibodies, monoclonal antibody fragments that bind to target cells, chimeric antibodies, and antibodies that bind to target cells chimeric antibody fragments that bind to target cells, domain antibodies, domain antibody fragments that bind to target cells, resurface reconstitution Shaped antibodies, single-chain resurfaced antibodies, resurfaced antibody fragments that bind to target cells, humanized antibodies antibody, resurfaced humanized antibody, single-chain humanized antibody, humanized antibody fragment that binds to target cells, Idiotypic (anti-Id) antibodies, CDRs, dimers, trimers, tetramers, miniantibodies, small molecule immunogens SIP, lymphokines, hormones, vitamins, growth factors, colony-stimulating Factors, nutrient transport molecules, or large molecular weight proteins are preferred.

13. The cell-binding molecules / agents according to claims 2, 3 and 11 are capable of binding to tumor cells, virus-infected cells, and the like. cells, microbially infected cells, parasite infected cells, autoimmune disease cells, activated tumor cells, bone marrow cells, Agents capable of targeting activated T cells, affected B cells, or melanocytes It can be said that:

14. The cell-binding molecule / agent according to claims 2, 3, and 11 binds to any of the following antigens or receptors: It can be any agent / molecule that is available to the body: CD3, CD4, CD5, CD6, CD7 , CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD 14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, C D22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, C D30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, C D38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, C D46, CD47, CD48, CD49b, CD49c, CD51, CD52, CD53 , CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD6 2L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, C D74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83 , CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96 , CD98, CD100, CD103, CD105, CD106, CD109, CD11 7, CD120, CD125, CD126, CD127, CD133, CD134, CD 135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD16 3, CD166, CD168, CD174, CD180, CD184, CDw186, C D194, CD195, CD200, CD200a, CD200b, CD209, CD2 21, CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4- 1BB, 5AC, 5T4 (trophoblast glycoprotein, TPBG, 5T4, Wnt activation inhibitor) WAIF1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor Body-like kinase 1, AFP, AKAP-4, ALK, α integrin, αvβ6, aminopeptide Putidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin Yetin 3, Annexin A1, Anthrax toxin protective antigen, Anti-transferrin receptor, A OC3 (VAP-1), B7-H3, Bacillus anthrax, BAFF (B-cell activating factor), B-reactive Lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA12 5 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9) , CALLA, CanAg, canine IL31, carbonic anhydrase IX, cardiac myosin, CCL1 1 (C-C motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD19 4), CCR5, CD3E (epsilon), CEA (carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (factor D), Ch4D5, cholecystokinin 2 (CCK2R), CLDN18 (claudin-18), clumping factor A, CRIP TO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2 receptor, CD115), CTLA4 (cytotoxic T cell proliferation factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), ... Cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (C D184), CXC chemokine receptor type 4, cADP ribose hydrolase, Cyclin n B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B , dabigatran, DLL4 (delta-like ligand 4), DPP4 (dipeptidyl peptidase 4) 4), DR5 (death receptor 5), E. coli Shiga toxin type 2, ED-B, EGFL7 ( Protein 7 containing EGF-like domain), EGFR, EGFRII, EGFRvIII, Doglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell cell adhesion molecule), EphA2, episialin, ERBB2 (epidermal growth factor receptor 2), ER BB3, ERG (TMPRSS2ETS fusion gene), E. coli, ETV6-AML, FA P (fibroblast activation protein α), FCGR1, α-fetoprotein, fibrin II, β chain, fibronectin ectodomain B, FOLR (folate receptor), folate receptor α , folate hydrolase, Fos-related antigen 1, respiratory syncytial virus F protein, Frizzle d receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface glycolipid antigen), GD3 idiotype, GloboH, glypican 3, N-glycolylneuraminic acid, G M3, GMCSF receptor α chain, growth differentiation factor 8, GP100, GPNMB (transmembrane protein Protein NMB), GUCY2C (guanylate cyclase 2C, guanylate cyclase C (G C-C), intestinal guanylate cyclase, guanylate cyclase-C receptor, heat-stable enteric apoptosis receptor (hSTAR), heat shock protein, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), IgG4, HGF / SF (stem cell growth factor / cell scatter factor), HHGFR, HIV-1, histone complex, HLA-DA (human leukocyte antigen ), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, H NGF, human scatter factor receptor kinase, HPV E6 / E7, Hsp90, hTER T, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, IGF-1R) insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, L-12, IL-13, IL-17, IL-17A, IL-20 , IL-22, IL-23, IL-31RA, ILGF2 (insulin-like growth factor 2), インテグリン(a4、a ＩＩＩｂ b ３ ,αvβ3,α ４ b ７ 、α5β1、α6β4、α7 β7, αIIβ3, α5β5, αvβ5), interferon-γ-inducible protein, ITA GA2, ITGB2, KIR2D, LCK, Le, legumain, Lewis-Y antigen, LFA -1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, Lipoteicho Acid, LIV1A, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE- 1, MAGE-2, MAGE-3, MAGEA1, MAGEA3, MAGEA4, MAR T1, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor (G IF), MS4A1 (transmembrane 4 domain subfamily A member 1), MSLN (membrane sothelin), MUC1 (Mucin 1, cell surface associated (MUC1) or Polymorphism c epithelial mucin (PEM)), MUC1-KLH, MUC16 ( CA125), MCP1 (monocyte chemotactic protein 1), MelanA / MART1, ML -IAP, MPG, MS4A1 (transmembrane 4 domain subfamily A), MYCN, Erin-related glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granules cytoplasmic antigen), Nectin-4 (ASG-22ME), NGF, neuroapoptosis control protein Proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein Quality), OY-TES1, P21, non-mutant p53, P97, Page 4, PAP,, anti-( N-glycolylneuraminic acid) paratopes, PAX3, PAX5, PCSK9, PDC D1 (PD-1, programmed cell death protein 1, CD279), PDGF-Rα , (platelet-derived growth factor receptor α), PDGFR-β, PDL-1, PLAC1, PLA P-like testicular alkaline phosphatase, platelet-derived growth factor receptor β, sodium phosphate cotransporter transporter, PMEL17, polysialic acid, proteinase 3 (PR1), prostate cancer, PS (ho Sphatidylserine), prostate cancer cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, rabies virus virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), Akage Rhesus factor, RANKL, PhoC, Ras mutant, RG 55, ROBO4, RS virus, RON, sarcoma metastasis breakpoint, SART3, Clerostin, SLAMF7 (SLAM family member 7), selectin P, SDC 1 (syndecan 1), sLe(a), somatomedin C, SIP (sphingosine-1-phosphate dehydrogenase phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (prostate 1 six-transmembrane epithelial antigen), STEAP2, STn, TAG-22 (tumor-associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor epithelial matrix metalloproteinase 1), TENB2, tenascin C (TN-C), TGF-α, TGF-β (tran Transforming growth factor β), TGF-β1, TGF-β2 (transforming growth factor β), factor β2), Tie (CD202b), Tie2, TIM-1 (CDX-014), TN , TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor stimulator TNFRSF13B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B (tumor necrosis factor receptor superfamily member 10B), Family member 13B), TPBG (trophoblast glycoprotein), TRAIL-R1 (tumor Necrosis-inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5) ), tumor-specific glycosylation of major-associated calcium signal transducer 2 (MUC1) lysation, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, tyrosinase , VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309) , VEGFR-1, VEGFR2, or vimentin, WT1, XAGE1, or any Cells expressing insulin growth factor receptor, or any epidermal growth factor receptor.

15. The tumor cells according to claim 13 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

16. The linker component R according to claim 1, 2, 3, and / or 4 １ and / or R ２ 6-Male Imidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (v al-cit), alanine-phenylalanine (ala-phe), lysine-phenylalanine Lanine (lys-phe), p-aminobenzyloxycarbonyl (PAB), 4-thio -pentanoic acid ester (SPP), 4-(N-maleimidomethyl)cyclohexane-1- Carboxylic acid ester (MCC), 4-thio-butyric acid ester (SPDB), maleimide ethyl 4-thio-2-hydroxysulfonyl-butyric acid ester (2-sulfo-S PDB), pyridinyl-dithiol (PySS), alkoxyamino (AOA), ethyl 4-methyl-4-dithioester-pentanoic acid (MPDP), azide (N ３ ), alkynyl, dithio, peptide, and / or (4-acetyl)aminobenzoic acid It may be composed of one or more linker components of (SIAB).

17. "Drug １ " and "Drug ２ " is a tubulysin analogue The conjugate compound of formula (II) is preferably one of the following: T01, T02, T03, T 3. The conjugated compound of claim 2, having the structure of T04, T05, T06, and T07: 【Chemistry 15】 【change】 During the ceremony: mAb is an antibody; Z ３ and Z' ３ are independently H, 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 sides, mannosides, glucuronosides, allosides, fructosides), NH-glycosides, S-glycosides Glycoside or CH ２ - is a glycoside; M １ and M ２ are independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and n is 1 to 30; 【Chemistry 16】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 do.

18. "Drug １ " and "Drug ２ " is calicheamicin ) analogues, and the conjugate compound of formula (II) preferably has the following structure C01: The conjugated compound of claim 2, 【Chemistry 17】 During the ceremony: mAb is an antibody; n is 1 to 30; [Chemistry 18] X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 do.

19. "Drug １ " and "Drug ２ " is maytansinoid The conjugate compound of formula (II) is preferably an analogue of the following structure M01:

3. The conjugated compound of claim 2: 【Chemistry 19】 During the ceremony: mAb is an antibody; n is 1 to 30; 【Chemistry 20】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 do.

20. "Drug １ " and "Drug ２ " is a taxane analogue, and The conjugate compound of (II) is preferably represented by the following structures Tx01, Tx02, and Tx03:

3. The conjugated compound of claim 2, wherein: 【Chemistry 21】 During the ceremony: mAb is an antibody; n is 1 to 30; 【Chemistry 22】 X １ , X ２ , R １ , and R ２ is the same as defined in claims 1 and 2.

21. "Drug １ " and "Drug ２ " is a CC-1065 analogue and / or duocarmycete The conjugate compound of formula (II) is preferably a duocarmycin analogue. The conjugate of claim 2, which is of the following structures CC01, CC02, and CC03: Compound: 【Chemistry 23】 During the ceremony: mAb is an antibody; n is 1 to 30; Z ４ and Z ４’ are independently H, PO(O)(OM １ ) (OM ２ ), C.H. ２ PO(O) (OM) １ )(OM ２ ), SO ３ M １ , H ３ N(CH ２ CH ２ ) ２ NC(O)-、O(C H ２ CH ２ ) ２ NC(O)-, R １ or a glycoside; X ３ and X ３’ are independently O, NH, NHC(O), OC(O), —C(O)O, or Or R １ or not present; 【Chemistry 24】 X １ , X ２ , R １ , R ２ , M １ , and M ２ is the same as defined in claims 1 and 2 That is correct.

22. "Drug １ " and "Drug ２ " is daunorubicin or a doxorubicin analogue, The conjugate compound of formula (II) is preferably the following Da01, Da02, Da03, and Da04, 【Chemistry 25】 During the ceremony: mAb is an antibody; n is 1 to 30; X ３ and X ３’ are independently H, O, NH, NHC(O), NHC(O)NH, C(O) ), R １ or OC(O) 【Chemistry 26】 X １ , X ２ , R １ , and R ２ is the same as defined in claims 1 and 2.

23. "Drug １ " and "Drug ２ " are auristatins and dolastatins (dolastatins) The conjugate compound of formula (II) is preferably AuO1 , Au02, Au03, Au04, and Au05. Compound: 【Chemistry 27】 During the ceremony: mAb is an antibody; n is 1 to 30; X ３ and X ３’ are independent, CH ２ ,O,NH,NHC(O),NHC(O)NH,C (O), or OC(O)R １ or absent; X ４ and X ４’ are independent, CH ２ , C(O), C(O)NH, C(O)N(R １ ), R １ , N.H.R. １ , N.R. １ , C(O)R １ or C(O)O; Z ３ and Z ３’ are independently H, R １ , OP(O)(OM １ ) (OM ２ ), N.H.R. １ , OCH ２ OP (O) (OM １ ) (OM ２ ), OSO ３ M １ , or O-glycosides (glucosides , galactosides, mannosides, glucuronosides, alosides, fructosides), NH-glycosides S-glycoside or CH ２ - is a glycoside; M １ and M ２ are independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and 【Chemistry 28】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claim 1 and 。

24. "Drug １ " and "Drug ２ " is a benzodiazepine dimer analog, and The conjugate compound of II) is preferably the following PB01, PB02, PB03, PB04, In the structures of PB05, PB06, PB07, PB08, PB09, PB10, and PB11 3. The conjugated compound of claim 2, which is: 【Chemistry 29】 【change】 【change】 During the ceremony: mAb is an antibody; n is 1 to 30; X ３ and X ３’ are independent, CH ２ ,O,NH,NHC(O),NHC(O)NH,C (O),OC (O),OC (O)(NR). ３ ),R １ 、NHR １ 、NR １ ,C(O)R １ 、 or C(O)O or absent; X ４ and X ４’ are independent, CH ２ , C(O), C(O)NH, C(O)N(R １ ), R １ , N.H.R. １ , N.R. １ , C(O)R １ or C(O)O; M １ and M ２ are independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and 【Transformation 30】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 In addition, R １ and / or R ２ may not be present.

25. The aforementioned "Drug １ " and "Drug ２ " is preferably a tubulysin, maytansin, oids, taxanoids (taxanes), CC-1065 analogs, daunorubicin, and doxorubicin compounds, benzodiazepine dimers (e.g., pyrrolobenzodiazepines (P BD), tomaymycin, anthramycin, indolinobenzodiazepines, imidazoline Dimers of benzothiadiazepines or oxazolidinobenzodiazepines), calicheamases Isin and enediyne antibiotics, actinomycin, azaserines, bleomycin epirubicin, tamoxifen, idarubicin, dolastatins, auristatins (e.g., monomethyl auristatin E, MMAE, MMAF, auristatin PYE, Auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, thiotepa, vincristines, hemiasterin nazumamides, microginins s), radiosumins, alterobactins bactins, microsclerodermicins s), theonellamides, esperamicins ramicsins), PNU-159682, and any of their analogs and derivatives 3. The conjugate of claim 2, wherein the two different cytotoxic agents are selected from the following combinations: is a compound, A more preferred structure of a conjugate containing two or more different cytotoxic agents via a bridge linker is: Below Z01, Z02, Z03, Z04, Z05, Z06, Z07, Z08, Z09, Z1 0, Z11, Z12, Z13, Z14, Z15, Z16, Z17, and Z18. Ru: 【Chemistry 31】 【change】 【change】 【change】 During the ceremony: mAb is an antibody; n is 1 to 30; X ３ and X ３’ are independent, 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 absent; X ４ and X ４’ are independently H, CH ２ , OH, O, C(O), C(O)NH, C(O ) N (R １ ), R １ , N.H.R. １ , N.R. １ , C(O)R １ or C(O)O; M １ and M ２ are independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and 【Chemistry 32】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 In addition, R １ and / or R ２ may not be present.

26. "Drug １ " and "Drug ２ " is a polyalkylene glycol analogue, The conjugate compound of (II) preferably has the following structure Pg01: Conjugate Compounds: 【Transformation 33】 During the ceremony: mAb is an antibody; n is 1 to 30; R' and R'' are independently H or CH ３ and m ３ and m ４ are independently 1 to 5000; 【Transformation 34】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 Ri, R ４ is OH, H, or R as defined in claim 1 １ Or R ３ is.

27. "Drug １ " and "Drug ２ " is a cell-binding ligand or receptor analog, The conjugate compound of formula (II) is preferably the following LB01 (PMSA ligand conjugate): LB02 (folate receptor conjugate), LB03 (somatostatin receptor conjugate), LB04 ( Octreotide, somatostatin analogue receptor conjugate), LB05 (lanreotide, somatostatin analogue receptor conjugate), tostatin analog receptor conjugate), LB06 (CAIX receptor conjugate), LB07 (CA IX receptor conjugate), LB08 (luteinizing hormone-releasing hormone (LH-RH) ligand and GnRH conjugate), LB09 (luteinizing hormone-releasing hormone (LH-RH) and G nRH ligand conjugate), LB10 (GnRH antagonist, abarelix conjugate) , LB11 (cobalamin, VB12 analog conjugate), LB12 (gastrin-releasing peptide receptor (GRPr), MBA conjugate), LB13 (α ｖ β ３ Integrin receptor, cyclic R GD pentapeptide conjugate), LB14 (hetero-bivalent peptide linker of VEGF receptor conjugate) Gand), LB15 (neuromedin B conjugate), LB16 (G protein-coupled receptor, bombesin conjugate), and LB17 (Toll-like receptor, TLR ２ The structure of the conjugate 3. The conjugated compound of claim 2: 【Chemistry 35】 【change】 【change】 【change】 【change】 During the ceremony: mAb is an antibody; n is 1 to 30; X ３ and X' ３ are independent, 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 absent; X ４ and X' ４ are independently H, CH ２ , OH, O, C(O), C(O)NH, C(O ) N (R １ ), R １ , N.H.R. １ , N.R. １ , C(O)R １ or C(O)O; M １ and M ２ are independently H, Na, K, Ca, Mg, NH ４ , N.R. １ R ２ R ３ and m ３ and m ４ are independently 0 to 5000; 【Transformation 36】 X １ , X ２ , R １ , R ２ , and R ３ is the same as defined in claims 1 and 2 In addition, R １ and / or R ２ may not be present.

28. A therapeutically effective amount of claims 2, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6 and / or 27, and pharmaceutically acceptable salts, carriers, diluents a method for treating cancer, autoimmune disease, or infectious disease, comprising administering to said patient an effective amount of a therapeutic agent, a therapeutic agent, or an excipient, or a combination thereof; A pharmaceutical composition for treating or preventing a disease.

29. In vitro, in vivo, or ex vivo x vivo) have a cell-killing activity.

28. The conjugate according to claim 2, 23, 24, 25, 26, and / or 27.

30. The linker component R １ and / or R ２ 1 to 20 units of natural or unnatural amino acid Acid peptide, p-aminobenzyl unit, 6-maleimidocaproyl unit, disulfide unit, thioether unit, hydrazone unit, triazole unit, or alkoxy unit Claims 2, 17, 18, 19, 20, 21, 22, and 23 may include any of the following:

28. The conjugate according to claim 24, 25, 26, and / or 27.

31. The linker component R １ and / or R ２ is cleavable by a protease, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and / or 27 Conjugate of.

32. A therapeutically effective amount of claims 2, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 29. A method for treating a chemotherapeutic agent, a radiotherapeutic agent, an immunotherapeutic agent, or a combination thereof, comprising administering to a patient a therapeutically effective amount of a compound according to claim 6, 27, or 28. co-administered with other therapeutic agents such as drugs for autoimmune diseases, anti-infective drugs, or other conjugates; A pharmaceutical composition for the treatment or prevention of cancer, autoimmune diseases, or infectious diseases, which is synergistically effective.

33. The synergist according to claim 32 is preferably selected from one or several of the following agents: Abatacept (Orencia), abiraterone acetate (Zytiga®), acetaminophen phenanthrene / hydrocodone, adalimumab, afatinib dimaleate (Gilotrif (Trademark), alemtuzumab (Campath®), alitretionin (Panretin®), (registered trademark), ado-trastuzumab (Kadcyla™), amphetamine mixed salts ( amphetamine / dextroamphetamine, or adderall XR), anastrozole (A rimidex®), aripiprazole, atazanavir, atezolizumab (MPDL3280A ), atorvastatin, axitinib (Inlyta®), belinstat (Beleod®) aq™), bevacizumab (Avastin®), cabazitaxel (Jevtana®), (Registered Trademark), cabozatinib (Cometriq™), bexarotene (Targretin® )), blinatumomab (Blincyto™), bortezomib (Velcade®), Bosutinib (Bosulif®), brentuximab vedotin (Adcetris®) )), budesonide, budesonide / formoterol, buprenorphine, capecitabine, Lufilzomib (Kyprolis®), celecoxib, ceritinib (LDK378 / Zykadia ), cetuximab (Erbitux®), cyclosporine, cinacalcet, crizotinib tinib (Xalkori®), dabigatran, dabrafenib (Tafinlar®) )), darbepoetin alfa, darunavir, imatinib mesylate (Gleevec®), Dasatinib (Sprycel®), denileukin diftitox (Ontak®) ), denosumab (Xgeva®), Depakote, dexlansoprazo acetaminophen, dexmethylphenidate, dinutuximab (Unituxin™), doxycycline Phosphorus, duloxetine, emtricitabine / rilpivirine / tenofovir disopro fumarate Xyl, emtricitabine / tenofovir / efavirenz, enoxaparin, Enzalutamide mide (Xtandi®), epoetin alfa, erlotinib (Tarceva®), Esomeprazole, eszopiclone, etanercept, everolimus (Afinitor (registered trademark) (Aromasin®), exemestane (Aromasin®), everolimus (Afinitor®), )), ezetimibe, ezetimibe / simvastatin, fenofibrate, filgrasti fluticasone, fingolimod, fluticasone propionate, fluticasone / salmeterol, fluticasone Vestruant (Faslodex®), gefitinib (Iressa®), and gracilis Ramar, goserelin acetate (Zoladex), imatinib (Gleevec), ibritumomab tiuki Cetan (Zevalin®), ibrutinib (Imbruvica™), idelalisib ( Zydelig®), infliximab, insulin aspart, insulin dete Mil, insulin glargine, insulin lispro, interferon beta-1a, inter Ferron beta 1b, lapatinib (Tykerb®), ipilimumab (Yervoy®) ipratropium bromide / salbutamol, lanreotide acetate (Somatuline (registered trademark)), (Trademark) Depot), lenaliomide (Revlimid®), lenvatinib mesylate (Lenvi ma™), letrozole (Femara®), levothyroxine, , lidocaine, linezolid, liraglutide, lisdexamfetamine, MEDI4736 (AstraZeneca, Celgene), memantine, methylphenidate, metoprolol, Modaf nilotinib (Tasigna®), nivolumab (Opdivo®), mometasone, nilotinib (Tasigna®), and (Arzerra®), ofatumumab (Arzerra®), obinutuzumab (Gazyva®), ), olaparib (Lynparza™), olmesartan, olmesartan / hydrochloride Thiazides, omalizumab, omega-3 fatty acid ethyl esters, oseltamivir, oxycodone, Palbociclib (Ibrance®), palivizumab, panitumumab (Vectibix®) (Registered Trademark), panobinostat (Farydak®), pazopanib (Votrient®), trademark), pembrolizumab (Keytruda®), pemetrexed (Alimta®), lutuzumab (Perjeta™), pneumococcal conjugate vaccine, pomalidomide (Pomalyst™) (Registered trademark), pregabalin, quetiapine, rabeprazole, radium 223 chloride (Xo figo®), raloxifene, raltegravir, ramucirumab (Cyramza®), trademark), ranibizumab, regorafenib (Stivarga®), rituximab (Ri tuxan®), rivaroxaban, romidepsin (Istodax®), rosuva Statins, ruxolitinib (Jakafi™), salbutamol, sevelamer, sildenafil Phil, siltuximab (Sylvant™), sitagliptin, sitagliptin / methicillin Formin, solifenacin, sorafenib (Nexavar®), sunitinib (Suten t (registered trademark), tadalafil, tamoxifen, telaprevir, temsirolimus (Tor isel®), tenofovir / emtricitabine, testosterone gel, thalidomide (Immunoprin, Talidex), tiotropium bromide, toremifene (Fareston (registered trademark), trametinib (Mekinist®), trastuzumab, tretinoin (Benib Sanoid®), ustekinumab, valsartan, vandetanib (Caprelsa®), (Registered Trademark), vemurafenib (Zelboraf®), vorinostat (Zolinza®), (registered trademark), divaflibercept (Zaltrap®), and Zostavax, and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents or the like therefor. is an excipient, or a combination thereof.

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