CN112826940A - Conjugates of cytotoxic molecules and cell-binding receptor molecules - Google Patents

Abstract

The conjugate of potent cytotoxic molecule and cell-binding receptor molecule has the structure of formula (I), wherein T, L, m, n, R, and¹，R²，R³，R⁴，R⁵，R⁶，R⁷，R⁸，R⁹，R¹⁰，R¹¹，R¹²and R¹³As defined herein. The conjugates are useful for treating cancer, immune disorders and infectious diseases.

Description

Conjugates of cytotoxic molecules and cell-binding receptor molecules

The present application is a divisional application of a patent application having an application date of 11/2014 and an application number of CN201410631737.3 and entitled "conjugate of cytotoxic molecule and cell binding receptor molecule".

Technical Field

The invention relates to a conjugate of high-efficiency cytotoxin molecules and cell surface receptors, which is suitable for targeted therapy, a synthetic method thereof and application of a conjugate structure containing the conjugate in the aspects of treating cancers, autoimmune system diseases and infectious diseases.

Technical Field

Many documents report methods for targeting various pathogenic cells, such as cancer cells, by conjugated binding of cell surface receptor binding molecules to cytotoxic molecules. Reporter cell surface receptor binding molecules are known to include: antibodies (sea et al in Immunoconjugates 189-216(C.Vogel, ed.1987); Ghose et al in Targeted Drugs 1-22(E.Goldberg, ed.1983); diene et al in Antibody mediated Delivery systems 1-23(J.Rodwell, ed.1988); Silverstein, Nat.Immunol.2004, 5, 1211-7; Fanning et al, Clin.Immunol.Immunol.1996, 79, 1-14; Ricart A.D., et al, Nature Clinical Practice 2007, 4, 245-255; Singh R.Rickson H.K., pharmaceutical Antibodies: Methods 147, 23-60; folic acid J.2009, 85-60); prostate specific membrane antigen binding ligand (PMSA) (Low, et al, WO 2009/026177 a 1); albumin polypeptide (Temming, et al, Bioconjugate chem.2006, 17, 1385-1394); cobalamin and proteins (Gupta, et al, Crit. Rev. therap. drug Carrier Syst.2008, 25, 347-79; Petrus, et al, Angew. chem. iht. Ed.2009, 48, 1022-8); carbohydrate molecules (Darbre, et al, curr. top. med. chem.2008, 8, 1286-93); bioactive macromolecules (Dhar, et al, proc.natl.acad.sci.2008, 105, 17356-61); dendritic polymers (Lee, et al, nat. Biotechnol.2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci.2009, 106, 685-90); nanoparticles to which binding ligands are attached (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, nat. Med.2007, 13, 372-7; Javier, et al, Bioconjugate chem.2008, 19, 1309-12); liposomes (Medinai, et al, curr. phar. des.2004, 10, 2981-9); viral capsids (Flenniken, et al, Virus Nanotechnol.2009, 327,71-93), and the like.

Many classes of cytotoxins have been used to form antibody drug conjugates with cell binding bodies, particularly antibodies (Wu, et al, nat. Biotechnol.2005, 23, 1137-1146.Ricart, et al, nat. Clin.Pract. Oncol.2007, 4, 245-. These cytotoxins include: calicheamicin derivatives (Giles, et al Cancer 2003, 98, 2095-, 8,2175-84; WO 2007102069), doxorubicin (Trail, et al, Science 1993, 261, 212-5; saleh et al, J Clin Oncol 2000, 18, 2282-92), and methotrexate, vincristine, vinblastine, daunorubicin, mitomycin C, melphalan, and chlorambucil derivatives.

The use of cell surface receptor binding molecules, particularly targeting antibodies which have a binding effect on cell surface antigens, allows the toxin molecules to be directly transported to the surface of or near pathogenic cells, thus enhancing the efficiency of the action of the toxin molecules and at the same time minimizing the side effects of such toxin molecules. To date, some biologically active short-chain peptide compounds have been isolated from nature, including tubulysin (structure as shown in the figure below). Tubulysin was initially isolated from the culture medium of the myxobacteria of Archangium gephyra by Hofle and Reischenbach (GBF Braunschweig) (F.Sasse et al.J.Antibiot.2000, 53, 879-885; WO9813375),

(structural formula of known tubulysin Compound)

Tubulysin is an antimitotic peptide that inhibits tubulin polymerization during cell division and thus induces apoptosis. Tubulysin is a promising lead compound for targeted therapy due to its significantly superior activity to vinblastine, taxol and epothilones (Wipf, et al, org. Lett.2004, 6, 4057-60; Peltier, et al, J.am. chem. Soc.2006, 128, 16018-9; Wipf, et al, org. Lett.2007, 9, 1605-. Structurally, Tubulysin consists of four peptide bonds, which include the N-terminal N-methylpiperidine acid (Mep), the second amino acid isoleucine (Ile), the third amino acid tubulivaline (Tuv), and either the two amino acids tubularis (Tut) or tubulylalanine (Tup), which may be C-terminal. Currently, although several tubulysins have been synthesized, they all produce significant toxic effects (> 20% weight loss) at doses that achieve the desired therapeutic effect (US patent appl.2010/0048490). There has been an ongoing effort to develop antibody drug conjugates, including those using tubulysin, with the goal of achieving the desired therapeutic effect while avoiding undesirable toxic side effects. For this reason, the problem to be solved is that solubility of Tubulysin is too low, which often leads to a large amount of antibody aggregation when they form conjugates with antibodies. In addition, simple Tubulysin homologues, such as phenylalanine (Phe) and tyrosine (Tyr) in place of Tup and Tut, respectively, have low stability in animals and consequently low biological activity (at least 300 times less than that of Tubulysin A and D). The present invention will describe the use of tubulysin-like molecules with better water solubility, higher stability and lower toxic side effects for the construction of cell binding molecule-drug conjugates and the corresponding use of such conjugates for the targeted treatment of cancer and immune diseases.

Summary of The Invention

An antibody drug conjugate of the present invention is a conjugate having the structural formula (I):

where T is a targeting or cell binding agent; l is a cleavable linker; represents a linkage linking L to an atom on the bracketed structure; n is equal to 1 to 20; m is equal to 1-10.

The structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Each represents C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(2-8 carbon atoms) heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl (having 3 to 8 carbon atoms); or two R groups, e.g. R¹R²，R²R³，R³R⁴，R⁵R⁶，R¹²R¹³Two together may be C₃-C₇A carbocyclic group (containing 3-7 carbons), a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R³And R⁴May be H, andR²may be absent.

R⁵、R⁶、R⁸And R¹⁰Are each H or C₁～C₄An alkyl group or a heteroalkyl group (containing 1 to 4 carbon atoms).

R⁷Can be independently selected from H and R¹⁴or-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵. Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkane, or C₃-C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; x¹Is O, S, S-S, NH or NR¹⁴。

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴Op(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

R¹¹Is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶、-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units.

R¹²Is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH₂OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶。R¹⁴Is C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl. R¹⁶Is H, OH, R¹⁴Or 1-4 amino acid units.

R¹³Is C₁-C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar denotes an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each of which contains from 4 to 10 carbon atoms, preferably from 4 to 6 carbon atoms. Heteroaryl refers to an aromatic ring in which one or more carbon atoms are replaced by a heteroatom, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms. Aryl Ar also refers to an aromatic or heteroaromatic ring substituted with one or more H atoms, the H atom-substituted groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂A glycoside or a pharmaceutically acceptable salt.

In addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)R¹⁸、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄-C₁₂(having 4 to 12 carbon atoms) glycoside or C₁-C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁-C₈Alkyl, carboxyalkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, or C₃-C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁-C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃-C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Respectively N or CH.

Or, when R is¹¹When it is the group¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁-C₆Alkyl or heteroalkyl.

In another embodiment, the linker used to link the antimitotic agent to the cell binding body has the formula-Ww- (Aa) r-Vv-, wherein-W-is an extender and W is 0 or 1; each-Aa-is an independent amino acid unit, r is from 0 to 12; -V-is spacer and V is 0, 1 or 2. The extensions W may independently comprise a spacer capable of autodecomposition, a peptide, a hydrazone bond, a disulfide bond or a thioether bond.

In another embodiment, the cell-binding agent T may be any form of cell-binding agent, such as a peptide or non-peptide structure. Generally, T is an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to a target cell, a chimeric antibody fragment that binds to a target cell, a domain antibody fragment that binds to a target cell, an engineered protein that mimics an antibody (e.g., fibronectin-binding adnectin), a predesigned ankyrin repeat protein (DARPin), a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transporting molecule (transferrin), or a binding peptide, protein, antibody or small molecule attached to albumin, a macromolecule, a dendrimer, a liposome, a nanoparticle, a vesicle or a (viral) microcapsule. The best choice of cell binding agent T is a monoclonal antibody.

As an application of the invention, the conjugates with the structural formulas I-VII and their pharmaceutically acceptable salts or solvates are all used for treating cancer, autoimmune diseases or infectious diseases of human or other animals.

Description of the figures

FIG. 1 shows the synthesis of conjugates of cytotoxins and cell binders using branched linkers.

FIG. 2 shows the synthesis of maleimide linkers and their use in antibody drug conjugates.

FIG. 3 shows the synthesis of bromomaleimide and bisbromomaleimide linkers and their use in antibody drug conjugates.

FIG. 4 shows the synthesis of Val-Cit amino acid linker and its use in antibody drug conjugates.

Figure 5 shows the synthesis of the Tuv component of the antimitotic agent.

Figure 6 shows the synthesis of the Tuv component of the antimitotic agent.

FIG. 7 shows the synthesis of a Boc-Tuv fragment and an antimitotic agent that can be used in conjugates.

FIG. 8 shows the synthesis of fragments of the antimitotic agents Tuv, Ile-Tuv and Mep-Ile-Tuv.

FIG. 9 shows the synthesis of fragments of the antimitotic agents Ile-Tuv, Mep-Ile-Tuv, Val-Ile-Tuv and Val-Ile-Tuv (O-alky).

Figure 10 shows the synthesis of an anti-mitotic agent and its conjugated linkage to an antibody.

FIG. 11 shows the synthesis of a Phe- (D) Lys amino acid linker.

Figure 12 shows the synthesis of antibody-antimitotic agent conjugates.

Figure 13 shows the synthesis of antibody-antimitotic agent conjugates.

FIG. 14 shows the synthesis of a cell binding body-antimitotic agent conjugate.

FIG. 15 shows the synthesis of a cell binding body-antimitotic agent conjugate.

Figure 16 shows the synthesis of an antibody-antimitotic conjugate.

Figure 17 shows the synthesis of an antibody-antimitotic conjugate.

Figure 18 shows the synthesis of hydrophilic Tut analogs for the synthesis of hydrophilic antimitotic agents (phosphate prodrugs).

Figure 19 shows the synthesis of an antibody-antimitotic conjugate.

FIG. 20 shows Boc format solid phase synthesis of an antimitotic agent capable of conjugation into a bond.

FIG. 21 shows Fmoc group based solid phase synthesis of an antimitotic agent capable of conjugation to form a bond.

FIG. 22 shows the synthesis of a hydrophilic anti-mitotic agent and other conjugated linkages to an antibody.

FIG. 23 shows the synthesis of Tuv derivatives and the solid phase synthesis of Mep-Ile-Tuv and NMe2-Val-Ile-Tuv fragments.

Figure 24 shows synthetic anti-mitotic agents and their conjugated linkages to antibodies.

Figure 25 shows the solid phase synthesis of anti-mitotic agents and their conjugation linkage to antibodies.

Figure 26 shows the solid phase synthesis of anti-mitotic agents and their conjugation to antibodies.

FIG. 27 shows the synthesis of trans-2-arylcyclopropylamine, trans-2-arylcyclopropylcarboxylic acid and trans-2-arylethylepoxycarboxylic acid.

FIG. 28 shows the synthesis of vinyl amino acids and alkyl epoxy amino acids.

FIG. 29 shows the synthesis of hydrophilic Tut analogs for the synthesis of hydrophilic antimitotic prodrugs.

Figure 30 shows the synthesis of hydrophilic antimitotic prodrugs conjugated and linked to cell binding agents.

Figure 31 shows the synthesis of hydrophilic anti-mitotic prodrugs conjugated to antibodies.

Figure 32 shows the synthesis of hydrophilic anti-mitotic prodrugs conjugated to antibodies.

FIG. 33 shows anti-CD in vitro assay₂₂Poisoning effect of antibody-antimitotic agent TZ 01-TZ 09 conjugates (drug/antibody molar ratio DAR 3.0-4.3) on Ramos (Burkitt lymphoma cells). The duration of action of the conjugate on the cells was 5 days and half the lethal dose IC₅₀The values of (a) are shown in the figure.

Figure 34 shows the poisoning effect of trastuzumab-antimitotic agent (TZ03, TZ04, and TZ07) conjugates (drug/antibody molar ratio DAR ═ 3.5-4.0) on KPL-4 cells (breast cancer cells). As can be seen, trastuzumab-TZ in the absence of unconjugated trastuzumab⁰³The conjugate has strong effect of inhibiting cancer cell proliferationAction, its semi-lethal dose IC₅₀Is 90 pM; in the presence of unconjugated trastuzumab (containing 1mmol of trastuzumab at the point of attachment to the saturating antigen), the corresponding amount of semi-lethal IC₅₀Greater than 20 nM. Therefore, the conjugate has a specific effect ratio of more than 222 (IC)₅₀＝20nM/IC₅₀＝0.09nM)。

FIG. 35 anti-CD₂₂Antibody-antimitotic agent (TZ)⁰³，TZ⁰⁴And TZ⁰⁷) Conjugates (drug/antibody molar ratio DAR ═ 3.8-4.2) and non-conjugated CDs₂₂Antibodies and CD₂₀Poisoning effect of antibody (rituximab) on BJAB cells (Burkitt lymphoma cells). As can be seen, the conjugate has a stronger inhibitory effect on the proliferation of cancer cells (the half-lethal dose IC of the conjugate) than the unconjugated antibody_5oSemi-lethal IC of unconjugated body, 5-19pM₅₀> 20 nM). When using non-conjugated antibody CD with the concentration of 1mmol₂₂Saturation of binding sites for antigen, huCD₂₂The specific specificity ratio of the TZ03 conjugate is greater than 660 (IC)₅₀＝3.3nM/IC₅₀0.005nM), the specificity ratio of huCD-TZ07 conjugate was greater than 790 (IC)₅₀＝15nM/IC₅₀＝0.019nM)。

TABLE 1 shows the structure of antimitotic drugs produced by solid phase synthesis and their ion peaks in mass spectra and their poisoning effect (IC) on Ramos cells (ATCC, human Burkitt lymphoma cells) in vitro tests₅₀) The value is obtained.

Table 2 shows the structural formulae of some antibody-antimitotic agent conjugates.

Disclosure of Invention

Definition of

Alkyl refers to linear or cyclic straight or branched aliphatic hydrocarbons containing 1 to 8 carbon atoms. Branched means that one or more lower alkyl groups, such as methyl, ethyl or propyl, are attached to a linear alkyl group. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2, 2-dimethylpentyl, 2, 3-dimethylpentylCyclopentyl, 3, 3-dimethylpentyl, 2, 3, 4-trimethylpentyl, 3-methylhexyl, 2, 2-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 3, 5-dimethylhexyl, 2, 4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, n-octyl and isooctyl. C₁-C₈Alkyl groups may be unsubstituted or substituted with one or more of the following, but are not limited to: c₁-C₈Alkyl radical, C₁-C₈Alkoxy, aryl, acyl, acyloxy, ester, -C (O) NH₂，-C(O)NHR’，-C(O)N(R’)₂，-NHC(O)R’，-S(O)₂R ', -S (O) R', -OH, halogen (-F, -Cl, -Br, -I), -N³，-NH²，-NHR’，-N(R’)₂and-CN; wherein R' denotes C₁-C₈Alkyl or aryl.

C₃-C₈Carbocyclic means a saturated or unsaturated nonaromatic cyclic compound containing 3, 4, 5, 6, 7, or 8 carbon atoms. Typical C₃-C₈Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1, 3, 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. C₃-C₈The carbocycle may be unsubstituted or substituted with one or more of the following, but is not limited to: c₁-C₈Alkyl radical, C₁-C₈Alkoxy, aryl, acyl, acyloxy, ester, -C (O) NH₂，-C(O)NHR’，-C(O)N(R’)₂，-NHC(O)R’，-S(O)₂R ', -S (O) R', -OH, halogen (-F, -Cl, -Br, -I), -N₃，-NH₂，-NHR’，-N(R’)₂and-CN; wherein R' is C₁-C₈Alkyl or aryl.

C₃-C₈Carbocyclyl means C as defined above₃-C₈A group resulting from substitution of one hydrogen atom on a carbocyclic ring by a chemical bond.

Alkenyl refers to straight or branched aliphatic hydrocarbons having one carbon-carbon double bond, with 2 to 8 carbon atoms in the carbon chain. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, 3-methyl-2-butenyl, n-pentenyl, hexenyl, heptenyl and octenyl.

Alkynyl refers to a straight or branched chain aliphatic hydrocarbon containing one carbon-carbon triple bond, with 2 to 8 carbon atoms in the carbon chain. Examples of alkynyls include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, hexynyl, heptynyl and octynyl.

Heteroalkyl means an alkyl group containing 2 to 8 carbon atoms and having 1 to 4 carbon atoms substituted with O, S or N.

Aryl or aryl refers to an aromatic or heteroaromatic hydrocarbon radical consisting of 3 to 14 carbon atoms (in most cases 6 to 10 carbon atoms) containing one or more rings. The heteroaromatic hydrocarbon group means an aromatic hydrocarbon group produced by substituting one or more carbon atoms (in most cases, 1, 2, 3 or 4 carbon atoms) with O, N, Si, Se, P or S (preferably O, S, N). Aryl or aryl also means an aromatic hydrocarbon radical in which one or more hydrogen atoms are substituted, these substituents being: r¹³，F，Cl，Br，I，OR¹³，SR¹³，NR¹³R¹⁴，N＝NR¹³，N＝R¹³，NR¹³R¹⁴，NO₂，SOR¹³R¹⁴，SO₂R¹³，SO₃R¹³，OSO₃R¹³，PR¹³R¹⁴，POR¹³R¹⁴，PO₂R¹³R¹⁴，OPO₃R¹³R¹⁴Or PO₃R¹³R¹⁴. Wherein R is¹³And R¹⁴Respectively H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl, carbonyl or pharmaceutically acceptable salts.

Halogen atom means fluorine, chlorine, bromine, iodine atom, preferably fluorine and chlorine.

Heterocyclic means an aromatic, nonaromatic or heterocyclic ring containing from 2 to 8 carbon atoms, wherein from 1 to 4 carbon atoms are replaced by a hetero element. These hetero elements are O, N, S, Se and P, preferably O, N and S. Useful heterocycles are also described in The Handbook of chemistry and Phvsics, 78 th edition, CRC Press, 1997-1998, pages 225 to 226. Suitable non-heteroaryl groups include, but are not limited to, epoxy, cycloazethyl, sulfoethyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl, and fused rings thereof with phenyl.

Heteroaryl refers to an aromatic monocyclic, bicyclic or polycyclic structure containing 5 to 14, preferably 5 to 10, atoms. For example, pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1, 2, 4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolinyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, N-oxopyridyl and fused rings thereof with phenyl.

The terms alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl also refer to alkylene, cycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, and heterocyclylene, among others, resulting from the loss of two hydrogen atoms from the corresponding hydrocarbon.

"pharmaceutically acceptable" or "pharmaceutically acceptable" means that the corresponding compound or composition of compounds does not produce deleterious, allergic, or other untoward effects in an animal or human.

Pharmaceutically acceptable adjuvants include all carriers, diluents, adjuvants or formers such as preservatives, antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like. In the field of medicine, it is common practice to add these adjuvants to active pharmaceutical ingredients. It can be said that it is not unreasonable to add adjuvants to pharmaceutical ingredients unless they are not compatible with the pharmaceutically active ingredient. Active auxiliary components may also be added to the pharmaceutical composition for good results.

In the present invention, pharmaceutically acceptable salts refer to salt derivatives of the compounds of the present invention. The compounds of the present invention may be modified appropriately to form the corresponding acid or base salts. Pharmaceutically acceptable salts include the common non-toxic salts or quaternary ammonium salts which can be prepared from the compounds of the present invention and the corresponding non-toxic inorganic or organic acids. For example, pharmaceutically acceptable salts can be prepared with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like and organic acids including acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid, oxalic acid, fumaric acid, lactic acid and the like. Other salts include ammonium salts such as tromethamine, meglumine, and pyrroleethanol, and metal salts such as sodium, potassium, calcium, zinc, and magnesium.

The pharmaceutically acceptable salts of the present invention may be prepared by conventional chemical methods. In general, these salts can be formed by adding other suitable equivalent amounts of base or acid to an aqueous or organic solution of the free acid or base of the compounds of the invention or a mixture of both. The reaction medium for the non-aqueous phase is generally diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of suitable salts is available in Remington's Pharmaceutical Sciences, 17 th edition Mack Publishing Company, Easton, PA, 1985, page 1418.

Conjugate structure of drug-linker-cell binding receptor molecule

As previously mentioned, the present invention describes an antibody drug conjugate having the structural formula (I):

where T is a targeting or cell binding agent; l is a cleavable linker; represents a linkage linking L to an atom on the bracketed structure; n is equal to 1 to 20; m is equal to 1-10.

The structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Each represents C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(2-8 carbon atoms) heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl (having 3 to 8 carbon atoms); or two R groups, e.g. R¹R²，R²R³，R³R⁴，R⁵R⁶，R¹²R¹³Two together may be C₃～C₇A carbocyclic group (containing 3-7 carbons), a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R³And R⁴May be H, and R²May be absent.

R¹、R²、R³And R⁴Are each H or C₁～C₄An alkyl group or a heteroalkyl group (containing 1 to 4 carbon atoms).

R⁷Can be independently selected from H and R¹⁴or-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵. Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkane, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; x¹Is O, S, S-S, NH or NR¹⁴。

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

R¹¹Is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶、-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units.

R¹²Is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH₂OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶。R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl. R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units.

R¹³Is C₁-C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar denotes an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each aromaticThe ring or heteroaromatic ring contains 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms. Heteroaryl refers to an aromatic ring in which one or more carbon atoms are replaced by a heteroatom, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms. Aryl Ar also refers to an aromatic or heteroaromatic ring substituted with one or more H atoms, the H atom-substituted groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、pR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt.

In addition, when R is¹⁰When not H, or when R¹³When the group structure is as follows, R¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂～C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Respectively N or CH.

Or, when R is¹¹When it is the group¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆Alkyl or heteroalkyl.

The antimitotic agent conjugates of the present invention may also have a structure represented by structural formula (II):

t is a targeting or cell binding body; l is a cleavable linker; n is equal to 1 to 20; m is equal to 1-10.

The structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Are respectively C₁-C₈Alkyl, heteroalkyl; c₂-C₈Heterocyclic ring,Carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃-C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl, or two R groups, e.g. R¹R²、R¹R³、R²R³、R³R⁴、R⁵R⁶；R¹²R¹³May be C₃-C₇A (3-7 carbon) carbocyclic group, a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R³And R⁴May be H, and R²May be absent.

R⁵、R⁶、R⁸And R¹⁰Are respectively H, C₁-C₄Alkyl or heteroalkyl.

R⁷Is H, R¹⁴、-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkyl, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl or alkylcarbonyl, X¹Is O, S, S-S, NH or NR¹⁴。

R⁹Is H, -OH, -OR¹⁴、-OC(＝O)R¹⁴、-OC(＝O)NHR¹⁴、-OC(＝O)R¹⁴SSR¹⁵、OP(＝O)(OR¹⁴) OR OR¹⁴OP(＝O)(OR¹⁵)₂Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl.

R¹¹Is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶or-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-N(R¹⁴)₂、-O-R¹⁴、-S-R¹⁴、-S(＝O)-R¹⁴or-NHR¹⁴；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units.

R¹²Is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴、-C(O)NH₂、-C(O)NHR¹⁴、-C(O)N(R¹⁴)(R¹⁶)、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH²OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶。R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl. R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units.

R¹³Is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each of which contains 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms. Heteroaryl refers to an aromatic ring in which one or more carbon atoms are replaced by a heteroatom, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms. Aryl Ar also refers to an aromatic or heteroaromatic ring substituted with one or more H atoms, the H atom-substituted groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO²、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt.

In addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Respectively N or CH.

Or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆Alkyl or heteroalkyl.

Some example structures of structural formula (II) are as follows:

wherein Aa refers to a natural or unnatural amino acid; n is equal to 1 to 20; q is equal to 1-5; x ', Y ' and Z ' are respectively CH, O, S, NH or NR²²；R²²And R²³Are respectively C₁-C₈Alkyl radical, C₂-C₈Alkylene, alkynylene, heteroalkyl, C₃-C₈Aryl, heterocyclyl, carbocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, arylalkyl, heteroalkylcycloalkyl, heteroarylalkyl, or- (OCH)₂CH₂) -; r 'and R' are each H or CH₃. Within the parentheses are antimitotic agents, within the square brackets are antimitotic agents and the linker attached.

The cell binding body-antimitotic agent conjugates of the present invention may also have a structure represented by structural formula (III):

wherein T, L, m, Y, R¹，R²，R³，R⁴，R⁵，R⁶，R⁸，R⁹，R¹⁰，R¹¹，R¹²，R¹³And n is as defined for formula (II).

R⁷Is R¹⁴，-R¹⁴C(O)X¹R¹⁵-or-R¹⁴X¹R¹⁵-。R¹⁴And R¹⁵Are respectively C₁-C₈Alkyl or heteroalkyl, C₂-C₈Alkylene, alkynylene, heterocyclyl, carbocyclyl, cycloalkyl, C₃-C₈Aryl, heterocyclylalkyl, heteroaralkyl, heteroalkylcycloalkyl or carbonylalkyl. X¹Is O, S, S-S, NH or NR¹⁴。

Some examples of compounds having structural formula (III) are shown in the following figures:

wherein Ar, n, q, X ', Y ', Z ', R²²，R²³R 'and R' are as defined for the compounds IIa to IIr.

The cell binding body-antimitotic agent conjugate of the present invention may also have a structure represented by structural formula (IV):

wherein T, L, m, Y, R¹，R²，R³，R⁴，R⁵，R⁶，R⁸，R¹⁰，R¹¹，R¹²，R¹³And n is as defined for formula (II).

R⁹is-O-, -OR¹⁴，-OC(O)R¹⁴，-OC(O)NHR¹⁴，-OC(O)R¹⁴SSR¹⁵OR-OP (O) (OR)¹⁴) O-, wherein R¹⁴And R¹⁵Are respectively C₁～C₈Alkyl or heteroalkyl, C₃～C₈Aryl, heteroaryl, heterocyclyl, carbocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, carbonylalkyl, or a pharmaceutical salt. In addition, R⁹Or may be absent.

Some examples of compounds having structural formula (IV) are shown in the following figures:

wherein Ar, Aa, n, q, X ', Y', R²²，R²³R 'and R' are as defined for the compounds IIa to IIr.

The cell binding body-antimitotic agent conjugate of the present invention may also have a structure represented by structural formula (V):

wherein T, L, m, Y, R¹，R²，R³，R⁴，R⁵，R⁶，R⁸，R⁹，R¹⁰，R¹²，R¹³And n is as defined for formula (II).

R¹¹Is R¹⁴，-R¹⁴C(＝O)R¹⁷-，-R¹⁴X²R¹⁷-，-R¹⁴X²R¹⁷-，-R¹⁴C(＝O)X²-, wherein R¹⁷Is H, OH, C₁-C₈Alkyl radical, C₂-C₈Alkylene, alkynylene, heteroalkyl, C₃-C₈Aryl, arylene, heterocyclyl, carbocyclyl, heterocycloalkyl, or one or two amino acid units; x²is-O-, -S-, -NH-, -NR-, -¹⁴-，-OR¹⁴-，-SR¹⁴-，S(＝O)R¹⁴-or-NHR¹⁴；R¹⁴Is C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkylene, alkynyl, C₃-C₈Aryl, heterocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl or carbonylalkyl.

Some examples of compounds having structural formula (V) are shown in the following figures:

wherein Ar, Aa, n, q, X ', Y', R²²，R²³R 'and R' are as defined for the compounds IIa to IIr.

The cell binding body-antimitotic agent conjugate of the present invention may also have a structure represented by structural formula (VI):

wherein T, L, m, Y, R¹，R²，R³，R⁴，R⁵，R⁶，R⁷，R⁸，R⁹，R¹⁰，R¹¹，R¹³And n is as defined for formula (II).

R¹²Is R¹⁴，-O-，-S-，-NH-，＝N-，＝NNH-，-N(R¹⁴)-，-OR¹⁴-，C(O)O-，C(O)NH-，C(O)NR¹⁴-，-SR¹⁴，-S(O)R¹⁴，-NHR¹⁴-，-CH₂OP(＝O)(OR¹⁵)-，-P(＝O)(OR¹⁵)-，-OP(＝O)(OR¹⁵) O-, or-SO₂R¹⁴。R¹⁴And R¹⁵Are respectively C₁-C₈Alkyl, heteroalkyl; c₂-C₈Alkylene, alkynyl; or C₃-C₈Aryl, heterocyclyl, carbocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylalkyl, heteroalkylcycloalkyl, or alkylcarbonyl.

Some examples of compounds having structural formula (VI) are shown in the following figures:

wherein Ar, Aa, n, q, X', R²²，R²³R 'and R' are as defined for the compounds IIa to IIr.

The cell binding body-antimitotic agent conjugates of the present invention may also have a structure represented by structural formula (VII):

wherein T, L, m, Y, R¹，R²，R³，R⁴，R⁵，R⁶，R⁸，R⁹，R¹⁰，R¹¹，R¹²，R¹³And n is as defined for formula (II).

R¹³Is C₁-C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl; wherein aryl means an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each containing from 4 to 10 carbon atoms, preferably from 4 to 6 carbon atoms. Heteroaryl refers to an aromatic ring in which one or more carbon atoms are replaced by a heteroatom, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms. Aryl Ar also refers to an aromatic or heteroaromatic ring substituted with one or more H atoms, the H atom-substituted groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、pR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂A glycoside or a pharmaceutically acceptable salt.

In addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄-C₁₂(having 4 to 12 carbon atoms) glycoside or C₁-C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁-C₈Alkyl, carboxyalkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, or C₃-C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁-C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃-C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Respectively N or CH.

Or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁-C₆Alkyl or heteroalkyl.

Some examples of compounds having structural formula (VII) are shown in the following figures:

wherein Ar, Aa, n, q, X', R²²，R²³R 'and R' are as defined for the compounds IIa to IIr.

By way of example, and not limitation, the synthesis of the antimitotic agents of the invention and their conjugates with the cell binding agents of the invention are shown in FIGS. 1-32.

As a specific conjugated linker of the invention, the cleavable linker (L) is a chemical chain containing C, N, O, S, Si or P atoms. In conjugates, the chemical chain is covalently linked to a cell binding agent (T) on one side and to an antimitotic agent on the other side. The linker may be of varying lengths, for example from 2 to 100 atoms in length. All of the atoms of the linker may be attached by any suitable chemical means, such as by an alkylene, alkenylene, alkyne, ether, polyoxyalkylene, ester, amine, imine, polyamine, hydrazine, hydrazone, amide, urea, semicarbazide, dihydrazinocarbon, alkoxyamine, urethane, amino acid, acyloxyamine, or hydroxamic acid. Of course, the atoms of these dissociable linkers may be saturated or unsaturated, may be free radicals, and may form a ring with each other to form a divalent ring structure, such as cycloalkane, cyclic ether, cyclic amine, arylene, heteroarylene, and the like.

A cleavable linker refers to a linker that contains at least one chemical bond that can be cleaved under physiological conditions, such as a chemical bond that is pH sensitive, acid labile, base labile, easily oxidized, easily metabolized, biochemically labile, or easily enzymatically cleaved. It should be noted that the physiological condition that breaks chemical bonds need not be a biological or metabolic process, and that conventional chemical processes may be entirely possible, such as hydrolysis or substitution reactions. Since endosomes have a lower pH than the cytosol, chemical bonds that hydrolyze under acidic conditions are a good choice. For the same reason, it is also feasible to exchange thiol groups for disulfide bonds, considering that the glutathione concentration in tumor cells is as high as millimolar.

The structure of the cleavable linker L of the present invention can be expressed as: -Ww- (Aa) r-Vv-, wherein

W-denotes an extender, W is 0 or 1, -Aa-is an amino acid unit, r is an integer from 0 to 12, -V-denotes a spacer, V is 1 or 2.

When the extension-Ww-is present, it serves to link the cell-binding body (T) to the amino acid unit-Aa-or spacer-V- (when-Aa-is absent). The extension W may contain an autodisintegrable spacer, polypeptide unit, hydrazone bond, disulfide bond or thioether bond. It is apparent that the cell binding agent (T) contains a group capable of bonding to a corresponding functional group on the extension. The ligatable functional groups that are originally present on the cell binding body or chemically generated include, but are not limited to: mercapto, amino, hydroxyl, carbonyl, anomeric hydroxyl of sugar, carboxyl and the like, and the most preferable functional groups are mercapto, carboxyl and amino. Sulfhydryl groups may be generated by reducing disulfide bonds within a cell binding body (e.g., an antibody) molecule. Alternatively, sulfhydryl groups may be generated by reaction with amino groups of lysine on the cell conjugate using either 2-iminothiolane (Traut's reagent) or thiolactones, or by other methods of sulfhydryl generation, such as first modifying the cell conjugate with a disulfide-containing linker or thioester, and then generating sulfhydryl groups by reduction or hydrolysis, respectively.

Some examples of W attached to T are shown in the following figures:

where R is²⁰，R²¹Is selected from-C₁～C₉Alkylene-, -C₁～C₇Carbocyclyl-, -O- (- (C)₁～C₈Alkyl) -, -arylene-, -C₁～C₉Alkylene-arylene-, -arylene, -C₁～C₉Alkylene group, -C₁～C₉Alkylene- (C)₁～C₈Carbocyclyl) -, - (C)₃～C₈Carbocyclyl) -C₁～C₉Alkylene-, -C₃～C₈Heterocyclyl-, - (C)₁～C₁₀Alkylene- (C)₃～C₈Heterocyclyl) -, - (C)₃～C₈Heterocyclyl) -C₁～C₉Alkylene-, - (CH)₂CH₂O)_k-，-(CH(CH₃)CH₂O)_k-and- (CH)₂CH₂O)_k-CH₂-; k is an integer between 1 and 20; r 'and R' are each H or CH₃。

The aforementioned covalent linkage of W and T can be achieved by various chemical reactions.

For example, formation of amide bonds:

wherein the extender unit contains an active reactive group E which can form an amide bond with a primary or secondary amine on the cell conjugate. Possible reactive sites E include, but are not limited to, hydroxysuccinimide esters (NHS and S-NHS, etc.), 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters (including S-tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, isothiocyanates, and the like.

The following are examples of linkages with thioether or disulfide bonds:

wherein the extender unit comprises a thiol group capable of participating in a reaction which forms a thioether or disulfide bond with the cell binding entity T. To form disulfide bonds, sulfhydryl groups on the cell binding body T can be generated either by reducing intramolecular disulfide bonds within it or by modifying the cell binding body T by other chemical processes.

The extension may also have a reactive group capable of reacting with an aldehyde or ketone group. Such aldehyde or ketone groups may be introduced into the appropriate location of the cell binding body T by appropriate chemical modification. For example, aldehyde or ketone groups may be formed on the cell-bound carbohydrate by oxidation with an oxidizing agent such as sodium periodate. For another example, an amine group on the N-terminal amino acid of an antibody (or a protein or polypeptide) can react with pyridoxal 5-phosphate (PLP) to introduce a keto group. These aldehyde or keto groups (-C ═ O) can react with reactive groups on the extender such as hydrazides, oximes, primary or secondary amines, hydrazines, thiosemicarbazones, hydrazine carboxylates or arylhydrazines, linking the two together.

The following are examples of linkages with hydrazones, oximes or imines:

wherein R is²⁰And R²¹As defined above, R²⁵Refers to an organic substitution of an amino acid.

The extension (which may contain a spacer V and/or an amino acid) may be linked to the cell-bound complex T before the cell-bound complex-extension fragment is conjugated to an antimitotic agent, and the linking may be carried out in an aqueous buffer. The following are some examples of conjugated linkages in two steps in this manner (the linkage is at R)¹⁶The cytotoxic molecule above is omitted):

wherein E includes, but is not limited to, hydroxysuccinimide ester (NHS, S-NHS, etc.), 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester (including S-tetrafluorophenyl ester), acid anhydride, acid chloride, sulfonyl chloride, isocyanate, isothiocyanate, etc. R 'and R' are each H or CH₃；R²⁰，R¹⁶And Ar is as defined above; r²⁶Is H, F or NO₂(ii) a J is F, Cl, Br, I, tosylate (TsO) or mesylate (MsO). Of course, in the above structure

Containing at least one antimitotic agent or other drug, e.g. structure

(

To omit the flag).

The extension may also be linked to the antimitotic agent first and then conjugated to the cell binding body T in an aqueous solution (which may contain up to 50% organic solvent) at a pH of 3-10 (preferably 5-8.5).

The following are some examples of conjugation in two steps in this manner:

wherein E includes, but is not limited to, hydroxysuccinimide ester (NHS, S-NHS, etc.), 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester (including S-tetrafluorophenyl ester), acid anhydride, acid chloride, sulfonyl chloride, isocyanate, isothiocyanate, etc. R 'and R' are each H or CH₃；R²⁰，R¹⁶And Ar is as defined above; r²⁶Is H, F or NO₂(ii) a J is F, Cl, Br, I, tosylate (TsO) or mesylate (MsO). Of course, in the above structure

Containing at least one antibioticMitotic agents or other drug constructs, e.g.

(

To omit the flag).

When the amino acid unit (- -Aa- -) is present, it links the extender and the spacer, when the spacer is absent, it links the extender and the antimitotic agent, and if both the extender and the spacer are absent, it directly links the cell-binding body T and the antimitotic agent. - (Aa) r-may be a natural or non-natural amino acid, and may be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit, r being an integer between 0 and 12. Herein, amino acids generally refer to aminoalkyl carboxylic acids, wherein the alkyl group may be an alkyl group substituted with a group such as an alkyl, acyl, hydroxyalkyl, mercaptoalkyl, aminoalkyl or carboxyalkyl group. The structures of natural and unnatural Amino acids and polypeptides are well described in the book "Amino acids and Peptides" (Cambridge University Press, 2004), by G.C. Barrett and D.T. Elmore. In addition, the amino acids also mean beta amino acids, gamma amino acids, and long-chain amino acids having a methyl group, a benzyl group, a hydroxymethyl group, a mercaptomethyl group, a carboxyl group, a carboxymethyl group, a guanidinopropyl group, or the like in the molecule. The most preferable amino acids are arginine, asparagine, aspartic acid, citrulline, cysteine, glycine, glutamic acid, leucine, lysine, glutamine, serine, ornithine, phenylalanine, threonine, tyrosine, valine and the like.

The amino acid units of the invention may be enzymatically cleaved by one or more enzymes, including proteolytic enzymes present in the tumor, to release the antimitotic agent. In one embodiment, it is protonated first in vivo and then becomes a protonated antimitotic agent.

When the spacer (-V-) is present, it links the amino acid unit and the antimitotic agent, and when the amino acid unit is absent, it links the extender and the antimitotic agent. When both the amino acid unit and the extender are missing, the spacer links the antimitotic agent and the cell binding molecule T. The spacer may contain functional groups that can be used to enhance the water solubility, biological transport, suitable renal clearance, uptake, absorption, biodistribution, and bioavailability of the conjugate. There are two basic types of separators: self-destructive and non-self-destructive. Non-self-immolative spacers refer to spacers in which part or all of the spacer remains attached to the antimitotic agent after an amino acid unit is cleaved, particularly enzymatically, from the antimitotic agent-linker-cell conjugate or the antimitotic agent-linker.

The following are some examples of self-destructing insulation:

wherein the atom marked with (—) is a spacer, a cleavable linker, an antimitotic agent or an access point to a cell binding body T; x, Y and Z³Are each NH, O or S; z²Is H, NH, O or S. v is 0 or 1; q is H, OH, C₁～C₆Alkyl group, (OCH)₂CH₂)_n，F，Cl，Br，I，OR¹⁷Or SR¹⁷，NR¹⁷R¹⁸，N＝NR¹⁷，N＝R¹⁷，NR¹⁷R¹⁸，NO₂，SOR¹⁷R¹⁸，SO₂R¹⁷，SO₃R¹⁷，OSO₃R¹⁷，PR¹⁷R¹⁸，POR¹⁷R¹⁸，PO₂R¹⁷R¹⁸，OPO(OR¹⁷)(OR¹⁸)，OCH₂PO(OR¹⁷(OR¹⁸) Or a glycoside, wherein R¹⁷And R¹⁸Are respectively H, C₁-C₈Alkyl radical, C₂-C₈Alkylene, alkynylene, heteroalkyl, C₃-C₈Aryl, heterocyclyl, carbocyclyl, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl or a pharmaceutically acceptable salt of a cationic salt.

The following are some examples of non-self-destructing insulation:

wherein the atom marked with (—) is an access point for a spacer, a cleavable linker, an antimitotic agent or a cell binding body; q is as described above, and m is 1-10; n is 1 to 20.

The cell binding agent T may be any molecule known to bind, complex or react with a fragment of the target cell that may be used for therapy or modification. By acting with specific target cells, the cell binding agent acts to deliver an antimitotic agent to such target cells.

Cell binding bodies include, but are not limited to, large molecular weight proteins such as whole antibodies (polyclonal or monoclonal); a single chain antibody; antibody fragments such as Fab, Fab ', F (ab ') 2, Fv [ Parham, J.Immunol.131, 2895-2902(1983) ], fragments produced by Fab expression libraries, anti-idiotypic antibodies (anti-Id), CDR's, any fragment of the above that specifically binds to an epitope of a cancer cell, a viral epitope or a microbial epitope; interferons (type I, II, III); a polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL-6, GM-CSF, or IFN- γ; hormones such as insulin, thyroid stimulating hormone releasing hormone (TRH), Melanocyte Stimulating Hormone (MSH), steroid hormones such as androgen, estrogen, or Melanocyte Stimulating Hormone (MSH); growth factors and colony stimulating factors such as epidermal growth factor (EFG), granulocyte macrophage colony stimulating factor (GM-CSF), Transforming Growth Factors (TGF) such as TGFa, TGF β, insulin and insulin-like growth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [ Burgess, Immunology Today, 5, 155-; vaccine Growth Factor (VGF); fibroblast Growth Factor (FGF); small molecular weight proteins, polypeptides, peptides and peptide hormones, such as bombesin, gastrin and gastrin releasing peptide; platelet-derived growth factor; interleukins and cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte macrophage colony stimulating factor (GM-CSF) and vitamins such as folic acid; apoproteins and glycoproteins such as transferrin [ O' Keefe et al, J.Bio.chem.260, 932-927(1985) ]; carbohydrate binding proteins or lipoproteins such as lectins; a cellular nutrient-transport molecule; and small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors, small molecule Tyrosine Kinase Inhibitors (TKI), non-peptides or other cell binding molecules or substances, e.g. bioactive macromolecules (Dhar, et al, proc.natl.acad.sci.2008, 105, 17356-61), dendrimers (Lee, et al, nat.biotechnol.2005, 23, 1517-26; Almutairi, et al; proc.natl.acad.sci.2009, 106, 685-90), nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, et al, nat.med.2007, 13, 372-7; Javier, et al, Bioconjugate chem.2008, 19, 1309-12), liposomes (inil, et al, currai.phar.10, 10, 299-81, 92, 2009, 92-71, rhino, 71-71). Generally, monoclonal antibodies are the best cell binding agents if appropriate.

The cell binding body may be first attached to a particular polypeptide, protein, drug molecule or other functional molecule by modification with a bifunctional-containing cross-linking agent prior to conjugation with the anti-mitotic agent. These dual-functional group-containing crosslinkers can be amine-nonselective functional group crosslinkers (succinimide (NHS) -cyclopropene (SDA), succinimide ester-azide), amine-sulfhydryl crosslinkers (NHS ester-maleimide, NHS ester-pyridinedithiol, NHS ester-haloacetyl), sulfhydryl-sugar crosslinkers (maleimide-hydrazide, pyridinedithiol-hydrazide), hydroxy-sulfhydryl crosslinkers (isocyanate-maleimide), amine-DNA crosslinkers (NHS ester/psoralen), and amine-carboxyl crosslinkers (carbodiimide), among others.

In the modification method using succinimide (NHS) -cyclopropene (SDA) crosslinker, the NHS ester on the crosslinker can first react with the amine group on the cell-bound backbone (in a buffer solution at pH 6-9), which will form a stable amide bond. The diazocyclopropene is then activated by irradiation with long waves at 330-370nm and produces a carbene reactive intermediate which reacts with amine groups on specific polypeptides, proteins or other functional molecules to complete the linkage. The order of these two reactions can also be changed: the amine on the functional molecule and the NHS ester on the cross-linking agent are reacted, and then the reaction is performed with the cell combination body under the irradiation of light (330-370 nm). Succinimide (NHS) -diazacyclopropene (SDA) crosslinkers can also be cleavable (like SDAD crosslinkers where disulfide bonds are present).

Wherein

Is a framework of cell-binding molecules,

is a functional molecule.

In the modification method using the NHS ester-azide crosslinker, the NHS ester on the crosslinker can first react with the amine groups on the cytoskeleton of the cell conjugate (in a buffered solution at pH 6-9), which will form a stable amide bond. Then, by Huisgen azide-alkyne cycloaddition, the alkynyl group on a particular polypeptide, protein or other functional molecule reacts with the azide at the other end of the crosslinker and forms a1, 2, 3-triazole linker. Alternatively, the NHS ester of the crosslinker can be reacted with the amine group on the functional molecule to form a stable amide bond (in a buffer solution with pH 6-9), and then the alkynyl group on the conjugate is reacted with the azide group at the other end of the crosslinker to form a1, 2, 3-triazole linker by Huisgen azide-alkyne cycloaddition.

In the modification method using the amino-sulfhydryl cross-linking agent, NHS ester on the cross-linking agent can firstly react with amino on the cell-associated body skeleton (in a buffer solution with the pH of 6-9), which will form a stable amido bond. And then, under the condition that the pH value is 4.5-8.5, the sulfydryl on the specific polypeptide, protein or other functional molecules reacts with maleimide, pyridine dithiol or halogenated acetyl at the other end of the cross-linking agent to form thioether or disulfide bonds. The order of such crosslinking reactions may also be varied as appropriate. For example, the amino group on the functional molecule can be reacted first with a cross-linking agent to form an amide bond, and then reacted with the thiol group on the cell conjugate. For example, under the condition that the pH is 4.5-7, the sulfydryl on the functional molecule can firstly react with the cross-linking agent to form thioether or disulfide bond, and then react with the amino on the cell combination body under the condition that the pH is 6-9 to form amido bond.

In the modification method using the thiol-sugar cross-linking agent, under the condition that the pH is 4.5-8, the thiol on the cell combination body can firstly react with maleimide or pyridine dithiol on the cross-linking agent to form thioether or disulfide bond. Then, the carbonyl group (aldehyde or ketone) on the functional molecule is reacted with hydrazide to generate hydrazone bond. In addition, under the condition that the pH value is 4.5-8, the sulfydryl on the functional molecule can be firstly reacted with a cross-linking agent to form thioether or disulfide bond, and then reacted with sugar, oxidized sugar or carbonyl (aldehyde or ketone) on the cell combination body to form hydrazone bond.

In the modification method using the hydroxyl-sulfhydryl cross-linking agent, under the condition that the pH value is 6-8, sulfhydryl on a cell combination body can firstly react with maleimide or pyridine dithiol on the cross-linking agent to generate thioether or disulfide bond. And then, under the condition that the pH value is 8-9, reacting hydroxyl on the functional molecule with isocyanate on the cross-linking agent to generate carbamate. In addition, the sulfydryl on the functional molecule can also react with a cross-linking agent to generate thioether or disulfide bonds under the condition that the pH value is 6-8. And then reacting with hydroxyl on the cell combination body at the pH of 8-9 to generate carbamate.

Another aspect of the invention is the production of antibodies. Including in vivo, in vitro production processes or combinations thereof. Methods for producing polyclonal antibodies against receptor peptides are well known, for example, in U.S. Pat. No. 4,493,795(Nestor et al). The classical method for preparing monoclonal antibodies is to immunize mice with specific antigens and to fuse the isolated splenocytes of the mice with myeloma cells (Kohler, G; Milstein, C.1975.Nature 256: 495-497). The detailed procedures are described in antibodies-A Laboratory Manual, Harlow and Lane, eds., cold spring harbor Laboratory press, new York (1988), which is incorporated herein by reference. In particular, specific monoclonal antibodies can be obtained by immunizing mice, rats, hamsters or other mammals with the antigen of interest. Wherein the antigens of interest include: intact cells, antigens isolated from cells, intact viruses, attenuated intact viruses, and viral proteins. Spleen cells were fused with myeloma cells using PEG 6000. The hybridomas obtained after the fusion are screened by using their sensitivity to HAT. Monoclonal antibodies produced by hybridoma cells play a role in practicing the invention by either immunoreacting with a particular target cell receptor or by inhibiting receptor activity.

The monoclonal hybridoma cells obtained after fusion can secrete monoclonal antibodies aiming at specific antigens. The monoclonal antibodies used in the present invention are enriched by culturing monoclonal hybridoma cells in a nutrient-rich medium. The culture conditions are such that the hybridoma cells have sufficient time to secrete the produced antibody into the culture medium. After the antibody-containing culture supernatant is collected, the antibody is purified by a well-known technique. The separation method comprises the following steps: protein a affinity chromatography; anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography (especially affinity chromatography and molecular sieve chromatography using antigen-crosslinked protein A); centrifuging; precipitation or other standard purification methods

Effective media as well as artificially synthesized media for hybridoma culture can be either technically synthesized or commercially available. Among these, typical synthetic media: DMEM (Dulbecco et al, Virol 8: 396(1959)) was supplemented with 4.5mg/L glucose, 20mM glutamine, 20% fetal bovine serum, and antifoam, such as:polyoxyethylene polyoxypropyleneCopolymer

In addition to cell fusion techniques, antibody-producing cell lines can be constructed by other methods, such as: direct transfection of B lymphocytes with tumorigenic DNA or introduction of oncogenic viral genes (e.g., EBV also known as HHV-4 or KSHV) into B lymphocytes, as described in U.S. patent Nos.: 4341761, respectively; 4399121, respectively; 4427783, respectively; 4444887, respectively; 4451570, respectively; 4466917, respectively; 4472500, respectively; 4491632, respectively; 4493890. monoclonal antibodies may also be prepared by anti-receptor polypeptides or polypeptides containing a carboxy terminus. See Niman et al, proc.natl.acad.sci.usa, 80: 4949 and 4953 (1983); geysen et al, Proc.Natl.Acad.Sci.USA, 82: 178-182 (1985); lei et al, Biochemistry 34 (20): 6675-6688(1995). In general, anti-receptor polypeptides or polypeptide analogs can be used alone or cross-linked to immunogenic carriers to produce monoclonal antibodies against receptor polypeptides as immunogens.

Antibodies of the invention as binding molecules have other well-established methods of production. Of particular interest are processes for the production of fully human antibodies. Phage display technology obtains fully human antibodies that specifically bind to a known antigen from a fully human antibody library by affinity screening. Phage display technology itself, vector construction and library screening are well documented in the literature. See Dente et al, Gene.148 (1): 7-13 (1994); little et al, Biotechnol adv.12 (3): 539-55 (1994); clackson et al, Nature 352: 264-628 (1991); huse et al, Science 246: 1275-1281(1989).

Monoclonal antibodies obtained from other species (e.g., mouse) using hybridoma technology are subject to humanization. The engineered antibody can greatly reduce the immune side effects of the heterologous antibody on the human body. Among the more common methods for humanization of antibodies are the transplantation and remodeling of the CDRs. See in detail: U.S. patent nos. 5859205 and 6797492; liu et al, Immunol Rev.222: 9-27 (2008); almagro et al, Front biosci.1; 13: 1619-33 (2008); lazar et al, Mol Immunol.44 (8): 1986-98 (2007); li et al, proc.natl.acad.sci.usa.103 (10): 3557-62(2006), which are incorporated herein by reference. Fully human antibodies can also be prepared by antigen immunization of transgenic mice, rabbits, monkeys, and other mammals that carry large amounts of human immunoglobulin light and heavy chains. Taking a mouse as an example: xenomouse (Abgenix, Inc.), HuMab-Mouse (Metarex/BMS), VelociMouse (Regeneron), see in detail: U.S. patent No.: 6596541, 6207418, 6150584, 6111166, 6075181, 5922545, 5661016, 5545806, 5436149 and 5569825. In the course of human therapy, chimeric antibodies constructed by integrating the genes of the variable regions of murine antibodies with the genes of the constant regions of human antibodies produce a much lower immunogenic response in humans than murine antibodies (Kipriyanov et al, Mol Biotechnol.26: 39-60 (2004); Houdbine, Curr Opin Biotechnol.13: 625-9(2002) incorporated herein by reference).

Antibodies immunospecific for malignant cell antigens may be obtained commercially or by a number of well-established techniques, such as: chemical synthesis or recombinant expression techniques. Genes encoding such antibodies are also available commercially, such as GenBank databases or other similar databases, published literature, or conventional sequencing by cloning methods.

In addition to antibodies, polypeptides or proteins may also bind as binding molecules, blocking, attacking or otherwise interacting with a receptor or epitope on the surface of a target cell. These polypeptides or proteins do not necessarily belong to the immunoglobulin family, as long as they are capable of specifically binding to a particular epitope or its corresponding receptor. These polypeptides can also be isolated by techniques similar to phage display antibodies (Szardenngs, J Recept Signal Transmission Res.2003; 23 (4): 307-49). Peptide fragments obtained from random polypeptide libraries are used similarly to antibodies and antibody fragments. Polypeptide or protein molecules can retain their antigen-binding specificity by linking their binding molecules to some macromolecule or medium. These macromolecules and mediators include: albumin, polymer, liposome, nanoparticle, or dendrimer.

In the treatment of cancer, autoimmune and infectious diseases, antibodies for coupling to anti-mitotic agents are exemplified by (but not limited to): 3F8 (anti-GD 2 antibody), abamectin (anti-CA-125 antibody), abciximab (anti-CD 41 antibody (integrin. alpha. -IIB), adalimumab (anti-TNF. alpha. -antibody), adalimumab (anti-EpCAM antibody, CD326), adalimumab (anti-TNF. alpha.)), Avermentil (anti-CD 20 antibody), Alacizumab pegol (anti-VEGFR 2 antibody), ALD518 (anti-IL-6 antibody), alemtuzumab (alias: Campath, Maampath, Campa, anti-CD 52 antibody), adalimumab (anti-CEA antibody), Anatuzumab (anti-TAG-72 antibody), Anrukinzumab (alias: IMA-638, anti-IL-13 antibody), aprezumab (anti-HLA-DR antibody), acimumab (anti-CEA antibody), abamectin-Sambumab (anti-TAG-72 antibody) (anti-TAG-62L) antibody, atlizumab (alternative names: Touzumab, Actemra, RoActemra, anti-IL-6 receptor antibody), Atorlimumab (anti-rhesus factor antibody), bapineuzumab (anti-beta-amyloid antibody), basiliximab (sulley, anti CD25 (alpha chain of IL-2 receptor) antibodyBaveximab (anti-phosphatidylserine antibody), betuzumab (alternative: Lymphoscan, anti-CD22 antibody), belimumab (alternative: Benlysta, Lymphostat-B, anti-BAFF antibody), Benralizumab (anti-CD 125 antibody), batimumab (anti-CCL 11 (eotaxin-1) antibody), bevacizumab (alternative: Scintiun, anti-CEA-related antigen antibody), bevacizumab (alternative: avastin, anti-VEGF-A antibody) biximab (alternative: FibriScint, anti-fibrin II beta chain antibody), Bivatuzumab (anti-CD 44 v6 antibody), blinatumumab (alternative: BiTE, anti-CD 19 antibody), Brentuximab (CAC10, anti-CD 30 TNFRSF8 antibody), Britinumumab (anti-IL-12, Brituzumab-23 antibody), anti-Cantuzumab (alternative: IL-242), carromumab, Katuzumab (alternative name: removab, anti-EpCAM, anti-CD 3 antibody), CC49 (anti-TAG-72 antibody), Cedelizumab (anti-CD 4 antibody), certolizumab (alternative name Cimzhia anti-TNF-. alpha.antibody), Cetuximab (alternative name: erbitux, IMC-C225, anti-EGFR antibody), cetuximab (anti-EpCAM antibody), Cixuumumab (anti-IGF-1 antibody), clexib (anti-CD 4 antibody), Clituzumab (anti-MUC 1 antibody), Conatumumab (anti-TRAIL-R2 antibody), CR 61 (anti-influenza A hemagglutinin antibody), Dacetuzumab (anti-CD 40 antibody), daclizumab (alternative name: Sainipipa, anti-CD 25 (alpha chain of IL-2 receptor) antibody), Dacuumumab (anti-CD 6229 (anti-CDK 38 antibody), anti-Gluconolab (anti-Gluconolab), anti-Gluconolab antibody), atorvastatin, Dorlixizumab, Ecromeximab (anti-GD 3 ganglioside antibody), eculizumab (alias: Soliris, anti-C5 antibody), erbitumumab (anti-endotoxin antibody), eculizumab (alias: Panorex, MAb17-1A, anti-EpCAM antibody), efletuzumab (alias: Raptiva, anti-LFA-1 (CD11A) antibody), efuzumab (alias: Mycogarb, anti-Hsp 90 antibody), Elotuzumab (anti-SLAMF 7 antibody), Isilelimumab (anti-IL-6 antibody), enromab (anti-ICAM-1 (CD54) antibody), Epitumomab (anti-epidialin antibody), eppauzumab (anti-CD 22 antibody), Erlizumab (anti-ITGB 5 (CD18) antibody, Erixon (alias: 48325/HER antibody), anti-Reunion antibody (anti-SLeMax/EPITU < 25 >), Epidex > EpsilozumabIntegrin α v β 3 antibody), esvimab (anti-hepatitis b surface antigen antibody), fanolesobab (alternative name: NeutroSpec, anti-CD 15 antibody), faraday mab (anti-interferon receptor antibody), farlettuzumab (anti-folate receptor 1 antibody), Felvizumab (anti-respiratory syncytial virus antibody), Fezakinumab (anti-IL-22 antibody), Figitumumab (anti-IGF-1 receptor antibody), Fontolizumab (anti-IFN- γ antibody), vorivivir e (anti-rabies glycoprotein antibody), Fresolimumab (anti-TGF- β antibody), galiximab (anti-CD 80 antibody), Gantenerumab (anti- β amyloid antibody), gavilimob (anti-CD 147 (baigin) antibody), gemtuzumab (anti-CD 33 antibody), Girentuximab (anti-carbonic anhydrase 9 antibody), glembatum (alias: CR011, anti-GPNMB antibody), golimumab (alias: SIMPONI, anti-TNF- α antibody), Gomiliximab (anti-CD 23(IgE receptor) antibody), Ibalizumab (anti-CD 4 antibody), ibritumomab (anti-CD 20 antibody), agovacizumab (alias: indinavis-125, anti-CA-125 antibody), infliximab (alias: myoscint, anti-cardiac myosin antibody), infliximab (alternative name: infliximab, anti-TNF- α antibody), Intetumumab (anti-CD 51 antibody), inomumab (anti-CD 25(IL-2 receptor α chain) antibody), Inotuzumab (anti-CD 22 antibody), yipriumumab (anti-CD 152 antibody), Iratumumab (anti-CD 30(TNFRSF8) antibody), Keliximab (anti-CD 4 antibody), Labetuzumab (alias: CEA-Cide, anti-CEA antibody), Lebrikizumab (anti-IL-13 antibody), Lemalesamumab (anti-NCA-90 (granulocyte antigen) antibody), ledelmumab (anti-TGF β -2 antibody), lexamumab (anti-TRAIL-R2 antibody), ribavirin (anti-hepatitis B surface antigen antibody), lintuzumab (anti-CD 33 antibody), Lucatumumab (anti-CD 40 antibody), luximab (anti-CD 23(IgE receptor) antibody), Mapatummab (anti-TRAIL-R1 antibody), Massumab (anti-T-cell receptor antibody), matuzumab (anti-EGFR antibody), merizumab (alias: bosatria, anti-IL-5 antibody), Metelimumab (anti-TGF β -1 antibody), Milatuzumab (anti-CD 74 antibody), minretumumab (anti-TAG-72 antibody), mitumumab (alias BEC-2, anti-GD 3 ganglioside antibody), morrolimumab (anti-rhesus factor antibody), mevizumab (alias: NUMAX, anti-respiratory syncytial virus antibody), molobumab-CD 3 (alternative name: OKT3 ORTHOCLONE, anti-CD 3 antibody)Tanacamab (anti-C242 antibody), tanacamab (anti-5T 4 antibody), natalizumab (alias: Tysabri, anti-integrin alpha 4 antibody), nebuckumab (anti-endotoxin antibody), Necituumab (anti-EGFR antibody), Nerelimomab (anti-TNF-alpha antibody), nimotuzumab (alias: Therascim, Theraloc, anti-EGFR antibody), Nofetumomab, ocrelizumab (anti-CD 20 antibody), Oxidermumab (alias: Afolimomab, anti-LFA-1 (CD11A) antibody), mumab (alias: Arzerra, anti-CD 20 antibody), Olaratumab (anti-PDGF-R alpha antibody), Oxamazumab (alias: Sorley, anti-IgE Fc region antibody), Oportuumab (anti-CAM antibody), GavoVOX (alias: Ovax, anti-Syntezus-125 antibody), Ovoximab (anti-Epzugazine virus antibody), Epidex virus (anti-Epzulene Gvab), Epidex-Epidex antibody (anti-Epidex 3), panitumumab (alternative name: victoria, ABX-EGF, anti-EGFR antibody), panobazumab (anti-pseudomonas aeruginosa antibody), Pascolizumab (anti-IL-4 antibody), pemtezomab (alternative name: Theragyn, anti-MUC 1 antibody), pertuzumab (alternative name: omnitag, 2C4, anti-HER 2/neu antibody), pekelizumab (anti-C5 antibody), pintumumab (anti-adenocarcinoma antigen antibody), priliximab (anti-CD 4 antibody), promitumumab (anti-vimentin antibody), PRO140 (anti-CCR 5 antibody), Racotumomab (alternative name: 1E10, anti- (N-glycolylneuraminic, NGNA) -ganglioside GM3) antibody, resiviru (anti-rabies glycoprotein antibody), ramumab (anti-2 antibody), ranibizumab (anti-veglis, veggis antibody), anti-VEGF antibody (anti-lucorkun antibody), anti-VEGF antibody (anti-VEGF antibody), anti-VEGF antibody protective antibody (anti-VEGF antibody), reslizumab (anti-IL-5 antibody), Rilutummab (anti-HGF antibody), rituximab (alternative names: rituximab, Rituxanmab, anti-CD 20 antibody), Robatimumab (anti-IGF-1 receptor antibody), Rontalizumab (anti-IFN-. alpha.antibody), Rovelizumab (alternative names: LeukArrest, anti-CD 11, CD18 antibody), Ruplizumab (alternative names: Antova, anti-CD 154(CD40L) antibody), Saltuzumab (anti-TAG-72 antibody), Severzumab (anti-cytomegalovirus antibody), Sibrotuzumab (anti-FAP antibody), Sifalimumab (anti-IFN-. alpha.antibody), Siltuximab (anti-IL-6 antibody), Siplizumab (anti-CD 2 antibody), (rt) 95 (anti-CD 33 antibody), Solanezumab (anti- β -amyloid antibody), Sonepcizumab (anti-sphingosine-1-phosphate antibody), sovizumab (anti-epitalin antibody), staumu (anti-myostatin antibody), thiovacizumab (alternative: leukascan, (anti-NCA-90 (granulocyte-antigen) antibody))), Tacatuzumab (anti- α -fetoprotein antibody), taduzumab (anti-integrin α IIb β 3 antibody), talizumab (anti-IgE antibody), taluzumab (anti-NGF antibody), taplizumab (anti-CD 19 antibody), tefibuzumab (alternative: aurexis, anti-aggregation factor a antibody), attentiomab, tenatumumab (anti-tenascin C antibody), tenecteximab (anti-CD 40 antibody), Teplizumab (anti-CD 3 antibody), TGN1412 (anti-CD 28 antibody), Ticilimumab (alias: tremelimumab (anti-CTLA-4 antibody), Tigatuzumab (anti-TRAIL-R2 antibody), TNX-650 (anti-IL-13 antibody), Tolizumab (alias Allizumab, Actemra, Roacttemra, (anti-IL-6 receptor antibody), Tolizumab (anti-CD 154(CD40L) antibody), Toximumab (anti-CD 20 antibody), trastuzumab (herceptin, (anti-HER 2/neu protein antibody), Tremelimumab (anti-CTLA-4 antibody), Tucotuzumab celluliukin (anti-EpCAM antibody), tuvirumab (anti-hepatitis B virus antibody), Urtoxuzumab (anti-E.coli antibody), Ulteus mab (alias: Stelara, anti-IL-12, IL-23 antibody), Valexizumab (anti-C3 (VAP-1) antibody, Vedolizumab (anti-CTLA-4. beta. antibody), Tigatuzumab (anti-AOlizumab (anti-CD-IL-3) antibody, Velizumab (anti-CD-C-3-2, anti-CD-C-3-antibody), vitaxin (anti-angiointegrin avb3 antibody), Volociximab (anti-integrin. alpha.5. beta.1), Votuzumab (alternative: HumaSPECT, anti-tumor antigen CTAA16.88 antibody), Zatuzumab (alternative: HUMAX-EGFR, (anti-EGFR antibody), Zanolimumab (alternative: HUMAX-CD4, anti-CD 4 antibody), Ziralimumab (anti-CD 147 (basic immunoglobulin) antibody), Azuolimumab (anti-CD 5 antibody), Enasicept (anti-CD 5 antibody)

Alfoseit (a)

Abiraypu

Lencept (ARCALYST), 14F7[ anti-IRP-2 (IRP-2) antibody]14G2a (anti-GD 2 ganglioside antibody for treatment of melanoma and solid tumors from nat. cancer inst.), J591 (anti-PSMA antibody for treatment of prostate cancer, Willcannel medical college), 225.28S [ anti-HMW-MAA (high molecular weight melanoma associated antigen) antibody, Soline Radiofarci SRL (Milan Italy) for treatment of melanoma]COL-1 (anti-CEACAM 3 antibody, CGM1, nat. cancer inst. for treatment of colorectal and gastric cancers), CYT-356 (c h)

For the treatment of prostate cancer), HNK20(OraVax for the treatment of respiratory syncytial virus), ImmuRAIT (from immunomedicine for the treatment of non-hodgkin lymphoma), Lym-1 (anti-HLA-DR 10 antibody, berfujic, for cancer), MAK-195F [ anti-TNF antibody (aka: tumor necrosis factor; TNFA, tumor necrosis factor- α; TNFSF2), Abert/Norel, for treating septic shock]MEDI-500[ alias: T10B9, anti-CD 3 antibody, TR α β (T cell receptor α/β), Complex, MedImmune for the treatment of graft versus host disease]RING SCAN [ anti-TAG 72 (tumor-associated glycoprotein 72 antibody), Neoprene group, for the treatment of breast, colon and rectal cancer. Avicidin (anti-EpCAM antibody (epithelial cell adhesion molecule), anti-TACTD 1 antibody (tumor associated calcium signaling 1), anti-GA 733-2 (gastrointestinal tumor associated Protein 2), anti-EGP-2 antibody (epithelial glycoprotein 2), anti-KSA antibody, KS1/4 antigen, M4S, tumor antigen 17-1A, CD326 from NeoRx for the treatment of colon, ovarian, prostate and non-Hodgkin's lymphoma, LymphoCide (IMMUNOMECIS Inc., NJ), smart ID10(Protein Denn Labs), Oncollagen (Techniclone Inc., Calif.), Allomum (Transplast, CA), anti-VEGF antibody (Genentech Inc., CA), CEAcide (IMNOMES Inc., ImNJ), IMC-1C11 (clone, NJ), and Cetussic, New Jexib, Inc., New Jersey).

Other antibodies for binding to an antigen include (but are not limited to): aminopeptidase N (CD13), annexin A1, B7-H3(CD276, various cancers), CA125, CA15-3 (cancer), CA19-9 (cancer), L6 (cancer), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA242, placental alkaline phosphatase (cancer), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, lymphoma other than Hodgkin's lymphoma, multiple myeloma), epsilon of CD3 (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B-cell malignancy), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30, CD33, CD37, CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia), CD51 (metastatic melanoma, sarcoma), CD52, CD56 (small cell lung carcinoma, ovarian cancer, Merkel cell carcinoma, as well as liquid tumors, multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD79, CD80 (lymphoma), CD98 (cancer), mucin (carcinoma), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (large intestine cancer, stomach cancer), ACACACAM 5 (carcinoembryonic antigen; CEA, CD66e) (breast cancer, colorectal cancer and lung cancer), DLL4 (delta-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4(CD184, heme 4 (tumor, endothelial 105), endothelial solid tumors (endothelial glycoprotein), solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head, neck, colon, prostate non-hodgkin lymphoma, and ovarian cancer), ERBB2 (epidermal growth factor receptor 2; lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (a cell surface antigen glyvolipid, melanoma), idiotypic GD3 (cancer), heat shock protein (cancer), HER1 (lung, gastric cancer), HER2 (breast, lung and ovarian cancer), HLA-DR10(NHL), HLA-DRB (non-hodgkin lymphoma, B cell leukemia), human chorionic gonadotropin (cancer), IGF1R (insulin-like growth factor 1 receptor, solid tumors, hematological cancers), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma), IL-6R (interleukin 6 receptor, multiple myeloma, rheumatoid arthritis, Castleman disease, IL6 dependent tumors), integrin (α v β 3, α 5 β 1, α 6 β 4, α ll β 3, α 5 β 5, α v β 5 cell attachment factor, to various cancers, MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE 4 (carcinoma), anti-transferrin receptor (carcinoma), P97 (melanoma), MS4A1 (transmembrane domain 4 subfamily A member 1, non-Hodgkin's B cell lymphoma, leukemia), MUCl or MUC1-KLH (breast cancer, ovarian cancer, cervical cancer, bronchial and gastrointestinal cancer), MUC16(CA125) (ovarian cancer), CEA (large intestine), GP100 (melanoma), MART1 (melanoma) MPG (melanoma), MS4A1 (transmembrane domain 4 protein A, small cell lung cancer, non-Hodgkin's lymphoma), nucleolus, neural oncogene products (carcinoma), P21 (carcinoma), anti- (N-glycolylneuraminic acid, breast cancer, melanoma), PLAP-like testis alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumor), PSA (prostate), ROBO4, TAG72 (tumor-associated glycoprotein 72, leukemia, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202B), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, non-Hodgkin's lymphoma, and other cancers, rheumatoid arthritis and systemic lupus erythematosus), TPBG (trophoblastic glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis-inducing apoptosis-ligand receptor 1, lymphoma, non-Hodgkin's lymphoma, large intestine cancer, lung cancer), VCAM-1(CD106, melanoma), vascular endothelial growth factor-A, VEGF-2(CD309) (various cancers). Some other tumor-associated antigens recognized by antibodies have been reviewed (Gerber et al, mAbs 1: 3, 247-253 (2009); Novellino et al, cancer immunological immunother.54(3), 187-207(2005) Franke et al, cancer bipher radiopharm.2000, 15, 459-76). Still many other antigens are: further different clusters (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CD 17, CD18, CD19, CD20, CD21, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD22, CD 3679, CD36, nucleolus, endoglin (CD105), ROBO4, aminopeptidase N, delta-like 4(DLL4), VEGFR-2(CD309), CXCR 49 CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPVE 6E 7, EGFRvIII, HER-2/neu, idiotype, MAGEA3, P53 nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Napi3B (NAPI-3B, NPTIIb, Tetratocaine-derived EGFP, EphhhHA, EpHb receptor, EPB, aminopeptidase N, HER-like 4 (DLL), receptor binding protein, receptor binding protein, receptor binding protein, receptor binding protein, receptor binding protein, receptor binding protein binding, mm42015, SEMA5B B, 5EMAG, semaphoring 5 bHlog, sdema domain, seven platelet repeats, cytoplasmic domain), PSCA, STEAP1(6 transmembrane epithelial prostate antigens), and STEAP2(HGNC 8639, IPCA-1, PCANP1, STAMP1, STEAP2, STMP, prostate), tyrosinase, survivin, hTERT, sarcoma translocation breakpoint, EphA2, PAP, ML-IAP, AFP, CAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic, MYCN, RhoC, TRP-2, GD3, fucosylganglioside, mesothelin, PSCA, MAGEA1, sLeNY (a), CYP1B 5, PLAC1, GM 24, XAC 3, BORIS-2, SAOB 3, SAOB-III-S597, HMC-S-597, SARG-9, SARG-S-849, HMLCE-7, SARG-S-7, SALCS-7, SAGE-S-7, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, Fos protein-related antigen 1.

Another particular application is the use of the antimitotic molecule-binding molecule conjugates (conjugates) of the invention in cancer therapy according to different combinations and methods. These cancers include, but are not limited to, adrenocortical carcinoma, rectal cancer, bladder cancer, brain tumors (adult: brain stem glioma, childhood, cerebellar astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, pineal, visual pathway and hypothalamic glioma), breast cancer, carcinoid tumors, gastrointestinal tract, unknown primary cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, ewing family tumor (PNET), extracranial malignant germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer (stomach), germ cell tumor, extragonadal, gestational cell tumor, head and neck tumor, hypopharynx cancer, islet cell cancer, kidney cancer (renal cell carcinoma), larynx cancer, leukemia (acute lymphocytes, acute myelocytes, chronic lymphocytes, chronic myelocytes, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, lymphoma (aids-related, central nervous system, cutaneous T cell, hodgkin's disease, non-hodgkin's disease, malignant mesothelioma, melanoma, merkel cell carcinoma, primary occult squamous neck metastasis cancer, multiple myeloma and other plasma cell tumors, mycosis fungoides, myelodysplastic syndrome, myeloproliferative diseases, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelial cells, germ cell tumors, low malignant potential tumors), pancreatic cancer (exocrine, islet cell cancer), sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary tumor, plasma cell tumor, prostate rhabdomyosarcoma, rectal cancer, renal cell cancer (kidney cancer), renal pelvis and ureter (transitional cells), salivary gland cancer, cerclage syndrome, skin cancer (dermatoid T cell lymphoma, Kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, gastric cancer, testis cancer, thymoma (malignant), thyroid cancer, urethra cancer, uterus cancer (sarcoma), children abnormal tumor, vaginal cancer, vulval cancer, and nephroblastoma.

Another particular application is that the antimitotic molecule-binding molecule conjugates of the invention are useful in the prevention and treatment of autoimmune diseases, according to various combinations and methods. Autoimmune diseases include, but are not limited to, achlorhydria autoimmune chronic active hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukolitis, addison's disease, globulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-glomerular basement membrane/tubule basement membrane nephritis, anti-phospholipid syndrome, anti-synthetase syndrome, arthritis, atopic allergy, allergic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral nervous system disease, autoimmune pancreatitis autoimmune polyendocrine disease I, II, III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Barlow's disease/Barlow's concentric sclerosis, Heiqitt syndrome, Berger's disease, Bickerstaff brainstem encephalitis, Blau syndrome, large bract pemphigoid, giant lymph node hyperplasia, American trypanosomiasis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, chronic Lyme disease, chronic obstructive pulmonary disease, allergic granulomatous vasculitis, cicatricial pemphigoid, abdominal diseases, cochlear vestibular syndrome, cold agglutinin disease, complement C2 deficiency, craniomaarteritis, acral scleroderma syndrome, Crohn's disease (an idiopathic inflammatory bowel disease), Cushing's disease, cutaneous leukocytic fragmented vasculitis, malignant atrophic papulosis, painful obesity, dermatitis herpetiformis, dermatomyositis, type 1 diabetes mellitus, diffuse cutaneous scleroderma, post-myocardial infarction syndrome, discoid lupus erythematosus, eczema, endometriosis, juvenile idiopathic arthritis, eosinophilic fasciitis, erythema nodosum, idiopathic mixed cryoglobulinemia, Evens syndrome, progressive ossified fibrous histodysplasia, fibromyalgia, fibromyositis, fibrositis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, Graves' disease, Guillain-Barre neuritis, Hashimoto encephalitis, lymphoma goiter, hemolytic anemia, hypersensitive purpura, herpes gestationis, hidradenitis suppurativa, Houseki syndrome (antiphospholipid antibody syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (autoimmune thrombocytopenic purpura), IgA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating disease, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, mucocutaneous lymph node syndrome, Lambertian myasthenia syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), amyotrophic lateral sclerosis, lupus-like hepatitis, lupus erythematosus, Magidyne syndrome, Meniere's syndrome, microscopic polyangiitis, Miller Fisher syndrome, mixed connective tissue disease, scleroderma, Mohal's disease, Wells's syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devick's disease), neuromuscular rigidity, ocular cicatricial pemphigoid, ocular contracture-myoclonus syndrome, ouder's thyroiditis, recurrent rheumatism, panda syndrome (childhood autoimmune neuropsychiatric abnormalities combined with streptococcal infection), neoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, progressive lateral atrophy, Bart's syndrome, pars plana, pemphigus vulgaris, pernicious anemia, periphlebitis, POEMS 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, Rossemens encephalitis, Raynaud's disease, recurrent polychondritis, Retter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, pyrexia, sarcoidosis, schizophrenia, schmidt's syndrome, schnithler's syndrome, scleritis, scleroderma, sjogren's syndrome, spondyloarthropathies, mucoid syndrome, still's disease, stiff man syndrome, subacute bacterial endocarditis, Susac's syndrome, acute febrile neutrophilic skin disease, sydenham's chorea, sympathetic ophthalmia, takayasu's arteritis, temporal arteritis (giant cell arteritis), painful ophthalmoplegia syndrome, transverse myelitis, ulcerative colitis (an idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathy, vasculitis, vitiligo, wegener's granulomatosis, wilson's syndrome, wiskott-aldrich syndrome.

In another embodiment, antigen binding molecules for conjugation for use in treating or preventing autoimmune diseases include, but are not limited to: an anti-elastin antibody; abys anti-epithelial cell antibodies; anti-basement membrane type IV collagen antibodies; antinuclear antibodies; anti-double-stranded DNA antibodies; anti-single-chain DNA antibody, anti-cardiolipin antibody IgM, IgG; anti-celiac (anti-celiac antibody) antibodies; anti-phospholipid antibodies IgK, IgG; anti-ribonucleoprotein antibodies; anti-mitochondrial antibodies; a thyroid antibody; microsomal antibodies, T-cell antibodies; thyroglobulin antibody, anti-scleroderma-70 antibody (anti-SCL-70); human anti-Jo antibody (anti-Jo); autoantibodies against systemic lupus erythematosus; anti-sjogren's syndrome antibody (Anti-La/SSB); anti-systemic lupus erythematosus antibody; anti-parietal cell antibodies; an anti-histone antibody; anti-ribonucleoprotein antibodies (anti-RNP); neutrophil cytoplasmic antibody (C-ANCA); perinuclear anti-neutrophil antibody (P-ANCA); anti-centromere antibodies; anti-nuclear fibrin antibodies, as well as Anti-glomerular basement membrane antibody (GBM), Anti-ganglioside (Anti-ganglioside) antibodies; anti-desmoglein 3 antibody (anti-desmogenin 3); anti-human P62 antibody; anti-human sp100 antibodies; anti-mitochondrial M2 antibody; antibodies to rheumatoid factor; anti-mutant citrullinated vimentin antibody (anti-MCV); an anti-topoisomerase antibody; anti-neutrophil cytoplasmic (CANCA) antibodies.

In certain preferred embodiments, the binding molecules used in the present invention for conjugate coupling can bind to a receptor or receptor complex expressed by activated lymphocytes associated with autoimmune diseases. Including immunoglobulin gene superfamily members (e.g., CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD70, CD79, CD90, CD152/CTLA-4, PD-1, or ICOS), TNF receptor superfamily members (e.g., CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, TRAIL 2/TRAIL-R Apo 1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and 30APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (type C, type S or type I), or complement regulatory proteins.

In another embodiment, a useful binding partner immunospecific for a viral or bacterial antigen is a human or human monoclonal antibody. The term "viral antigen" as used herein includes, but is not limited to: any viral peptide, polypeptide protein (e.g., HIV Nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, human T lymphocyte virus infection regulatory factor tax, herpes simplex virus glycoproteins (e.g., gB, gC, gD and gE) and hepatitis B surface antigen) that can elicit an immune response. The term "bacterial antigen" as used herein includes, but is not limited to: any microbial peptide fragment, polypeptide protein, saccharide, polysaccharide, lipid molecule (e.g., bacteria, fungi, pathogenic protozoa, yeast polypeptides including, for example, lipopolysaccharide and capsular polysaccharide 5/8) capable of eliciting an immune response. Useful type I antibodies useful for treating viral or bacterial infections include, but are not limited to: palivizumab, a human-derived anti-respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody for use in the treatment of HIV infection; ostavir, a human antibody for the treatment of hepatitis b virus; PROTVIR, a humanized antibody IgG.sub.1 for the treatment of cytomegalovirus, and also anti-lipopolysaccharide (anti-LPS) antibodies.

The binding molecules-antimitotic agent conjugates of the invention can be used to treat infectious diseases. These infectious diseases include, but are not limited to: acinetobacter infection, actinomycosis, african sleeping sickness (african trypanosomiasis), aids (acquired immunodeficiency syndrome), amebiasis, microsporidiosis, anthrax, cryptococcus haemolyticus infection, argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesiosis, bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, bacteroidal infection, venocytosis, ascariasis, BK viral infection, black knot disease, human blastocyst protozoal infection, blastomycosis, borlivia hemorrhagic fever, borrelia infection, poisoning (and botulism) in infants, brazilian hemorrhagic fever, brucellosis, bursitis, brucellosis, bunyavirus infection, calicivirus and saporovirus, campylosis, candida (candidiasis, thrush), candidiasis, cellulitis, Chagas disease (trypanosomiasis americana), chancroid, chicken pox, Chlamydia pneumoniae infection, cholera, chromomycosis, liver trematosis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever, common cold (acute viral nasopharyngitis; acute rhinitis), Creutzfeldt-Jakob disease, Crimeria-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis, cutaneous larva migration, Cyclosporidiosis, cysticercosis, cytomegalovirus infection, dengue fever, Armicaba disease, diphtheria, schizocephala, Longlinear, Ebola hemorrhagic fever, echinococcosis, Ehrlichiosis, enterobiasis (enterobiasis), enterococcus infection, Enterovirus infection, epidemic typhus, typhoid fever (fifth disease), juvenile acute eruption, fascioliasis, familial insomnia, filariasis, Clostridium perfringens induced food poisoning, non-parasitic amebiasis infection, a clostridial infection, gas gangrene (clostridial myonecrosis), a dirofilariosis, costermann-schterilsus syndrome, giardiasis, meliodiosis, palatine nematodiasis, gonorrhea, inguinal granulomatosis, a group a streptococcal infection, a group B streptococcal infection, haemophilus influenzae infection, hand-foot-and-mouth disease (HFMD), hantavirus lung syndrome, helicobacter pylori infection, hemolytic uremic syndrome, hemorrhagic fever nephrotic syndrome, hepatitis a, hepatitis B, hepatitis c, hepatitis d, hepatitis e, herpes simplex, histoplasmosis, hookworm infection, human abamectin infection, human eilerian disease, human granulocytic anaplasmosis, human metapneumovirus infection, human monocytic ehrlichiosis, human papillomavirus infection, human parainfluenza virus infection, membranous taeniasis, epstein-barr virus infection (mononucleosis virus), influenza, isosporosis, kawasaki disease, keratitis, aureobacerium infection, kuru, lassa fever, legionnaires disease (chard army association membership disease), legionellosis (pomtie fever), leishmaniasis, leprosy, leptospirosis, listeriosis, lyme disease (lyme borreliosis), lymphofilariasis (elephantiasis), lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever, measles, rhinoid (hutermore's disease), meningitis, meningococcosis, posterior genital fluke disease, microsporidiosis, molluscum contagiosum, mumps, typhus (endemic typhus), mycoplasmal pneumonia, footplate mycosis, myiasis, neonatal conjunctivitis (neonatal ophthalmia), crook-asia syndrome (vCJD, nvd), nocardia, onchocerciasis (river inflammation), paracoccidioidomycosis (southern American blastomycosis), paragonimiasis, pasteurellosis, pediculosis capitis (head louse), pediculosis corporis (body louse), pediculosis pubis (pubic louse), pelvic inflammatory disease, pertussis, plague, pneumococcal infection, pneumocystic pneumonia, poliomyelitis, prevotella infection, primary amoebic meningitis, progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, rat bite fever, respiratory syncytial virus infection, nosema disease, rhinovirus infection, rickettsia, rift valley fever, rocky mountain spotted fever, rotavirus infection, rubella, salmonellosis, SARS (Severe acute respiratory syndrome), scabies, schistosomiasis, sepsis, dysentery (mycosis), herpes zoster (shingles), smallpox (dysentery), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, nematodes, syphilis, taeniasis, tetanus (trismus), tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea manuum, pityriasis rubra, tinea pedis, onychomycosis, tinea versicolor, toxocariasis (eye larva migratory disorder), toxocariasis (visceral larva migratory disorder), toxoplasmosis, trichinosis, trichomoniasis, trichiasis (trichuris infection), tuberculosis, tularemia, ureolytic urea mycoplasma infection, venezuelan equine encephalitis, venezuelan hemorrhagic fever, viral pneumonia, west nile fever, white hairy sarcoidosis (white hairy sarcoidosis), pseudotuberculosis infection, yersinia, yellow fever, zygomycosis.

The binding molecules for the antibodies previously described in this patent are useful against pathogenic strains, including, but not limited to: acinetobacter baumannii, Actinomyces israeli, Actinomyces gordonii and Propionibacterium propionate, Trypanosoma brucei, HIV (human immunodeficiency virus), Endomonas histolytica, Anaplasmata, Bacillus anthracis, Cryptococcus hemolyticus, Hunningvirus, ascaris, Aspergillus, Plantago virus family, Babesia, Bacillus cereus, Polyporus, Bacillarioides, Bellisrelatively ascaris, BK virus, Rhizobium hirsutus, Protozoa hominis, Blastomyces dermatitidis, arenavirus, Borrelia, Clostridium botulinum, Sinomenii, Brucella, Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerosa, Caliciviridae family, Campylobacter, Candida albicans and other Candida species, Bartonella, group A streptococci and staphylococci, Trypanosoma cruzi, Debrevix, varicella Zoster Virus (VZV), Chlamydia trachomatis, Chlamydia pneumoniae, Vibrio cholerae, Pythium ultimum, Clonorchis sinensis, Clostridium difficile, Coccidioides and Coccidioides posadasii, Colorado tick fever virus, rhinovirus, coronavirus, prion Creutzfeldt-Jakob disease, Crimia-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium sp, Uncaria felis; multiple parasites, cyclosporins, taenia solium, cytomegalovirus, dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), arboviruses, dicamba fragilis, corynebacterium diphtheriae, schizocephalum, Melilongoides, Ebola, Echinococcus, Elekia, Enterobacter, Enterovirus, Rickettsia pulcherrima, parvovirus B19, human herpesvirus type 6 and human herpesvirus type 7, fasciola brunetti, fasciola hepatica and fasciola magnus, FFI prion, Hyperfamily Trichostera capsulata, Aerobacter aerogenes, Clostridium perfringens, other Clostridia, Geotrichum gangrenosum, GSS prion, Giardia intestinalis, Burkholderia rhinobylonica, Acanthopanax japanoralis and Acanthopanax japannicus, Neisseria gonorrhoeae, Mycoplasma pyogenes, Streptococcus agalactis, haemophilus influenzae, enteroviruses, most coxsackie a viruses and enterovirus type 71, the sinova virus, helicobacter pylori, escherichia coli O157: h7, family 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, histoplasmosis, ancylostoma duodenale and American aphrodisiae, Haemophilus influenzae, human bocavirus, Erwinia ehrlichi, anaplasma, human metapneumovirus, Serratia arenicola, human papillomavirus, human parainfluenza virus, Micromembranella and Epimedium minitans, EB virus, Orthomyxoviridae, Belleville, King's bacillus, Klebsiella pneumoniae, Klebsiella ozaea ozaenas, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophiladensis, Lepidium, Mycobacterium leprae and Mycobacterium diffusible, Leptospira, Listeria, Borrelia and other Borrelia, spanish and Maleinia, lymphocytic choriomeningitis virus (LCMV), Plasmodium, Marburg, measles, Burkholderia farinosa, Neisseria meningitidis, retrograduate, Microsporophyl, Molluscum Contagiosum (MCV), mumps, Rickettsia, Mycoplasma pneumoniae, various bacteria (M.Dora mycosis) and fungi (M.Dora mycosis), parasitic dipteran maggot, Chlamydia trachomatis and gonococcus, vCJD prion, Nocardia asteroides and other Nocardia species, Spiraptera circinelloides, Braziliana, paragonia and other paragonimus, Pasteurella, tinea capitis, Yersinia, crab louse, Bordetella pestis, Lawsonia, Streptococcus pneumoniae, Pneumocystis, poliovirus, Lewy, Omithelitis, and resistant Michelia, JC virus, Chlamydia psittaci, Rickettsia, rabies virus, Streptococcus candidus and Treponema pestis, respiratory syncytial virus, Siberian nosema, rhinovirus, Rickettsia, Leptosporidium, Leptosphaeria gonorrhoeae, Rickettsia, rotavirus, rubella virus, Salmonella, atypical pneumonia coronavirus, sarcoptic mite, schistosoma, Shigella, varicella zoster virus, smallpox or smallpox, Trichosporon scherianum, Staphylococcus aureus, Streptococcus pyogenes, strongyloides, treponema pallidum, Taenia, Clostridium tetani, Trichophyton trichophyton, Trichophyton, Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes, Exophyton venenum, Trichophyton, Malassezia, Toxophytes or Toxoplasma ascaris, trichina, trichomonas vaginalis, trichuris, mycobacterium tuberculosis, francisella tularensis, mycoplasma urealyticum, venezuelan equine encephalitis virus, vibrio cholerae, citrullinator, west nile virus, trichotheca alba, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever virus, mucorales (mucormycosis) and entomomycetales (entomophthora), pseudomonas aeruginosa, campylobacter fetus (vibrio), aeromonas hydrophila, edwardsiella tarda, yersinia, shigella shigae, shigella flexneri, salmonella typhimurium, Treponema, borneospora poensis, borrelia borgpetersenii, leptospira icterohaemorrhagiae, pneumocystis carinii, brucella suis, brucella typhi, Rickettsia, utsugumushi, pathogens; pathogenic fungi (aspergillus, candida albicans, histoplasma capsulatum); protozoa (entamoeba histolytica, trichomonas vaginalis, trichomonas humanus, trypanomonas gamniense, trypanosoma rhodesiense, leishmania donovani, leishmania tropicalis, leishmania brasiliensis, pneumocystis pneumonia, plasmodium vivax, plasmodium falciparum, malaria falciparum); or helminth (Schistosoma japonicum, Schistosoma mansoni, Schistosoma Egypti and hookworm)

Other antibodies that are binding ligands of the invention are useful for treating viral diseases including, but not limited to: antibodies against antigens of pathogenic viruses include the following examples but are not limited to: smallpox virus, herpesvirus, adenovirus, papovaviridae, enteroviridae, picornaviridae, parvoviridae, reovirus, retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, arbovirus, rhabdovirus, arenaviridae, Non-a/Non-B hepatitis virus, rhinovirus, coronavirus, rotaviridae, oncoviruses [ e.g., hepatitis B virus (hepatocellular carcinoma), human papilloma virus (cervical carcinoma, anal carcinoma), kaposi's sarcoma-associated herpesvirus (kaposi's sarcoma), EB virus (nasopharyngeal carcinoma, burkitt lymphoma, primary central nervous system lymphoma), MCPyV (merkel cell carcinoma), SV40 (simian virus 40), hepatitis c virus (hepatoma), human T-lymphotropic virus type 1 (adult T cell leukemia/lymphoma), immune dysregulation causes the virus: [ such as human immunodeficiency virus (AIDS) ]; central nervous system virus: [ e.g., JCV (progressive multifocal leukoencephalopathy), MeV (subacute sclerosing panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitis, Orthomyxoviridae (possible) (narcolepsy), RV (rabies), vesicular stomatitis-India virus genus, herpesviral meningitis, Lamzici Hunter syndrome type II; poliomyelitis (polio, post-polio syndrome), human T-lymphotropic virus type 1 (tropical spastic paraplegia) ]; cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)); cardiovascular disease viruses [ such as coxsackie virus (pericarditis, myocarditis) ]; respiratory system/acute nasopharyngitis virus/viral pneumonia: [ Epstein-Barr virus (herpes virus type 4 infection/infectious mononucleosis), cytomegalovirus; SARS coronavirus (severe acute respiratory syndrome) orthomyxovirus: influenza a/B/C (influenza/avian influenza), paramyxovirus: human parainfluenza virus (parainfluenza), respiratory syncytial virus (human respiratory syncytial virus), pneumovirus ]; digestive system virus [ MUV (mumps), cytomegalovirus (cytomegalovirus esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus; HBV (hepatitis b virus), coxsackie virus, hepatitis a (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) ].

According to a further object, the present invention also includes the above-mentioned conjugated pharmaceutical compositions in combination with other possible pharmaceutical carriers as therapeutic agents for cancer and autoimmune diseases. The methods of the invention for treating cancer and autoimmune diseases include in vitro, in vivo or ex vivo therapy. Examples of the application of in vitro therapy include drug treatment of in vitro cultured cells to kill all cells except cells not expressing the antigen of interest; or killing cells that express undesired antigens. As an example of a method of treatment for ex vivo therapy: hematopoietic stem cells are treated in vitro to kill diseased or malignant cells before being returned to the patient. For example, cancer and autoimmune diseases are treated clinically by ex vivo treatment to remove tumor cells or lymphocytes from the bone marrow and then transfused back to the original patient, or T cells and other lymphocytes from the bone marrow are removed prior to transplantation to prevent an immune antagonistic response of the graft. The implementation method comprises the following steps: bone marrow cells are harvested from a patient or other individual and cultured in a serum-containing medium to which the conjugate drug of the present invention is added at 37 ℃ in a drug concentration range of 1pM to 0.1mM for a period of about 30 minutes to about 48 hours. The specific concentration of the drug and the incubation time are determined by experienced clinicians. After the culture is finished, the bone marrow cells are washed by serum-containing culture medium and then are infused back into the human body through intravenous injection. If the patient is to receive other treatments, such as ablative chemotherapy or total body radiation, prior to the bone marrow cell harvesting and reinfusion treatment, the treated bone marrow cells can be stored in a qualified liquid nitrogen medical facility.

For in vivo clinical use, the conjugate drugs of the present invention will be provided in the form of a solution or a lyophilized solid that can be dissolved in sterile water and then injected. Examples of suitable methods of administering conjugated drugs are as follows: the conjugate drug was injected intravenously once a week for 4-12 weeks. A single dose is administered by dissolving in 50 to 500ml of physiological saline to which human serum albumin (e.g., 0.5 to 1ml of 100mg/ml concentrated human serum albumin) may be added. The dose of the drug is approximately 50 μ g to 20mg per kg of body weight per week, i.v. injection (10 ug to 200mg/kg of body weight per injection). After the treatment for 4-12 weeks is finished, the patient can receive a new treatment. The detailed treatment method including administration route, excipient, diluent, pharmaceutical dose, treatment time and the like can be decided by a skilled surgeon.

Examples of diseases that can be treated by selectively killing the cell population by in vivo or ex vivo methods include any kind of malignancy, autoimmune disease, transplant rejection and infection (including viruses, bacteria or parasites).

The amount of conjugated drug required to achieve the desired biological effect will vary depending upon a number of factors including the nature of the compound, its therapeutic effect and bioavailability of the conjugated drug, the type of disease, the race of the patient, the diseased state of the patient, the route of administration, all of which together determine the timing and manner of administration.

In summary, the conjugated drugs of the present invention can be administered parenterally by dissolving in physiological buffer in a mass to volume ratio of 0.1 to 10%. Typical drug doses range from 1ug to 0.1g per kilogram of body weight per day; recommended doses of the drug range from 0.01mg to 20mg per kilogram of body weight per day or equivalent doses of the child. The recommended amount to be administered depends on a number of variables, including the type of disease or disorder, the general health status of the individual patient, the relative biological activity of the conjugate drug, the dosage form of the compound, the mode of administration (intravenous, intramuscular, or other), the pharmacokinetic properties of the selected mode of administration, as well as the rate of administration (single injection or continuous instillation) and the schedule of administration (number of administrations over a period of time).

The conjugate of the present invention can also be administered in the form of a unit dose, where "unit dose" refers to a dose administered once to a patient, and the unit dose of drug can be packaged and used simply and conveniently, and the unit dose of drug is an active conjugate drug itself which remains physically and chemically stable, or a pharmaceutically acceptable mixture as described later. Typical daily dosages range from 0.01 to 100mg per kg body weight. Generally, the unit dosage for a human per day ranges from 1 to 3000 mg. Recommended unit doses are from 1mg to 500mg administered four times a day, or from 10mg to 500mg once a day. The conjugate medicine can be prepared into a medicinal preparation by adding one or more pharmaceutically acceptable auxiliary materials. The unit dose of the drug may be administered orally, such as in the form of a tablet, a simple capsule or a soft gel capsule; or intranasally, such as a powder, nasal drops, or spray; or by dermal administration, such as ointments, creams, lotions, gels or sprays or dermal patches. The medicament may conveniently be administered in unit dosage form and may be prepared by any known pharmaceutical method, such as Remington: the Science and Practice of pharmacy, 21th ed.; lippincott Williams & Wilkins: philadelphia, PA, 2005.

Pharmaceutical dosage forms comprising the compounds of the invention include pharmaceutical compositions, preferably oral or parenteral administration. For oral administration, such as tablets, powders, capsules, tablets (lozenges) and the like may comprise one or more of the following materials or other compounds of similar nature: binders such as microcrystalline cellulose or tragacanth; diluents such as starch or lactose; dispersing agents such as starch and cellulose derivatives; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavour enhancer such as peppermint or methyl salicylate. The capsules may be in the form of hard or soft capsules, typically mixed optionally with a plasticizer from a gelatin mixture, as may starch capsules. In addition, the physical form of the unit dose can be varied by the addition of a variety of different materials, for example, sugar coatings, shellacs, or enteric agents. Other oral dosage forms such as syrups or elixirs may contain sweetening agents, preserving agents, colouring agents and flavouring agents. In addition, the active compounds can be formulated by various treatments and formulations to provide fast-dissolving, slow-release or sustained-release formulations, of which sustained-release formulations are preferred. Tablets are preferably formulated with a combination of lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate, talc, and the like.

Liquid preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. The liquid agent may also contain a binder, a buffer, a preservative, a chelating agent, a sweetening agent, a flavoring agent, a coloring agent, and the like. Non-aqueous solvents include ethanol, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic lipids such as ethyl oleate. The aqueous solvent comprises a mixture of water, ethanol, a buffering agent and a salt, in particular, a biocompatible, degradable lactide polymer, a lactide/glycolide polymer or a polyethylene glycol/polyglycerol copolymer can be used as an adjuvant for controlling the release of the active drug. Excipients for intravenous injection may include liquid and nutritional supplements, electrolyte supplements, and linoglycol-based excipients, and the like. Other possible parenteral delivery systems for the active agents of the invention include microparticles of ethylene-vinyl acetate copolymer, implantable osmotic pumps and liposomes.

Other possible modes of administration include inhalation, including dry powders, aerosols and drops. The inhalant may be a solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, deoxycholate or oil, and may be administered in the form of nasal drops, intranasal gel. The buccal preparation comprises lozenge, candy lozenge, etc., and optionally flavoring agent such as sucrose, acacia, and other adjuvants such as glycocholate, etc. Suppositories are suitable in unit dosage form, with solids such as cocoa butter as the carrier, and salicylic acid may also be added. The topical preparation for skin is preferably selected from plaster, emulsion, lotion, patch, gel, spray, aerosol or oil. Petrolatum, lanolin, polyethylene glycols, alcohols and mixtures thereof may be used as the pharmaceutical carrier. The formulations for dermal administration may be in the form of patches, emulsions, buffered solutions, dissolved or dispersed in polymers or adhesives.

In particular, the conjugate of the present invention may be used in combination with other known or unknown therapeutic agents, such as chemotherapeutic agents, radiation therapy, immunotherapy agents, autoimmune disease agents, anti-infective agents or other antibody drug conjugates, to achieve a synergistic effect. The co-drug or radiation therapy may be administered or administered before or after administration of the conjugate drug of the invention. It may be 1 hour, 12 hours, a day, a week, a month, or several months before or after administration of the conjugate of the invention.

In other embodiments, the drugs that act synergistically include, but are not limited to:

1) chemotherapeutic agents: a) alkylating agent: such as [ nitrogen mustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, cyclophosphamide chloroacetate); nitrosoureas: (carmustine, lomustine); alkyl sulfonate salt: (busulfan ); triazenes: (dacarbazine); platinum-containing compounds: (Carboplatin, cisplatin, oxaliplatin)](ii) a b) Plant alkaloid: such as [ vinca alkaloids: (vinblastine, vincristine, vindesine, vinorelbine); a taxane compound: (Taxol, taxotere)](ii) a c) DNA topoisomerase inhibitor: such as [ epipodophyllotoxin: (9-aminocamptothecin, camptothecin, clinatot, etoposide phosphate, irinotecan, teniposide, topotecan,); mitomycin: (mitomycin C)](ii) a d) An antimetabolite: such as { [ antifolate: dihydrofolate reductase inhibitors: (methotrexate, trimetrexate); IMP dehydrogenase inhibitors (mycophenolic acid, formamidothiazole, ribavirin, EICAR); ribonucleotide reductase inhibitors (hydroxyurea, deferoxamine)](ii) a [ pyrimidine analogs: uracil analogs: (5-fluorouracil, doxifluridine, raltitrexed (touude)); cytosine analogues: (cytarabine, fludarabine); purine analogues: (azathioprine, mercaptopurine, guanine)]}; e) hormones: such as { receptor antagonist: [ anti-estrogen: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goserelin, leuprorelin acetate); anti-androgens: (bicalutamide, flutamide)](ii) a Tretinoin/deltoid: [ vitamin D3 analogues (CB 1093, EB 1089 KH 1060, cholecalciferol, vitamin D2); photodynamic therapy: (for this, phthalocyanine photosensitizer, PC4, deoxyorine); cytokines: (interferon-alpha, interferon-gamma, tumor necrosis factor) Human proteins containing TNF domains]F) kinase inhibitors, such as bibw 2992 (anti-EGFR/Erb 2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, pinatinib, vandetanib, flumatinib, e7080 (anti-VEGFR 2), molitinib, melitinib, meditinib, ponatinib (ap 245631), HQP1351, barfitinib (ino-406), bosutinib (SKI-606), sunitinib, cabobovinib, voritinib, vegium, Iniparib, ruxolitinib, CYT387, axitinib, tivozanib, bevacizumab, sorafenib, trastuzumab, cetuximab, lanitumumab, panitumumab, isapinib; g) other classes: such as gemcitabine, epoxomicins (e.g., lenalidomide), bortezomib, lenalidomide, pomalidomide, tosedodes, zybrestat, PLX4032, sta-9090, Stimuvax, allovivin-7, xegeva, Provenge, Yervoy, prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), pingycins (e.g., bleomycin A2, B2, pelomycin), anthracyclines (e.g., erythromycin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitors (e.g., verapamil), Ca²⁺Inhibitors of ATPase (e.g., thapsigargin), inhibitors of histone deacetylase (vorinostat, romidepsin, panobinostat, valproic acid, motinostat (MGCD0103), belinostat, PCI-24781, entinostat, SB939, remininostat, givinostat, AR-42, one, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazones, epigallocatechin gallate, 5 disulfiram, salinosporamide a.

2) Anti-autoimmune agents include, but are not limited to: cyclosporin, cyclosporin A, azathioprine, aminocaproic acid, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, glucocorticoids (e.g., hormones, betamethasone, budesonide, flunisolide, fluticasone propionate, hydrocortisone, dexamethasone, fluconazole, triamcinolone acetonide, beclomethasone propionate), dehydroepiandrosterone, etanercept, hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenolate mofetil, sirolimus, tacrolimus, prednisone

3) Anti-infectious disease agents include, but are not limited to: a) aminoglycosides: amikacin, wuyimycin, gentamicin (netilmicin, sisomicin, isepamicin), hygromycin, kanamycin (amikacin, arbekacin, aminoxykanamycin, dibekacin, tobramycin) neomycin (neomycin B, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin, clarithromycin; b) amide alcohols: azidochloramphenicol, chloramphenicol, florfenicol, thiamphenicol; c) ansamycins: geldanamycin, herbimycin; d) carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin, meropenem, panipenem; e) cephalo species: cephem (chlorocepham), cephalosporacetonitrile, cefaclor, cephradine, cefadroxil, ceforanine, ceftazidime, cephalothin or cephalosporins, cephalexin, ceflaicin, cefamandole, cefapirin, ceftriaxone, cefazedone, cefazolin, cefbuperazone, cefcapene, cefdaxime, cefepime, cefminox, cefoxitin, cefprozil, cefixime, ceftezole, cefuroxime, cefixime, cefdinir cefditoren, cefepime, cefetamet, cefepime, cefodizime, cefonicid, cefoperazone, cefotaxime, cefotiam, cefozopran, cefalexin, cefepime, cefpodoxime, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, cefoselin, ceftizoxime, cepham, ceftriaxone, cefuroxime, ceftizome, cephamycin (cefoxitin, cefotetan, cefmetazole) oxacephem (flomoxef, latamoxef); f) a glycopeptide: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin) ramoplanin, daptomycin; g) glycyl: such as tigecycline; h) a beta-lactamase inhibitor: penicillane (sulbactam, tazobactam), clavulane (clavulanic acid); i) lincosamides: clindamycin, lincomycin; j) a lipopeptide: daptomycin, a54145, Calcium Dependent Antibiotic (CDA); k) macrolides: azithromycin, quinomycin and quinomycin, clarithromycin, dirithromycin, erythromycin, fluoromycin, josamycin, ketolides (telithromycin, quinomycin and quinomycin) medecamycin, meoxakacin, oleandomycin, rifamycin (rifampin, rifabutin, rifapentine), natamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506), oleandomycin acetate, tulithromycin; 1) a monocyclic β -lactam antibiotic: aztreonam, tigemonam; m) oxazolidinones: linezolid; n) penicillins: amoxicillin, ampicillin (pivampicillin, hydracillin, bacampicillin, maytansillin, phthalazinocillin) azicillin, azlocillin, penicillin, benzathine, phenoxybenzathine, cloxacillin, procaine penicillin, carbenicillin (cairinillin), cloxacillin, dicloxacillin, cephalomycin, flucloxacillin, mexacillin (diazamidine penicillin diester), mezlocillin, methicillin, nafcillin, oxacillin, acemetacillin, penicillin, necillin, piperacillin, hydrocillin, sulbenicillin, temocillin, ticarcillin; o) a polypeptide: bacitracin, polymyxin E, polymyxin B; p) quinolone drugs: alafloxacin, balofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, Floxin, gatifloxacin, gemifloxacin, grepafloxacin, trovafloxacin carnot, levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) a bacterin: pristinamycin, quinupristin/dalfopristin); r) sulfonamides: mafenide, chlordanese, sulfacetamide, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (compound sulfamethoxazole); s) steroid antibacterial drugs: such as fusidic acid; t) tetracyclines: doxycycline, chlortetracycline, demeclocycline, lymecycline, chloromethycin, methacycline, minocycline, oxytetracycline, piperacycline, rolicycline, tetracycline, glycyl (e.g., tigecycline); other types of antibiotics: annonaceae, arsinamine, bacterpenol inhibitors (bacitracin), Dadal/AR inhibitors (cycloserine), dictyostatin, discodermolide, exenatide, epothilones, ethambutol, etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalamide, metronidazole, mupirocin, mycolactines, NAM synthesis inhibitors (such as fosfomycin), nitrofurantoin, taxol, platemycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin), tazobactam tinidazole, annosquamoside;

4) antiviral drugs: a) entry/fusion inhibitor: aplaviroc, maraviroc, viriviroc, gp41 (enfuvir), PRO140, CD4 (Ibalizumab); b) an integrase inhibitor: latilatavir, eltiravir, globoidnanA; c) a maturation inhibitor: berevir lima, vivocon; d) neuraminidase inhibitor: oseltamivir, zanamivir, peramivir; e) nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cidofovir clavvudine, dexelvucitabine, didanosine (DDI), elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3 '-fluoro substituted 2', 3 '-dideoxynucleoside analogs (e.g., 3' -fluoro-2 ', 3' -dideoxythymidine (FLT) and 3 '-fluoro-2', 3 '-dideoxy (FLG) fomivirse, ganciclovir, idoside, lamivudine (3TC), L-nucleosides (e.g., β -L-thymidine, β -L-2' -deoxycytidine), penciclovir, Racivir, ribavirin, stampidine, vudine (d 894 of d 4T), taliravirin (imidine), telbivudine, tenofovir, valacyclovir trifluorothymidine, valganciclovir, zalcitabine (DDC), zidovudine (AZT); f) non-nucleosides: amantadine, atevirdine, carprivrine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphoryl formic acid), imiquimod, interferon alpha, lovirine, lodenosine, tazobactam, nevirapine, NOV-205, peginterferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), aceticadamantane; g) a protease inhibitor: amprenavir, atazanavir, borrelivir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950) tipranavir; h) other types of antiviral drugs: abzyme, arbidol, calanolides a, cerulenin, cyanobacterial-N, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taliverine (velvamidine), hydroxyurea, KP-1461, miltefosine, pleconaril, synthesis inhibitors, ribavirin, seliciclib;

5) other immunotherapeutic drugs: such as imiquimod, interferons (e.g., alpha, beta), granulocyte colony stimulating factor, cytokines, interleukins (IL-1-IL-35), antibodies (e.g., trastuzumab, pertuzumab, bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab), protein binding drugs (e.g., Abraxane), AN antibody binding drug selected from the group consisting of a calicheamicin derivative, a maytansine derivative (DM1 and DM4), CC-1065 and a duocarmycin minor groove agent, AN effective paclitaxel derivative, doxorubicin, AN ari antimitotic drug (e.g., trastuzumab-DM 1, Inmotuzumab, brentuximab vedotin, Glembutuzumab vedotin, loratadin, Lortuzumab mertansine, AN-LMB 2, VB 3638, VB 4-4, Cantuzumab 34133, Cantussie-36969, AVE 9682), IMGN388, IMGN529, IMGN853, milatuzumab-doxorubicin, SGN-75 (anti-CD 70), anti-CD 22-MCC-DM 1).

As a further object of the present invention, the present invention also relates to a process for producing the antibody drug conjugate therein. The conjugates of the invention can be prepared by a variety of methods well known in the art, for example, the antimitotic agents in the conjugates of the invention can be synthesized according to the methods described below or modified methods described below. These modifications are well known and obvious methods readily available to those skilled in the art from the scientific literature. In particular, there are many descriptions in Comprehensive Organic Transformations (R.C. Larock, 1999, Wiley-VCH, 2 nd edition).

During the reaction described herein, it is sometimes necessary to protect reactive functional groups such as hydroxyl, amino, imino, mercapto and carboxyl groups that may participate in the reaction to avoid side reactions. A common method of use of conventional Protective functional Groups is described in "Green's Protective Groups in Organic Synthesis", by P.G.Wuts and T.W.Greene (2006, Wiley-Interscience, 4 th edition). Some reactions may be carried out in a suitable acid or base containing solution. The acid, base and solvent for such reaction are not particularly limited, and any conventional acid, base and solvent may be used herein as long as they have no adverse effect. Moreover, these reactions can be carried out over a wide range of temperatures. In general, however, the reaction temperature is generally between-80 ℃ to 150 ℃ (preferably between room temperature and 100 ℃). The time required for the reaction can likewise vary widely, depending, of course, on a number of factors, in particular the reaction temperature and the nature of the solvent used. In general, reaction times of from 3 to 20 hours are suitable for a more ideal reaction.

The operation treatment after the completion of the reaction can be carried out by a conventional method. For example, the reaction product can be recovered by distilling off the solvent from the reaction system. Alternatively, after the solvent is distilled off, the residue may be poured into water and then extracted with a water-immiscible organic solvent, if necessary. Finally, after the extraction solvent has been distilled off, the reaction product is obtained. In addition, if higher purity is desired, it can be further purified by various conventional methods, such as recrystallization, sedimentation, or various chromatographic methods. Generally, column chromatography and preparative thin-plate chromatography are more commonly used. The synthesis of the antimitotic agents of the invention and conjugates thereof is shown in figures 1-28.

In the examples below, the cell binding body-antimitotic agent conjugates of the present invention will be described in further detail. Of course, the present invention is not limited to the following examples.

Experimental part:

mass spectral data were taken from a Bruker Esquire 3000 system and nuclear magnetic data were taken from a Bruker AVANCE300 spectrometer. Chemical shifts were accurate to parts per million and were internal normalized with tetramethylsilane. The uv spectral data were taken from a Hitachi U1200 spectrophotometer. High performance liquid chromatography data were obtained from an Agilent 1100 HPLC system equipped with a fraction collector and a variable wavelength detector. Thin-plate chromatography was performed using Analtech GF silica gel TLC thin-layer chromatography plates. Amino acids and their derivatives and pre-filled resins are available from Merck chemical International, Synthetech, Peptides International, Inc., Chembridge International, or Sigma-Aldrich. Partial cross-linkers such as NHS esters/maleimides (AMAS, BMPS, GMBS, MBS, SMCC, EMCS or Sulfo-EMCS, SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM (PEG)4, SM (PEG)6, SM (PEG)8, SM (PEG)12, SM (PEG) 24); NHS ester/pyridinedimercapto (SPDP, LC-SPDP or Sulfo-LC-SPDP, SMPT, Sulfo-LC-SMPT); NHS ester/haloacetyl (SIA, SBAP, SIAB or Sulfo-SIAB); NHS ester/cyclopropene (SDA or Sulfo-SDA, LC-SDA or Sulfo-LC-SDA, SDAD or Sulfo-SDAD); maleimide/hydrazide (BMPH, EMCH, MPBH, KMUH); pyridinedimercapto/hydrazide (PDPH); and isocyanate/maleimide (PMPI) from Thermo Fisher Scientific. SPDB and SPP were prepared according to the literature (Cumber, A.et al, Bioconjugate chem., 1992, 3, 397-. The humanized anti-CD22 antibody was purchased from Santa Cruz Biotechnology Ltd. Trastuzumab was purchased from Genentech. All other chemicals and anhydrous solvents were purchased from Sigma-Aldrich.

EXAMPLE 1 bis (2-hydroxyethyl) amino-4-oxobutanoic acid methyl ester (3)

Dimethyl succinate (20.0g, 136.9mmol) and dihydroxyethylamine(7.20g, 68.7mmol) was dissolved in a mixed solution of anhydrous toluene (500ml) and pyridine (50ml), and refluxed at 150 ℃ for 28 hours. Concentrating the mixed solution and then passing through SiO₂Column chromatography (from EtOAc/DCM ═ 5% to 25% to EtOAc) afforded the title compound (12.5g, 83%). ESI MS m/z + ion peak: c₉H₁₇NaNO₅Calculated (M + Na) 242.2, experimental 242.4.

EXAMPLE 2 bis (2- (methylsulfonyloxy) ethyl) amino-4-oxobutanoic acid methyl ester (4)

Methyl bis (2-hydroxyethyl) amino-4-oxobutanoate (12.0g, 49.56mmol) was dissolved in anhydrous pyridine (350ml) and methanesulfonyl chloride (20.0g, 175.4mmol) was added. After overnight reaction the solution was concentrated and then diluted with EtOAc (350mL) and cooled 1M NaH₂PO₄The solution (2X 300ml) was washed with MgSO₄Drying, filtration and spin-drying gave the crude product (18.8g, 101%). The crude product was used directly in the next reaction without purification. ESI MS m/z + ion peak: c₁₁H₂₁NaNO₉S₂Calculated (M + Na) 398.2, experimental 398.4.

EXAMPLE 3 bis (2- (Thioacetyl) ethyl) amino-4-oxobutanoic acid methyl ester (5)

Methyl bis (2- (methylsulfonoxy) ethyl) amino-4-oxobutanoate (freshly prepared, 90% pure, 8.5g, ca. 20mmol) was dissolved in DMA (350ml) and triethylamine (30ml, 215mmol) and thioacetic acid (10ml, 134mmol) were added sequentially at 0 ℃. Stirred at room temperature overnight, concentrated, diluted with EtOAc (350mL) and then sequentially with NaHCO₃Saturated solution (300ml), NaCl saturated solution (300ml) and 1M NaH₂PO₄The solution (300ml) was washed. Then, the organic phase was washed with Na₂SO₄Drying, filtering, spin drying, and treating with SiO₂Column chromatography of EtOAc/hexane (10% -25% EtOAc) afforded the title compound (5.1g, 76%). ESIMS m/z + ion peaks: c₁₃H₂₁NaNO₅S₂Calculated (M + Na) 358.1, experimental 358.2.

Example 4.4- (bis (2- (pyridin-2-yldithio) ethyl) amino) -4-oxobutanoic acid (6)

Methyl bis (2- (thioacetyl) ethyl) amino-4-oxobutanoate (5.0g, 14.9mmol) was dissolved in THF (150ml) and aqueous NaOH (5.0g, 125mmol) was added (100 ml). After stirring for 35 minutes at room temperature, the mixture is washed with H₃PO₄Neutralized to pH 7. Then, a solution of 1, 2-dipyridyl disulfide (Aldrithiol-2, 26.0g, 118mmol) in THF (100ml) was added and stirred for 4 hours. Concentrating, and treating with SiO₂Column chromatography of MeOH/DCM/HOAc (1: 20/1%) gave the title compound (5.8g, 85.6%). ESI MS m/z + ion peak: c₁₈H₂₁NaN₃O₃S₄Calcd for (M + Na) 478.0, experimental 478.2.

Example 5, 5-dioxopyrrolidin-1-yl-bis (2- (pyridin-2-yldithio) ethyl) amino-4-oxobutyrate (7)

4- (bis (2- (pyridin-2-yldithio) ethyl) amino) -4-oxobutanoic acid (5.2g, 11.5mmol) was dissolved in DMA (100ml) and NHS (1.6g, 13.9mmol) and EDC (5.0g, 26.1mmol) were added. Stirring overnight, spin-drying, and passing through SiO₂Column chromatography (from EtOAc/DCM ═ 5% to 15% to EtOAc) afforded the title compound (5.8g, 85.6%). ESI MS m/z + ion peak: c₂₂H₂₄NaN₄O₅S₄Calcd for (M + Na) 575.1, experimental 575.2.

EXAMPLE 6 Exo-3, 6-epoxy-Delta-tetrahydrophthalimide (12)

A solution of maleimide (10.0g, 103.0mmol) in toluene (200ml) was added to furan (10.0ml, 137.4mmol), and the mixture was placed in a 1L autoclave and heated to 100 ℃ for reaction for 8 hours. After cooling to room temperature, the pot contents were washed with methanol, concentrated and recrystallized from a mixed solution of ethyl acetate and n-hexane to give the title compound (16.7g, 99%).¹H NMR(CDCl₃): 11.12(s, 1H) (NH), 6.68-6.64(m, 2H), 5.18-5.13(m, 2H), 2.97-2.92(m, 2H). MS m/z + ion peaks: c₈H₇NaNO₃Calcd for (M + Na) 188.04, experimental 188.04.

Example 7.4- ((2- ((3aR, 4R, 7S, 7aS) -1, 3-dioxo-3 a, 4, 7,7 a-tetrahydro-1H-4, 7-oxiranoindol-3H) -yl) ethyl) (2- ((4R, 7S, 7aS) -1, 3-dioxo-3 a, 4, 7,7 a-tetrahydro-1H-4, 7-oxiranoindol-2 (3H) -yl) ethyl) amino) -4-oxobutyric acid methyl ester (13)

Methyl 4- (bis (2-methanesulfonyl) ethoxyamine) -4-oxobutanoate (4, freshly made, 90% pure, 8.5g,. about.20 mmol) was dissolved in DMA (350mL) and 3, 6-oxo- Δ -tetrahydrophthalimide (10.2g, 61.8mmol), sodium carbonate (8.0g, 75.5mmol) and sodium iodide (0.3g, 2.0mmol) were added in that order. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, ethyl acetate (350mL) was added, and the mixture was successively washed with a saturated aqueous sodium bicarbonate solution (300mL), a saturated brine (300mL) and 1M NaH₂PO₄(300mL) washed. Drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating the filtrate; the product was purified by silica gel column chromatography (10% to 30% ethyl acetate/n-hexane) to give the desired compound (7.9g, 77%). ESI MS m/z +: c₂₅H₂₇NaN₃O₉(M + Na): calculated values: 536.2, experimental value: 536.4.

example 8.4- (bis (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrolidine) ethylamine) -4-oxobutanoic acid (14)

Compound 13(3.0g, 5.8mmol) and trimethyltin hydroxide (4.8g, 26.4mmol) were dissolved in 1, 2-dichloroethane (150mL) and refluxed at 80 ℃ for 8 hours. After cooling to room temperature, the reaction solution was filtered through silica gel and rinsed with dichloromethane/methanol to remove residual trimethyltin hydroxide. The filtrates were combined, concentrated under reduced pressure, added with N, N-dimethylacetamide and toluene, and stirred under reflux at 120 ℃ overnight. Purification by silica gel column chromatography (5% to 10% methanol/dichloromethane) gave the title compound (1.62g, 76%). ESI MS m/z + C₁₆H₁₇NaN₃O₉(M + Na): calculated values: 386.1 of the total weight of the steel; experimental values: 386.2.

example 9.2, 5-Dioxopyrrolidine-4- (bis (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrolidine) ethylamine) -4-oxobutanoic acid ethyl ester.

Compound 14(16.0g, 4.4mmol) was dissolved in DMA (100mL) and NHS (0.76g, 6.61mmol) and EDC (1.70g, 8.90mmol) were added. The reaction mixture was stirred at room temperature overnight. The reaction was concentrated and purified by silica gel column chromatography (5% to 15% ethyl acetate/dichloromethane) to give the title compound (1.72g, 85.0%). ESI MS m/z + C₂₀H₂₀NaN₄O₉(M + Na): calculated values: 483.1, respectively; experimental values: 483.2.

example 10.5- (3 ', 6' -Oxo-. DELTA. -tetrahydrophthalimide) pentanoic acid tert-butyl ester

Tert-butyl 5-hydroxypentanoate (10.0g, 57.4mmol) was dissolved in pyridine (60mL) and methanesulfonyl chloride (8.0mL, 103.3mmol) was added. The reaction mixture was stirred at room temperature for 6 h. The reaction was concentrated, diluted with ethyl acetate and diluted with cold 1M NaH₂PO₄(pH 6) Washing the solution, drying the solution by anhydrous magnesium sulfate, filtering the solution, and concentrating the mother solution under reduced pressure to obtain the mesylate. This was added to compound 12(9.90g, 60.0mmol) and Na₂CO₃(8.5g, 80.1mmol) in DMF (80 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated, ethyl acetate was added, and the mixture was washed with saturated brine and 1M NaH₂PO₄The solution was washed with water (pH 6), dried over anhydrous magnesium sulfate, filtered, and the mother liquor was concentrated and purified by silica gel chromatography (ethyl acetate/dichloromethane ═ 1: 12) to give the title compound (14.01g, 76%). ESI MS m/z + C₁₇H₂₃NaNO₅(M + Na): calculated values: 344.16, respectively; experimental values: 344.16.

EXAMPLE 11.5 Maleimidopentanoic acid (21b)

Compound 17(5.0g, 15.57mmol) was dissolved in 1, 4-dioxane (40mL), concentrated hydrochloric acid (10mL) was added at 4 deg.C, and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated to dryness to give 5- (3 ', 6' -oxo-. DELTA. -tetrahydrophthalimide) pentanoic acid (4.08g, 99%). The above compound was dissolved in N, N-dimethylacetamide/toluene (1: 1, 40mL) and reacted under reflux for 6 h. The reaction mixture was concentrated and recrystallized from ethanol/ether/n-hexane to obtain the objective compound (2.76g, 90%). ESI MS m/z + C₉H₁₂NO₄(M + H): calculated values: 198.07, respectively; experimental values: 198.07.

example 12.5- (Maleimido) hexanoic acid succinimidyl ester (23b) (DMPS linker)

5-Maleamidopentanoic acid 21b (2.0g, 10.1mL) was dissolved in dichloromethane (20mL), then NHS (1.50g, 13.0mmol) and EDC (7.0g, 36.4mmol) were added and the reaction mixture was stirred at room temperature overnight. Concentrating the reaction solution to dryness, and separating by silica gel chromatography (ethyl acetate/diethyl acetate)Methyl chloride 1: 10) to give the title compound 23b (2.43g, 82%). ESI MS m/z + C₁₃H₁₄NaN₂O₆(M + Na): calculated values: 317.09, respectively; experimental values: 317.09.

example 13.5- (3 ', 6' -Oxo-. DELTA. -tetrahydrophthalimide) pentanoic acid hydrazide tert-butyl formate (25a-a)

5- (3 ', 6' -Oxo-. DELTA. -tetrahydrophthalimide) pentanoic acid (1.0g, 3.77mmol) was dissolved in DMF (30mL), followed by addition of tert-butylcarbamate (0.60g, 4.53mmol) and EDC (2.0g, 10.4mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness, and then purified by silica gel chromatography (ethyl acetate/dichloromethane ═ 1: 10) to obtain the objective compound (1.18g, 83%). ESI MS m/z + C₁₈H₂₅NaN₃O₆(M + Na): calculated values: 402.17, respectively; experimental values: 402.18.

example 14.5- (Maleimido) pentanoic acid hydrazide (25a-b)

Compound 25a-a (1.18g, 3.11mmol) was dissolved in N, N-dimethylformamide/toluene (1: 1, 20mL) and reacted at reflux for 6 h. The reaction was concentrated, 1, 4-dioxane (20mL) was added, HCl (5mL, 36%) was added at 4 ℃ and stirred for 30 min. The reaction mixture was concentrated to dryness, and recrystallized from ethanol/diethyl ether/n-hexane to give the objective compound (577mg, 88%). ESI MS m/z + C₉H₁₄N₃O₃(M + H): calculated values: 212.10,; experimental values: 212.10.

example 15.3 ' -bromo-maleimide compounds 39 and 40, and 3 ', 4 ' -dibromo-maleimide compounds 43 and 44

Compound 37 or 38 (6 g) was dissolved in DMF (60mL), then bromomaleic anhydride (1eq) or 2, 3-dibromo-maleic anhydride (1eq) was added and the reaction mixture was stirred overnight. The reaction solution is concentrated to dryness to obtain pure trans-acid. Acetic acid (50mL) and acetic anhydride (2-4 g) are added into the trans-acid, and the reaction mixture is refluxed for 6-12 h at 120 ℃. The reaction mixture was concentrated and separated by silica gel chromatography (ethyl acetate/dichloromethane: 1: 10 to 1: 1) to obtain 3 ' -bromo-maleimide compounds 39 and 40, and 3 ', 4 ' -dibromo-maleimide compounds 43 and 44 (61% to 87%) were obtained in the same manner.

5- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoic acid

ESI MS m/z+C₉H₁₁BrNO₄(M + H): calculated values: 275.98, respectively; experimental values: 275.98.

3- (2- (2- (2- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) -propionic acid.

ESI MS m/z+C₁₃H₁₉BrNO₇(M + H): calculated values: 380.03, respectively; experimental values: 380.03.

5- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoic acid

ESI MS m/z+C₉H₁₀Br₂NO₄(M + H): calculated values: 353.89, respectively; experimental values: 353.89.

3(2- (2- (2- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) -propionic acid

ESI MS m/z+C₁₃H₁₈Br₂NO₇(M + H): calculated values: 457.94, respectively; experimental values: 457.94.

example 16N-hydroxysuccinimide etherates 41 and 42 of 3 ' -bromo-maleimide, and N-hydroxysuccinimide etherates 45 and 46 of 3 ', 4 ' -dibromo-maleimide

R＝C₁～C₈ alkyl or C₂H₄(OC₂H₄)n，n＝1～20；X³＝H or Br；41，42，X³＝H；45，46，X³＝Br

3 ' -bromo-maleimide compounds 39 and 40(1eq), or 3 ', 4 ' -dibromo-maleimide compounds 43 and 44 were dissolved in DMA (. about.0.15 ml), NHS (1.1eq) and EDC (2-4 eq) were added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated and separated by silica gel chromatography (ethyl acetate/dichloromethane ═ 1: 20 to 1: 5) to obtain N-hydroxysuccinimide etherates 41 and 42 of 3 ' -bromo-maleimide and N-hydroxysuccinimide etherates 45 and 46 (70% to 93%) of 3 ', 4 ' -dibromo-maleimide.

2, 5-dioxopyrrolidin-1-yl 5- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoate (41)

ESI MS m/z+C₁₃H₁₃BrN₂NaO₇(M + Na): calculated values: 395.00, respectively; experimental values: 395.00.

2, 5-dioxopyrrolidin-1-yl 5- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoate (45)

ESI MS m/z+C₁₃H₁₂Br₂N₂NaO₆(M + Na): calculated values: 472.91, respectively; experimental values: 472.91.

2, 5-dioxopyrrolidin-1-yl 3- (2- (2- (2- (3-bromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propanoate (42)

ESI MS m/z+C₁₇H₂₁BrN₂NaO₉(M + Na): calculated values: 499.04, respectively; experimental values: 499.04.

2, 5-dioxopyrrolidin-1-yl 3(2- (2- (2- (3, 4-dibromo-2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propanoate (46)

ESI MS m/z+C₁₇H₂₀Br₂N₂NaO₉(M + Na): calculated values: 576.95, respectively; experimental values: 576.95.

example 17.4- (2-Pyridyldithio) -4-methylpentanoic acid

4-mercapto-4-methylpentanoic acid (Goff, D. et al, Bioconjugate chem.1990, 1, 381-386) (4.67g, 31.5mmol) was dissolved in methanol (15mL), and then a methanol solution (80mL) of 2, 2-dithiodipyridine (30.0g, 136.2mmol) and 100mM sodium phosphate buffer (pH 7.5, 70mL) were added to the reaction solutionStirring for 6 h. The reaction mixture was concentrated, extracted with ethyl acetate/n-hexane (1: 1), and the aqueous layer was adjusted to pH 3 with 1M HCl and extracted with ethyl acetate (3X 100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Chromatography on silica gel (methanol/dichloromethane/acetic acid ═ 1: 15: 0.01) afforded the title compound (7.05g, 87%). ESI MS m/z + C₁₁H₁₆NO₂S₂(M + H): calculated values: 258.05, respectively; experimental values: 258.05.

example 18.4- (2-Pyridinedithio) -4-Methylpentanoic acid succinimidyl ester (243) (SMDP linker)

4- (2-Pyridinithio) -4-methylpentanoic acid (2.0g, 7.78mmol) was dissolved in dichloromethane (20mL) and NHS (1.10g, 9.56mmol) and EDC (4.0g, 20.8mmol) were added and the reaction mixture was stirred overnight. The reaction solution was concentrated and separated by silica gel chromatography (ethyl acetate/dichloromethane 1: 10) to obtain the objective compound (2.48g, 90%). ESI MS m/z + C₁₅H₁₈NaN₂O₄S₂(M + Na): calculated values: 377.07, respectively; experimental values: 377.08.

EXAMPLE 19 (3aR, 4R, 6S, 6aR) -6- ((R) -2, 2-dimethyl-1, 3-dioxolane) -2, 2-dimethyltetrahydrofuran [3, 4-d ] [1, 3] dioxo-4-ol (62)

Compound D-gulonolide (20.01g, 112.37mmol) and anhydrous copper sulfate (25.0g, 157.22mmol) were dissolved in dry acetone (450mL) and concentrated sulfuric acid (1.6mL) was added and the reaction mixture was stirred at room temperature for 24 h. And (3) adjusting the pH value of the reaction liquid to 7 by using calcium hydroxide, filtering the suspension, and concentrating the mother liquor to obtain a light yellow syrupy diacetone compound which can be directly used for the next reaction without purification. The diacetone compound obtained above was dissolved in THF (300mL), cooled to-78 deg.C, and then slowly dropped1M DIBAL-H in toluene (180mL, 180mM), the reaction mixture was stirred at-78 deg.C for 1H. After the reaction solution was quenched with water (50mL), it was filtered, and the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel chromatography (n-hexane/ethyl acetate 5: 1) gave the title compound (25.27mmol, 83% of two-stage overall yield) as a colorless syrup. ESI MS m/z + C₁₂H₂₀NaN₂O₆S (M + Na): calculated values: 283.12, respectively; experimental values: 283.12.

EXAMPLE 20 (3aR, 4R, 6S, 6aR) -6- ((R) -2, 2-dimethyl-1, 3-dioxolane) -2, 2-dimethyl-N- (2-methylpropylidene) tetrahydrofuran [3, 4-d ] [1, 3] dioxo-4-aminium oxide (64)

Compound 62(10.0g, 38.4mmol) and hydroxylamine hydrochloride (25.01g, 360.87mmol) were dissolved in pyridine (150mL) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated, water (250mL) was added, extraction was performed with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to silica gel chromatography (ethyl acetate) to obtain a colorless glassy compound 2, 3: 5, 6-O-diisopropyl-D-guloxime (63) (10.34g, 98%, without purification, was used directly in the next reaction). The resulting oxime (63) (10.30g, 37.43mmol), isobutyraldehyde (3.00g, 41.66mmol) and magnesium sulfate (3g, 25mmol) were stirred at room temperature overnight. The reaction solution was filtered, and the mother liquor was concentrated and then subjected to silica gel chromatography (ethyl acetate) to obtain a white solid compound (11.57g, 94%). ESI MS m/z + C₁₆H₂₇NO₆(M + H): calculated values: 329.18, respectively; experimental values: 329.18.

EXAMPLE 21 (3R, 5R) -2- ((3aR, 4R, 6S, 6aR) -6- ((R) -2, 2-dimethyl-1, 3-dioxolane) -2, 2-dimethyl-N- (2-methylpropylidene) tetrahydrofuran [3, 4-d ] [1, 3] dioxole) -3- [ (-) -10 ', 2' -camphora ] N-propylisoxazole-5-carboxamide (65)

Mixing (3aR, 4R, 6S, 6aR, Z) -6- ((R) -2, 2-dimethyl-1, 3-dioxolane) -2, 2-dimethyl N- (2-methylpropylidene) tetrahydrofuran [3, 4-d ]][1，3]Dicyclopenten-4-amine (6.00g, 18.22mmol) and (2R) -N- (acryloyl) bornane-10, 2-sultam (5.10g, 18.95mmol) were dissolved in dichloromethane (50mL) and then heated at reflux for 37 h. The reaction mixture was concentrated, and recrystallized from ethanol (30mL) to give the objective compound (8.72g, 80%). The mother liquor after recrystallization was separated by column chromatography on silica gel (n-hexane/ethyl acetate 7: 3) to obtain more of the title compound (0.47g, 4%). ESI MS m/z + C₂₉H₄₆N₂NaO₉S (M + Na): calculated values: 621.28, respectively; experimental values: 621.28.

EXAMPLE 22 (3R, 5R) -2- (tert-butoxycarbonyl) -3-isopropyloxazole-5-carboxylic acid formamide (67)

Mixing (3R, 5R) -2- (3aR, 4R, 6S, 6aR) -6- ((R) -2, 2-dimethyl-1, 3-dioxolane) -2, 2-dimethyl tetrahydrofuran [3, 4-d ]][1，3]Dioxole) -3- [ (-) -10, 2-camphora]N-isopropyloxazole-5-carboxamide (30.0g, 48.2mmol) was dissolved in THF (100mL) and methanol (60mL), an aqueous solution (60mL) of lithium hydroxide (5.0g, 208.7mmol) was added at 45 ℃, the mixture was stirred for 1 hour, the mixture was concentrated, water (150mL) was added, the pH was adjusted to 9 with 4M HCl, after extraction with ethyl acetate, the aqueous layer was adjusted to pH 3 with 4M HCl, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting product was stirred with n-hexane to give (3R, 5R) -2- { (3aR, 4R, 6S, 6aR) -6- [ (R) -2, 2-dimethyl-1, 3-dioxolane]-2, 2-dimethyltetrahydrofuran [3, 4-d, 1, 3]Dicyclopentene-3-isopropyloxazole-5-carboxamide acid (66a) (17.1g, 88%). The product can be directly used for the next reaction without purification. ESI MS m/z + C₁₉H₃₁NNaO₈(M + Na): 424.19. the compound (8) obtained above was reacted with0g, 19.95mmol) was dissolved in acetonitrile (80mL) and 60% HClO was added at 45 deg.C₄Aqueous solution (6.0mL, 35.77mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction was concentrated and dissolved in 1, 4-dioxane (40mL) and NaHCO was added at 4 deg.C₃(25g, 297mmol) in aqueous solution (32mL) and Boc₂O (4.80g, 22.00mmol) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated, water and ethyl acetate/n-hexane (1: 1) were added, the layers were separated, the aqueous layer was adjusted to pH 3 with 4M HCl, extracted with ethyl acetate, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and stirred with n-hexane to give (3R, 5R) -2- (t-butoxycarbonyl) -3-isopropyloxazole-5-carboxamide acid (4.91g, 95%). ESI MS m/z + C₁₂H₂₁NNaO₅(M + Na): calculated values: 282.13; experimental values: 282.13.

EXAMPLE 23 methyl (R) -2- [ (3R, 5R) -2- (tert-butoxycarbonyl) -3-isopropyloxazole-5-carbamoylic acid ] -3- (triphenyl) propionate (68)

(3R, 5R) -2- (tert-Butoxycarbonyl) -3-isopropyloxazole-5-carboxamide acid (1.01g, 3.89mmol) and 1- (S) -Tr-cysteine methyl ester hydrochloride (1.76g, 4.27mmol) were dissolved in dichloromethane (15mL) and iPr was added at 4 deg.C₂NEt (0.75mL, 4.31mmol) and TBTU (2.50g, 7.78mmol), the reaction mixture was stirred at room temperature overnight. The reaction solution was poured into NaHCO₃The aqueous solution was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel chromatography (n-hexane/ethyl acetate 1: 2) gave the title compound (2.10g, 87%). ESI MS m/z + C₃₅H₄₂N₂NaO₆S (M + Na): calculated values: 641.27, respectively; experimental values: 641.26.

EXAMPLE 24 (3R, 5R) -tert-butyl-3-isopropyl-3- (4- (methoxycarbonyl) thiazole) isopropyloxazole-2-carboxylic acid methyl ester (69)

Ph is₃P ═ O (4.10g, 14.74mmol) was dissolved in dichloromethane (40mL), and Tf was added₂O (2.0mL, 12.0mmol), the reaction mixture was stirred at-10 ℃ for 1h, and (R) -2- [ (3R, 5R) -2- (t-butoxycarbonyl) -3-isopropyloxazole-5-carboxamide acid was added thereto at that temperature]A solution of methyl-3- (triphenyl) propionate (68) (4.00g, 6.47mmol) in dichloromethane (20mL) and the reaction mixture stirred at room temperature for 6 h. Then, the reaction solution was added to saturated NaHCO at 4 deg.C₃The aqueous solution was extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The corresponding thiazolidine derivative was obtained by silica gel chromatography (n-hexane/ethyl acetate 3: 2). The compound obtained above was dissolved in methylene chloride (60mL), and MnO was added₂(5.80g, 66.7mmol) and the reaction mixture stirred at room temperature for 24 h. The reaction mixture was filtered, concentrated, and separated by silica gel chromatography (n-hexane/ethyl acetate 3: 2) to give the title compound (69) (1.75g, 75%). ESI MS m/z + C₁₆H₂₄N₂NaO₅S (M + Na): calculated values: 379.13, respectively; experimental values: 379.14.

EXAMPLE 25.2- ((1R, 3R) -3- (tert-Butoxycarbonyl) -1-hydro-4-methylaminopentyl) thiazole-4-carboxylic acid methyl ester (70)

(3R, 5R) -tert-butyl-3-isopropyl-3- (4- (methoxycarbonyl) thiazole) isopropyloxazole-2-carboxylic acid methyl ester (69) (1.00g, 2.81mmol) was dissolved in acetonitrile (20mL) and H₂To O (2mL), Mo (CO) was added₆(1.10g, 3.12mmol) and the reaction mixture was stirred at 70 ℃ for 16 h. After concentrating the reaction mixture, ethyl acetate (50mL) and 10% citric acid aqueous solution (50mL) were added, and NaIO was added thereto₄Until the aqueous layer became clear, it was extracted with ethyl acetate. The organic phase is treated with 10% Na₂S₂O₃Washing the aqueous solution with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and separating with silica gel column (n-hexane/ethyl acetate 3: 2) to obtain colorless extractSolid compound (906mg, 90%). ESI MS m/z + C₁₆H₂₆N₂NaO₅S (M + Na): calculated values: 381.14, respectively; experimental values: 381.14.

EXAMPLE 26.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylic acid methyl ester (71)

Compound 70(900mg, 2.51mmol) was dissolved in pyridine (15mL), acetic anhydride (0.5mL, 5.29mmol) was added, the mixture was stirred overnight, the reaction solution was concentrated, and the mixture was separated by silica gel chromatography (n-hexane/ethyl acetate 4: 1) to give a colorless solid compound (950mg, 95%). ESI MS m/z + C₁₈H₂₈N₂NaO₆S (M + Na): calculated values: 423.15, respectively; experimental values: 423.16.

example 27.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylic acid (72)

Compound 71(940mg, 2.35mmol) is dissolved in THF (15mL), NaH (120mg, 3.0mmmol, 60%) is added at 4 deg.C, the reaction mixture is stirred for 2h and CH is added₃I (0.155mL, 2.49mmol) and the reaction mixture was stirred overnight. The reaction was concentrated, redissolved in ethyl acetate, filtered, and concentrated to dryness to give pure methyl 2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy-methylamine) -4-pentyl) thiazole-4-carboxylate (73 a). The compound (73a) obtained above was dissolved in 1, 2-dichloroethane (20mL), trimethyltin hydroxide (620mg, 3.43mmol) was added, and the reaction mixture was stirred at 80 ℃ overnight. The reaction mixture was concentrated, dissolved in methanol/dichloromethane/acetic acid (1: 5: 0.01, 20mL), filtered, concentrated, and the mother liquor was concentrated again after adding toluene to give a dry compound. Then, the compound obtained above was dissolved in pyridine (15mL), acetic anhydride (0.4mL, 4.23mmol) was added, and the reaction mixture was stirred overnight. Concentrating the reaction solution, and passing through silica gelColumn chromatography (methanol/dichloromethane/acetic acid 1: 10: 0.01) gave the compound as an anhydrous solid (735mg, 78%). ESI MS m/z + C₁₈H₂₈N₂NaO₆S (M + Na): calculated values: 423.15, respectively; experimental values: 423.16.

example 28.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1, 3-dioxoisoindoline) -4-methylamine-pentyl) thiazole-4-carboxylic acid methyl ester (86)

Compound 70(850mg, 2.37mmol) was dissolved in THF (15mL), NaHH (100mg, 2.5mmol, 60%) was added at-20 ℃ and stirred at that temperature for 20min, N- (3-bromopropyl) phthalimide (655mg, 2.4mmol) was added, and the reaction mixture was stirred at-20 ℃ for 30min and then warmed to room temperature for 4 h. The reaction was quenched by addition of methanol (0.5mL), dichloromethane (60mL) was added, filtered and concentrated to afford methyl 2- (1R, 3R) -3- (tert-butoxy-methylamine) -1- (3- (1, 3-dioxoisoindoline-2-propoxy-4-pentyl) thiazole-4-carboxylate 85, which was used directly for the next step without purification the compound 85 was dissolved in THF (10mL) and NaH (170mg, 4.25mmol, 60%) was added at room temperature, stirred for 45min and CH was added₃I (0.20mL, 3.21mmol), the reaction mixture was stirred at room temperature overnight. The reaction solution is added with NaH₂PO₄After quenching (2.0M, 2mL), DMA (5mL) was added, concentrated under reduced pressure, and separated by silica gel chromatography (ethyl acetate/dichloromethane ═ 1: 10 to 1: 4) to give the title compound (921mg, 69%). ESI MS m/z + C₂₈H₃₇N₃NaO₇S (M + Na): calculated values: 582.22, respectively; experimental values: 582.22.

example 29.2- ((1R, 3R) -3- (tert-butoxy (methyl) amine) -1- (3- (1, 3-dioxoisoindoline) -4-methylamine-pentyl) thiazole-4-carboxylic acid (87)

Dried compound 86(910mg, 1.63mmol) was dissolved in 1, 2-dichloroethane (20mL), trimethyltin hydroxide (400mg, 2.21mmol) was added, and the reaction mixture was stirred at 80 ℃ overnight. The reaction solution was concentrated and subjected to silica gel chromatography (methanol/dichloromethane/acetic acid ═ 1: 10: 0.01) to give the objective compound (756mg, 85%). ESI MS m/z + C₂₇H₃₇N₃O₇S (M + H): calculated values: 546.22, respectively; experimental values: 546.22.

example 30.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy (3- (1, 3-di-oxoisoindoline-2-pentylamine) -4-methylamine) -4-pentyl) thiazole-4-carboxylic acid methyl ester (89)

Compound 71(800mg, 2.00mmol) was dissolved in THF (30mL), NaH (150mg, 3.75mmol, 60%) was added at room temperature, stirring was performed for 45min, and N- (3-bromopropyl) phthalimide (655mg, 2.4mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction solution is added with NaH₂PO₄After quenching (2.0M, 2mL), DMA (5mL) was added, concentrated under reduced pressure, and separated by silica gel chromatography (ethyl acetate/dichloromethane ═ 1: 10 to 1: 4) to give the title compound (971mg, 82%). ESI MS m/z + C₂₉H₃₇N₃NaO₈S (M + Na): calculated values: 610.22, respectively; experimental values: 610.22.

example 31.2- ((1R, 3R) -1-acetoxy-3- (tert-butoxy (3- (1, 3-di-oxoisoindoline-2-pentylamine) -4-methylamine) -4-pentyl) thiazole-4-carboxylic acid (90)

Dried compound 89(900mg, 1.53mmol) was dissolved in 1, 2-dichloroethane (35mL), trimethyltin hydroxide (400mg, 2.21mmol) was added, and the reaction mixture was stirred at 80 ℃ overnight. The reaction mixture was concentrated, the resulting product was dissolved in pyridine (20mL), acetic anhydride (3mL) was added, and the reaction mixture was stirredOvernight. The reaction solution was concentrated, and then separated by silica gel chromatography (methanol/dichloromethane/acetic acid: 1: 10: 0.01) to obtain the objective compound (755mg, 86%). ESI MS m/z + C₂₈H₃₅N₃NaO₈S (M + Na): calculated values: 596.20, respectively; experimental values: 596.20.

EXAMPLE 32 Ethyl (S) -5- (4- (benzyloxy) phenyl) -4- (tert-butoxycarbonylamino) -2-methylpentenoate (185)

Diisobutylaluminum hydride (40ml, 40mmol, 1.0M) was slowly added to a solution of (S) -3- (4- (benzyloxy) phenyl) -2- (tert-butoxycarbonylamino) -propionic acid methyl ester 184(8.00g, 20.76mmol) in DCM (250ml) at-78 deg.C, reacted for 2 hours, and quenched by addition of MeOH (5 ml). After the reaction mixture was warmed to room temperature, 1M HCl was added to adjust pH4, and the solution was separated. The aqueous layer was extracted with DCM (2X 150ml), the organic layers were washed with water, the organic layers were combined and washed with anhydrous Na₂SO₄Drying, filtering and concentrating to obtain the primary aldehyde intermediate product. This crude product was dissolved in DCM, a ylide solution synthesized from a solution of 1- (1-ethoxycarbonylethyl) triphenylphosphonium bromide (18.0g, 40.64mmol) and KOtBu (5.00g, 44.64mmol) in dichloromethane (80ml) was added at room temperature, the mixture was reacted overnight and then concentrated, and column chromatography (ethyl acetate/n-hexane, 1: 8-1: 4) gave the target product (185) (6.90g, 76%), ESI: m/z: [ M + Na ]]+, ion peak: c₂₆H₃₃NNaO₅462.22, experimental value, 462.22.

EXAMPLE 33 (4R) -4- (tert-Butoxycarbonylamino) -5- (4-hydroxyphenyl) -2-methylpentanoic acid ethyl ester (186)

Ethyl (S) -5- (4- (benzyloxy) phenyl) -4- (tert-butoxycarbonylamino) -2-methylpentenoate (185) (6.70g, 15.26mmol), methanol (150ml), 10% Pd/C (0.3g) were charged to a hydrogen reaction flask and reacted under 30psi of hydrogen for 6hIn this case, the residue was filtered through celite, concentrated, and recrystallized from ethanol/n-hexane to give the desired product (186) (4.61g, 86%). ESI: m/z: [ M + Na ]]+, ion peak: c₁₉H₂₉NNaO₅374.20, experimental value, 374.30.

EXAMPLE 34 Ethyl (4R) -4- (tert-butoxycarbonylamino) -5- (4-hydroxy-3-nitrophenyl) -2-methylpentanoate (187)

To compound (186) (4.50g, 12.81mmol) in anhydrous CH₂Cl₂(200ml) adding Ac to the solution₂O (2ml, 21.16mmol) and fuming nitric acid (0.65ml, 14.07mmol) were stirred at room temperature for 4 hours, diluted with water (150ml), separated and the aqueous phase extracted with ethyl acetate. The organic layers were combined and washed with anhydrous Na₂SO₄Drying, filtration, concentration and column chromatography (ethyl acetate/dichloromethane ═ 1: 10) afforded the desired product (187) (4.21g, 83%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₉H₂₈N₂NaO₇419.19, experimental value, 419.20.

EXAMPLE 35.4- (tert-Butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) pentanoic acid ethyl ester (188)

Compound (187) (4.00g, 10.09mmol) was dissolved in acetonitrile (70ml) and N, N-dimethylacetamide (30ml), cooled to 0 ℃ and N, N' -diisopropylethylamine (4.00ml, 23.00mmol) was added, and after two minutes phosphorus oxychloride (2.00ml, 21.45mmol) was added. Stirring at room temperature for 8 h, cooling to 0 deg.C, slowly adding a solution of sodium bicarbonate (3.5g, 41.60mmol) and water (20ml), stirring at 0 deg.C overnight, concentrating, purifying with C-18 column (20X4cm) under gradient elution conditions: 25ml/min, A: 0.5% acetic acid, B: methanol, 100% a for 10 minutes, then 45 minutes to 75% a and 25% B. Collecting the target fraction, and concentrating to obtainTarget compound (188) (3.89g, 81%). ESI: m/z: [ M-H ]]^-’Ion peak: c₁₉H₂₈N₂O₁₀P, 475.16, experimental value, 475.20.

EXAMPLE 36 (4R) -4- (tert-Butoxycarbonylamino) -2-methyl-5- (3-nitro-4-phosphonoxyphenyl) pentanoic acid (189)

To a solution of compound (188) (3.75g, 7.87mmol) in THF (100ml) was added a solution of lithium hydroxide (5.0g, 208.7mmol) in water (60ml), reacted at 0 ℃ for 4 hours, adjusted to pH6 with 4M hydrochloric acid, concentrated, purified on a C-18 column, gradient eluted, 25ml/min, A: 0.5% acetic acid, B: methanol, 100% a for 10 minutes, then 45 minutes to 75% a and 25% B. The target fraction was collected and concentrated to give the target compound (189) (2.82g, 80%). ESI: m/z: [ M-H ]]Ion peak: c₁₇H₂₄N₂O₁₀P, 447.12, experimental value, 447.20.

EXAMPLE 37 (4R) -5- (3-amino-4-phosphonoxyphenyl) -4- (tert-butoxycarbonylamino) -2-methylpentanoic acid (190)

Compound (189) (2.6g, 5.80mmol), methanol (80ml) were added to a hydrogen reaction flask in this order,

10% Pd/C (0.2 g). The reaction was carried out under 35psi of hydrogen pressure for 6 hours. The mixture was filtered through celite and concentrated to give the crude product (190) (2.18g, 90%) which was used in the next reaction without further purification. ESI: m/z: [ M-H ]]^-Ion peak: c₁₇H₂₆N₂O₈P, 417.15, experimental value, 417.15.

EXAMPLE 38 (S) -2- (tert-Butoxycarbonylamino) -3- (4-hydroxy-3-nitrophenyl) -propionic acid methyl ester (196)

Add acetic anhydride (4mL, 42.32mmol) and fuming nitric acid (0.85mL, 18.40mmol) to a solution of (S) -methyl 2- (tert-butoxycarbonylamino) -3- (4-hydroxyphenyl) -propionate (195) (4.5g, 15.24mmol) in dry dichloromethane (240 mL). After stirring at room temperature for 4 hours, it was diluted with water (150ml), separated and the aqueous phase extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (ethyl acetate/dichloromethane, 1: 10) to give the desired product (196) (4.30g, 83%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₅H₂₀N₂NaO₇363.13, experimental value, 363.20.

EXAMPLE 39 (S) -2- (tert-Butoxycarbonylamino) -3- (3-nitro-4-phosphonoxyphenyl) -propionic acid methyl ester (197)

To a solution of compound (196) (4.10g, 12.05mmol) in acetonitrile (90ml) at 0 ℃ was added N, N' -diisopropylethylamine (4.00ml, 23.00mmol), and after stirring for 2 minutes, phosphorus oxychloride (2.00ml, 21.45mmol) was added, and after the reaction mixture was stirred at room temperature for 8 hours, it was cooled to 0 ℃ and a solution of sodium bicarbonate (3.5g, 41.60mmol) and water (20ml) was slowly added thereto, and stirring was continued at 0 ℃ overnight, followed by concentration, purification with gradient elution, 25ml/min, a: 0.5% acetic acid, B: methanol, 100% a for 10 minutes, then 45 minutes to 75% a and 25% B. The target fractions were collected and concentrated to give the target compound (197) (4.20g, 83%). ESI: m/z: [ M-H ]]^-Ion peak: c₁₅H₂₀N₂O₁₀P, 419.08, experimental value, 419.10.

Example 40.3- (3-amino-4-phosphonoxyphenyl) - (2R) -2- (tert-butoxycarbonylamino) -propionic acid (198)

The dried compound (197) (4.0g, 9.52mmol) was dissolved in a mixed solvent of 1, 2-dichloroethane (50ml) and N, N-dimethylacetamide (60ml), and trimethyltin hydroxide (4.00g, 22.1mmol) was added to the solution. The mixture is reacted for 6 hours at 80 ℃, concentrated and subjected to column chromatography (water/acetonitrile 1: 4), the target component is collected and concentrated to obtain (S) -2- (tert-butoxycarbonylamino) -3- (3-nitro-4-phosphonoxyphenyl) -propionic acid. The resulting compound was charged into a hydrogen reaction flask together with N, N-dimethylacetamide (70ml) and 10% Pd/C (0.3 g). The reaction was carried out under 35psi of hydrogen pressure for 6 hours. The mixture was filtered through celite, concentrated, and recrystallized to give the desired product (198) (2.86g, 80%) which was used in the next reaction without further purification. ESI: m/z: [ M-H ]]^-Ion peak: c₁₄H₂₀N₂O₈P, 375.10, experimental value, 375.10.

Example 41.3- (4-benzyloxyphenyl) - (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -propionic acid benzyl ester (200)

Add sodium hydride (430mg, 10.75mmol, 60% oil) to a solution of benzyl 3- (4-benzyloxyphenyl) - (2R) -2- [ (tert-butoxycarbonylamino) -propionate (4.0g, 8.67mmol) in tetrahydrofuran (60mL), stir at room temperature for 1 hour, add methyl iodide (1.82g, 12.82mmol), stir the mixture overnight, quench the reaction with methanol (0.5mL), concentrate, and column chromatographe (ethyl acetate/dichloromethane, 1: 10) to give the title product (200) (3.83g, 93%). MS ESI: m/z: [ M + Na ]]⁺Ion peak: c₂₉H₃₃NNaO₅498.24, experimental value, 498.24.

EXAMPLE 42 (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -3- (4-hydroxy-2-nitrophenyl) propanoic acid (201)

Compound (200) (3.8g, 8.00mmol), methanol (80ml) were added to a hydrogen reaction flask in this order,

10% Pd/C (0.3 g). After reacting under 35psi of hydrogen for 6 hours, the reaction mixture was filtered through Celite and concentrated to give a crude product (2R) -2- [ (tert-butoxycarbonyl) -methylamino]-3- (4-hydroxyphenyl) propionic acid (201a), which compound was not further purified. To a solution of compound (201a) in anhydrous dichloromethane (240ml) at-25 ℃ was added a solution of tin tetrachloride (1.5ml, 12.75mmol) and fuming nitric acid (0.60ml, 12.98mmol) in dichloromethane (40ml) and the mixture was stirred at-25 ℃ for 75 minutes, quenched with saturated sodium bicarbonate, adjusted to pH 3-4, the aqueous phase extracted with ethyl acetate, the combined organic layers dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed (MeOH/DCM/HOAc 1: 8: 0.01) to afford the title compound (201) (1.98g, 73%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₅H₂₀N₂NaO₇363.13, experimental value, 363.13.

EXAMPLE 43 (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] -3- (3-nitro-4-phosphonooxyphenyl) propionic acid (202)

Compound (201) (1.98g, 5.82mmol) was dissolved in acetonitrile (30ml) and N, N-dimethylacetamide (30ml), and the resulting solution was added with N, N' -diisopropylethylamine (2.00ml, 11.50mmol) at 0 ℃ and after reacting for two minutes at that temperature, phosphorus oxychloride (1.10ml, 11.79mmol) was added. After stirring the reaction mixture at room temperature for 8 hours, the reaction mixture was cooled to 0 ℃, a solution of sodium bicarbonate (2.0g, 23.80mmol) and water (10m1) was slowly added thereto, stirring was continued at 0 ℃ overnight, the mixture was concentrated, purified by C-18 column (20 × 4cm) and eluted with gradient, 25ml/min, a: 0.5% acetic acid, B: methanol, 100% a for 10 minutes, then 45 minutes to 75% a and 25% B. The target fractions were collected and concentrated to give the target compound (202) (1.96, 80%). ESI: m/z: [ M-H ]]^-Ion peak: c₁₅H₂₀N₂O₁₀P, 419.09, experimental value, 419.09.

Example 44.3- (3-amino-4-phosphonoxyphenyl) - (2R) -2- [ (tert-butoxycarbonyl) -methylamino ] propanoic acid (203)

Compound (202) (1.96g, 4.67mmol), N, N-dimethylacetamide (60ml), and 10% Pd/C (0.2g) were sequentially added to a hydrogen reaction flask. The reaction was carried out under 30psi of hydrogen for 6 hours. The mixture was filtered through celite and concentrated to give the crude product (203) (1.74g, 95%)) which was used in the next reaction without further purification. ESI: m/z: [ M-H ]]-，calcd for C₁₅H₂₂N₂O₈P，389.12，Found，389.12.

EXAMPLE 45 phenyl-2- (2R) -tert-butoxycarbonylamino-1-propanone (204)

(1S, 2R) - (+) -demethylephedrine (7.0g, 46.29mmol) was added to a mixture of tetrahydrofuran (40ml) and 1M sodium bicarbonate (100ml), a solution of di-tert-butyl dicarbonate (10.15g, 46.53mmol) in tetrahydrofuran (60ml) was slowly added over 45 minutes at a temperature of 4 ℃, the reaction mixture was stirred at room temperature for 6 hours, concentrated, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, column chromatographed (EtOAc/Hexane 1: 2), the desired fraction was collected, concentrated to give crude (1S) -phenyl-2- (2R) -tert-butoxycarbonylamino-1-propanol (204b) (10.81, 93%), the crude was purified, MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₁₄H₂₁NaNO₃274.15, experimental value, 274.15. Adding a dichloromethane (180ml, 0.3M) solution of dess-martin reagent to a dichloromethane (50ml) solution of compound (204b), stirring for one hour, adding a frozen sodium hydroxide solution (1M, 100ml) to the mixture, separating, adjusting the organic phase to pH6 with 1M sodium dihydrogen phosphate (100ml), drying over anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography (EtOAc/Hexane 1: 5) to obtain the desired productSubstance 204(9.34g, 81% in two steps). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₁₄H₁₉NaNO₃272.14, experimental value, 272.14.

EXAMPLE 46 ethyl (1R, 3R) -3- ((2S, 3S) -N-methyl-3-methyl-2- ((R) -1-methylpiperidine-2-carboxamido) -penta-mino) -4-methyl-1- (4- (1-oxo-1-phenylpropan-2-yl) carbamoylmethyl) thiazol-2-yl) -pentanoate (205)

To a solution of compound 204(180mg, 0.722mmol) in 1, 4-dioxane (4ml) was added concentrated hydrochloric acid (1.0ml, 37%) at 4 deg.C, and the mixture was stirred at room temperature for 40 minutes, then concentrated, added toluene and concentrated to dryness. The dried solid was dissolved in N, N-dimethylacetamide (7ml), to the solution were added compound 106(251mg, 0.466mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (305mg, 1.56mmol), and N, N' -diisopropylethylamine (0.13ml, 0.747mmol) in that order, the reaction mixture was stirred for 8 hours, concentrated, and column chromatographed (EtOAc/CH)₂Cl₂1: 4) to yield the target product 205(255.3mg, 82%). ESI: m/z +: [ M + Na ]]⁺Ion peak: c₃₅H₅₁NaN₅O₆S, 692.36, experimental value, 692.36.

EXAMPLE 47 ethyl (1R, 3R) -3- ((2S, 3S) -N, 3-dimethyl-2- (R) -1-methylpiperidine-2-carboxamido) -pentanoylamino) -1- (4- ((R) -1- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -12-oxo-15-phenyl-3, 6, 9-trioxa-13, 14-diazepan-14-enyl-16-carbamoyl) thiazol-2-yl) -4-methylpentanoate (206)

Add Compound 12(50mg HCl salt, 0.126mmol) and acetic acid (3ul, 0.052mmol) to Compound 205(75mg, 0.112mmol) in MeOH (5mL) and react the mixture overnight before adding N, N' -diisopropylethylamine (23ul, 0.132mmol) was neutralized, concentrated, and column chromatographed (EtOAc/CH)₂Cl₂1: 4-1: 3) to give the title compound 206(79.3mg, 70%). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₅₀H₇₄NaN₈O₁₂S, 1033.51, experimental value, 1033.50.

EXAMPLE 48 (1S, 2R) -2- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -1-phenylpropyl-3- (2- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) -propionic acid ethyl ester (211)

Add dicyclohexylcarbodiimide (122mg, 0.591mmol) and 2, 2-dimethylolpropionic acid (25mg, 0.204mmol) to a solution of compound 208a (95mg, 0.141mmol) and 3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionic acid (55mg, 0.182mmol) in dichloromethane (5ml), stir the mixture overnight, concentrate, and column chromatographe (EtOAc/CH2Cl2 ═ 1: 3) to give the title compound 211(95.1mg, 71%). MS ESI: m/z +: [ M + Na ]]+, ion peak: c₄₈H₇₀NaN₆O₁₂S, 977.47, experimental value, 977.47.

EXAMPLE 49 ethyl 2, 5-dioxopyrrolidin-1-yl 2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxylate (234)

Add N-Hydroxysuccinimide (202.0mg, 1.756mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (980mg, 5.104mmol) to a solution of Compound 106(788.1mg, 1.464mmol) in N, N' -dimethylformamide (10ml), stir the mixture overnight, concentrate itColumn chromatography (EtOAc/CH)₂Cl₂1: 3) to give the title compound 234(762.8mg, 82%). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₃₀H₄₅NaN₅O₈S, 658.30, experimental value, 658.30.

EXAMPLE 50 (4R) -4- (tert-Butoxycarbonylamino) -5- (3- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (235)

Add 2, 5-dioxopyrrolidin-1-yl-5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoic acid ethyl ester (23d) (711mg, 2.417mmol), N, N '-diisopropylethylamine (0.250ml, 1.438mmol) to a solution of compound 190(825.1mg, 1.973mmol) in N, N' -dimethylformamide (7ml), stir the mixture overnight, concentrate, and column chromatography on C-18 column (4X25cm, v 15ml/min, from 100% 1% HOAc to 75% 1% HOAc/25% MeOH in 45min) to afford the title compound 235(895.7mg, 76%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₂₆H₃₅N₃O₁₁P, 596.21, experimental value, 596.21.

EXAMPLE 51 (4R) -4- (tert-Butoxycarbonylamino) -5- (3- (3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (236)

Add Ethyl 2, 5-dioxopyrrolidin-1-yl-3- (2- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propanoate (24C) (727mg, 1.826mmol), N, N '-diisopropylethylamine (0.250ml, 1.438mmol) to a solution of Compound 190(632.5mg, 1.512mmol) in N, N' -dimethylformamide (7ml), concentrate after stirring the mixture overnight, C-18 column chromatography (4X25cm, v ═ 15ml/min from 100% strength silica gel)1% HOAc to 75% 1% HOAc/25% MeOH in 45min) gave the title compound 236(763.2mg, 72%). MS ESI: m/z-: [ M-H ]]Ion peak: c₃₀H₄₄N₃O₁₄P, 700.25, experimental value, 700.25.

EXAMPLE 52 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (5- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) pentanoylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (239)

To a solution of compound 235(102mg, 0.171mmol) in 1, 4-dioxane (4ml) was added concentrated hydrochloric acid (1.0ml, 37%) and the mixture was stirred for 30 minutes and then concentrated to dryness to give crude product (237). The crude product (237) was dissolved in N, N-dimethylacetamide (5ml), and to this solution was added in the order compound 234(110mg, 0.173mmol), N, N' -diisopropylethylamine (30ul, 0.172mmol), and the mixture was stirred overnight, concentrated, and column-chromatographed (1% HOAc in water/acetone, 1: 9-1: 4) to give the title compound 239(123.2mg, 71%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₄₇H₆₇N₇O₁₄PS, 1016.42, experimental value, 1016.42.

EXAMPLE 53 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -3- (3- (2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethoxy) propionylamino) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (240)

To a solution of compound 236(108mg, 0.154mmol) in 1, 4-dioxane (4ml) was added concentrated hydrochloric acid (1.0m1, 37%) and the mixture was stirred for 30 minutes and then concentratedTo dryness to give crude product (238). The crude product (238) was dissolved in N, N-dimethylacetamide (5ml), to which was added in the order compound 234(110mg, 0.173mmol), N, N' -diisopropylethylamine (30ul, 0.172mmol), and the mixture was stirred overnight, concentrated, and column chromatographed (1% HOAc in water/acetone, 1: 9-1: 4) to give the title compound 240(131.2mg, 76%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₅₁H₇₅N₇O₁₇PS, 1120.47, experimental value, 1120.48.

EXAMPLE 54 (4R) -4- (tert-Butoxycarbonylamino) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridine-2-dithio) butyrylamino) phenyl) pentanoic acid (244)

Add Succinimidyl 4- (pyridine-2-dithio) butanoic acid (550.2mg, 1.687mmol) and N, N '-diisopropylethylamine (0.18ml, 1.03mmol) to a solution of Compound 190(548.3mg, 1.311mmol) in N, N' -dimethylformamide (10ml), stir the mixture overnight, concentrate, column chromatographe (1% HOAc in water/acetone, 1: 9-1: 4) to give the title compound 244(660.2mg, 80%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₂₆H₃₅N₃O₉PS₂628.16, experimental value, 628.16.

EXAMPLE 55 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -2-methyl-5- (4- (phosphonooxy) -3- (4- (pyridine-2-dithio) butyrylamino) phenyl) pentanoic acid (248)

To a solution of compound 244(110.5mg, 0.175mmol) in 1, 4-dioxane (4ml) was added concentrated hydrochloric acid (1.0ml, 37%) at 4 deg.C, the mixture was stirred for 30min and then concentrated to dryness to give the crude product (246). The crude product (246) was dissolved in NN-dimethylacetamide (5ml), to this solution were added in this order compound 234(110mg, 0.173mmol), N' -diisopropylethylamine (30ul, 0.172mmol), and the mixture was stirred overnight, concentrated, and subjected to column chromatography (1% HOAc in water/acetone, 1: 9-1: 4) to give the title compound 248(129.1mg, 71%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₄₇H₆₇N₇O₁₂PS₃1048.38, experimental value, 1048.38.

EXAMPLE 56 (4R) -4- (2- ((1R, 3R) -1-acetoxy-3- ((2S, 3S) -N, 3-dimethyl-2- ((R) -1-methylpiperidine-2-carboxamido) pentanoylamino) -4-methylpentyl) thiazole-4-carboxamido) -5- (3- (4-mercaptobutanamido) -4- (phosphonooxy) phenyl) -2-methylpentanoic acid (248b)

Compound 248(30mg, 0.0285mmol) was added to N, N-dimethylacetamide (2ml) and sodium dihydrogen phosphate (0.1M, pH 7), and dithiothreitol (20mg, 0.129mmol) was added to the mixture. The mixture was stirred for 2 hours, concentrated and subjected to column chromatography (1% HOAc in H)₂O/MeCN, 1: 9-1: 4) to give the title compound 248b (22mg, 85%). MS ESI: m/z-: [ M-H ]]^-Ion peak: c₄₂H₆₄N₆O₁₂PS₂939.38, experimental value, 939.38.

Example 57.4- (4-bromobutyl) -10-oxa-4-azatricyclo [5.2.1.0^ 2, 6 ] dec-8-ene-3, 5-dione (271)

Reacting 10-oxa-4-azatricyclo [5.2.1.0^ 2, 6}]Dec-8-ene-3, 5-dione (6.0g, 36.35mmol) and sodium hydride (60% oil, 1.50g, 37.50mmol) were added to N, N-dimethylacetamide (60ml), and after stirring for 1 hour, 1, 4-dibromobutane (35.0g, 162.10mmol) and sodium iodide (0.50g, 3.33mmol) were added. The mixture was stirred overnight, quenched with methanol (0.5ml) and concentratedColumn chromatography (EtOAc/Hexane ═ 1: 8) afforded the title compound 271(9.34g, 86%). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₁₂H₁₄BrNaNO₃322.02, experimental value, 322.02.

EXAMPLE 58 methyl 2- ((1R, 3R) -3- (tert-butoxycarbonylamino) -1- [4 ' - (3 ', 6 ' -endoxy-tetrahydrophthalimido) butoxy ] -4-methylpentyl) thiazole-4-carboxylate (272)

Methyl 2- ((1R, 3R) -3- (tert-butoxycarbonylamino) -1-hydroxy-4-methylpentyl) thiazole-4-carboxylate (70) (1.0g, 2.79mmol) and sodium hydride (120mg, 3.00mmol, 60% oil) were added to tetrahydrofuran (30ml), and after stirring for 30 minutes, compound 271(1.00g, 3.34mmol) and sodium iodide (50mg, 0.33mmol) were added. The mixture was stirred overnight, quenched with methanol (0.5ml), concentrated, and column chromatographed (EtOAc/CH)₂C1₂1: 10) to yield the target compound 272(1.36g, 84%). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₂₈H₃₉NaN₃O₈S, 600.25, experimental value, 600.25.

EXAMPLE 59 methyl 2- ((1R, 3R) -3- (N, N-tert-butoxycarbonylmethylamino) -1- [4 ' - (3 ', 6 ' -endoxy-tetrahydrophthalimido) butoxy ] -4-methylpentyl) thiazole-4-carboxylic acid methyl ester (273)

Compound 272(1.30g, 2.25mmol) and sodium hydride (108mg, 2.70mmol, 60% oil) were added to N, N-dimethylformamide (80ml), and after stirring for 1 hour, iodomethane (460mg, 3.24mmol) was added. The mixture was stirred overnight, concentrated, and column chromatographed (EtOAc/CH)₂Cl₂1: 12-1: 8) to yield 273(1.01g, 76%) of the title compound. MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₂₉H₄₁NaN₃O₈S，614.26，Found，614.26。

EXAMPLE 60- ((1R, 3R) -3- (N, N-tert-butoxycarbonylmethylamino) -1- [4 ' - (3 ', 6 ' -endoxy-tetrahydrophthalimido) butoxy ] -4-methylpentyl) thiazole-4-carboxylic acid (274)

The dried compound 273(900mg, 1.52mmol) was dissolved in a mixed solvent of 1, 2-dichloroethane (30ml) and toluene, and trimethyltin hydroxide (400mg, 2.21mmol) was added. The mixture was stirred at 100 ℃ overnight, concentrated and subjected to column chromatography (MeOH/CH)₂Cl₂HOAc ═ 1: 10: 0.01) yielded target compound 274(730mg, 94%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₂₈H₃₉N₃NaO₈S, 600.22, experimental value, 600.22.

EXAMPLE 61 methyl 2- ((1R, 3R) -1-acetoxy-3- (N, N- (tert-butoxycarbonyl) (4' - (3 ", 6" -methano-tetrahydro) butyl) amino) -4-methylpentyl) -thiazole-4-carboxylic acid (275)

Compound 71(1.50g, 3.74mmol) and sodium hydride (180mg, 4.50mmol, 60% oil) were added to N, N-dimethylformamide (80ml), and after stirring for 1 hour, compound 271(1.48g, 4.94mmol) and iodomethane (70mg, 0.467mmol) were added. The mixture was stirred overnight, concentrated, and column chromatographed (EtOAc/CH)₂Cl₂1: 10-1: 6) to give the title compound 275(1.60g, 69%). MS ESI: m/z +: [ M + Na ]]⁺Ion peak: c₃₀H₄₁NaN₃O₉S, 642.26, experimental value, 642.26.

EXAMPLE 62.2- ((1R, 3R) -1-acetoxy-3- (N, N- (tert-butoxycarbonyl) (4' -maleimidobutyl) amino) -4-methylpentyl) -thiazole-4-carboxylic acid (276)

The dried compound 275(800mg, 1.29mmol) was dissolved in a mixed solvent of 1, 2-dichloroethane (40ml) and toluene, and trimethyltin hydroxide (400mg, 2.21mmol) was added. The mixture was stirred at 100 ℃ overnight, concentrated and subjected to column chromatography (MeOH/CH)₂Cl₂1: 5,/HOAc: 0.01), collecting components, concentrating and drying. The resulting crude product was dissolved in pyridine (15ml), acetic anhydride (0.3ml, 3.17mmol) was added to the solution at 0 deg.C, the mixture was stirred at room temperature overnight, concentrated, and column chromatographed (MeOH/CH)₂Cl₂HOAc ═ 1: 10: 0.01) gave target compound 276(578.4mg, 74%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₂₉H₃₉N₃NaO₉S, 628.24, experimental value, 628.24.

Example 63.1- (2-methyl-2-oxiranyl) -2-phenylethylamine

To 1- (2-methyl-2-oxiranyl) -2-phenylethyl) carbamic acid tert-butyl ester (Sun, l.et al, j.mol.catalysis a: to a solution of chem, 2005, 234(1-2), 29-34) (300mg, 1.08mmol) in 1, 4-dioxane (8mL) was added concentrated hydrochloric acid (37%, 2mL) and stirring was continued for 1 hour at this temperature, TLC showed no starting reaction material. The resulting mixture was diluted with toluene (10ml), concentrated, and recrystallized from EtOH/Hexane to give the hydrochloride salt of the objective compound (201mg, 87%). ESI: m/z: [ M + H ]]⁺Ion peak: c₁₁H₁₆NO, 178.12, experimental value, 178.12.

EXAMPLE 64 (S) -5-phenyl-4- (tert-butoxycarbonylamino) -2-methylpentenoic acid ethyl ester (327)

At-78 deg.CDiisobutylaluminum hydride (40ml, 40mmol, 1.0M) was slowly added to methyl (S) -3-phenyl-2- (tert-butoxycarbonylamino) -propionate 326(5.60g, 20.05mmol) in CH at the temperature of (D)₂Cl₂To a solution (80ml), after 45 minutes of reaction, a ylide solution formed of a solution of 1- (1-ethoxycarbonylethyl) triphenylphosphonium bromide (18.0g, 40.64mmol) and KOtBu (5.00g, 44.64mmol) in dichloromethane (80m1) was added at that temperature, and after the mixture had reacted at-78 ℃ for 1 hour, it was stirred at room temperature overnight, to the mixture was further added 1 liter of a sodium dihydrogen phosphate solution, vigorously stirred, separated, the aqueous phase was extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (ethyl acetate/n-hexane ═ 1: 7-1: 5) to obtain the objective product 327(5.50g, 83%), ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₉H₂₇NNaO₄356.19, experimental value, 356.20.

EXAMPLE 65 (S) -5-phenyl-4- (tert-butoxycarbonylamino) -2, 3-epoxyethyl-2-methylpentanoic acid ethyl ester (328)

To a solution of compound 327(5.0g, 15.0mmol) in dichloromethane (80ml) was added 3-chloroperoxybenzoic acid (5.5g, 22.3mmol), the mixture was stirred overnight, and a solution of sodium bicarbonate (25ml, sat.) was diluted, the aqueous phase was separated, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (ethyl acetate/n-hexane ═ 1: 4) to give the title compound 328(4.71g, 90%). ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₉H₂₇NNaO₅372.19, experimental value, 372.20.

EXAMPLE 66 (S) -5-phenyl-4- (tert-butoxycarbonylamino) -2, 3-epoxyethyl-2-methylpentanoic acid (329)

To an aqueous solution (60mL) of lithium hydroxide (5.0g, 208.7mmol) was addedA solution of compound 328(4.70g, 13.45mmol) in tetrahydrofuran (100mL) was added, stirred for 1 hour, concentrated, added with water (150mL) and adjusted to pH4 with 4M hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (MeOH/DCM/HOAc ═ 1: 10: 0.01) to give the title compound 329(3.97g, 92%), ESI: m/z: [ M + Na ]]⁺Ion peak: c₁₇H₂₃NNaO₅，344.16，Found 344.16。

EXAMPLE 67 (S) -5-phenyl-4- (9-fluorenylmethoxycarbonylamino) -2, 3-epoxyethyl-2-methylpentanoic acid (331)

Trifluoroacetic acid (10ml) was added to a solution of compound (329) (3.90g, 12.14mmol) in dichloromethane at a temperature of 0 ℃ and stirred at that temperature for 30 minutes, diluted with toluene, concentrated and dried to give the crude trifluoroacetate salt 330. Sodium carbonate (5.0g, 47.16mmol) was added to a mixed solvent of water (60mL) and ethanol (60mL), to which was added compound 330 and 9-fluorenylmethoxycarbonyl chloride (3.70g, 14.30 mmol). After stirring for 6 hours, concentrate, add water (150mL) and adjust to pH4 with 4M hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (MeOH/DCM/HOAc ═ 1: 10: 0.01) to give the title compound (3.87g, 72%, yield in two steps), ESI: m/z: [ M + Na ]]⁺Ion peak: c₂₇H₂₅NNaO₅，466.17，Found 466.17。

Example 68 general procedure for polypeptide condensation

The hydrochloride salt of the amine is dissolved in dichloromethane or N, N-dimethylformamide (0.2M), cooled to 4 ℃ and the tert-butoxycarbonyl protected amino acid (1.3eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2eq), or O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (2eq), or tripyrrolidinylphosphonium bromide hexafluorophosphate (2eq), 1-hydroxybenzotriazole (1.5eq), N, N-diisopropylethylamine (3.5eq) are added in sequence. The reaction was slowly warmed to room temperature and continued for 15 hours, diluted with ethyl acetate, and then washed successively with 1M aqueous hydrochloric acid, saturated sodium bicarbonate, water and saturated aqueous sodium chloride. The combined organic phases are dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (0% to 20% MeOH: CH2Cl2) to yield the tert-butoxycarbonyl-protected polypeptide.

EXAMPLE 69 general procedure for the removal of tert-butoxycarbonyl

The tert-butoxycarbonyl protected amino acid was dissolved in 20% trifluoroacetic acid in dichloromethane or 4M hydrochloric acid in 1, 4-dioxane and stirred for 30 minutes or followed by TLC to determine completion of the reaction. Concentrating under reduced pressure to obtain corresponding polypeptide compound of trifluoroacetate or hydrochloride. The polypeptide containing trifluoroacetate salt can be concentrated 3-4 times with a dichloromethane/toluene solution containing 2% hydrochloric acid to form the corresponding hydrochloride compound.

Example 70 general procedure for Solid Phase Peptide Synthesis (SPPS)

The tert-butoxycarbonyl protected SPPS uses Merrifield resin or modified PAM resin or MBHA resin. The 9-fluorenylmethoxycarbonyl protected SPPS used was Wang resin or 2-chlorotrityl chloride resin, or HMPB, MBHA resin. The resin was pre-treated (pre-expanded) and loaded with the amino compound according to the resin manufacturer's instructions. The amino acid protected with t-butoxycarbonyl group on the resin was deprotected with 20% trifluoroacetic acid in dichloromethane or 4M hydrochloric acid in 1, 4-dioxane (stirred for 30 minutes) and washed sequentially with N, N-dimethylformamide, methanol, 50% N, N-diisopropylethylamine in dichloromethane and pure dichloromethane, and for the deprotection step involving multiple free amines this step was repeated one more time before acylation to ensure completion of the reaction. The free amine was suspended in a solution consisting of protected amino acid (3 equivalents of free amine), O-benzotriazol-N, N' -tetramethyluronium tetrafluoroborate or trispyrrolidinylphosphonium bromide hexafluorophosphate (3 equivalents of free amine) and N, N-diisopropylethylamine (5 equivalents of free amine), mixed for 4 hours and washed with N, N-dimethylformamide, methanol and dichloromethane in sequence. For reactions involving acylation of multiple free amines, the coupling step prior to deprotection needs to be repeated once more to ensure completion of the reaction. These steps are generally repeated until the desired peptide is synthesized.

EXAMPLE 71 general procedure for cleaving peptides from Wang resin or 2-Chlorotriphenylmethyl chloride resin

The Wang resin bound with peptide is mixed with dichloromethane containing 50% trifluoroacetic acid and triisopropylsilane (1-5%), or the peptide bound with 2-chlorotrityl chloride resin is mixed with dichloromethane containing 1% trifluoroacetic acid, the mixture is filtered after 2 hours, the mixture is sequentially eluted by dichloromethane (3x30ml) and methanol (3x30ml), the filtrates are combined, concentrated, dried and then added with cold ether, and the obtained precipitate is the deprotected peptide.

EXAMPLE 72 general procedure for cleaving peptides from Merrifield resin, modified PAM resin or MBHA resin

Combining the peptide-bound resin with HF/Me₂S/anisole (10: 1) or CH₃SO₃H/Me₂S/anisole (20: 1) mix (for cysteine containing peptides HF/anisole/Me)₂S/p-thiocresol (10: 1: 0.2), after 2 hours, concentrated under nitrogen, diluted with trifluoroacetic acid and filtered. Then eluting the resin with dichloromethane (3x30ml) and methanol (3x30ml) sequentially, combining the filtrates, concentrating, drying, and adding cold ether to obtain precipitate as deprotected peptide.

EXAMPLE 73 chromatographic purification

The crude polypeptide mixture is purified by column chromatography on silica gel (10% to 25% methanol in dichloromethane) or by reverse phase HPLC (gradient elution from 0% to 70% methanol in water (preferably with the addition of 1% acetic acid), the reaction is completed within 1 hour, the target fractions are mixed and the sample is collected after evaporation.

Example 74 preparation of conjugates

Antibodies, as a class of binding molecules, can be conjugated to the cleavage inhibitors via amide, thioether, or disulfide bonds. The antibody was diluted (> 5mg/mL) with 50mM sodium borate in PBS buffer (pH8.0), dithiothreitol (10 mM final concentration) was added, and the antibody released free thiol groups by treatment at 35 ℃ for 30 minutes. Approximately 8 thiol groups were coupled to each antibody as determined by Ellman's reagent [5, 5' -dithiobis (2-nitrobenzoic acid) ] after gel filtration chromatography on a G-25 column (1 mM EDTA in PBS buffer). The antibody can also release a thiol group with Traut' S reagent (2-iminothiophene) (Jue, R., et al. biochem.1978, 17 (25): 5399- & 5405), or can react with a different linker such as SATP (N-succinimide-S-acetylthiopropionate) or N-succinimide-S-acetyl (thiotetraenoic acid) (SAT (PEG)4) at pH 7-8, followed by hydroxylamine to form a thiol group (Duncan, R, et al, anal. biochem. Burm. 1983, 132, 68-73, Fuji, N.et al, chem. pharm. Bull.1985, 33, 362- & 367). Basically, 5-8 sulfydryl molecules are connected to each antibody molecule.

A drug containing a maleimide or bromoacetamide group (requiring 0.5M sodium borate solution (pH 9) to promote alkylation of antibody with bromoacetamide) was added to ice-cold Dimethylacetamide (DMA) of an antibody having free thiol groups (2-20% v/v) at 4 deg.C (the molar ratio of the attached drug to thiol groups should be 1.2-1.5: 1). After 1-2 hours, adding excessive cysteine to terminate the reaction; the concentrated coupling product is obtained by ultrafiltration, gel chromatography (G-25, buffer PBS) and sterile filtration. The protein concentration and the number of drugs attached to each antibody were determined by measuring the absorbance at 280nm and 252 nm. Size exclusion HPLC can be used to determine the specific gravity of the monomeric form of the linker, while less than 0.5% of unbound drug can be determined using RP-HPLC. For monomeric drugs formed by thioether linkages, an average of 3.2-4.2 small molecules of mitotic inhibitor will be attached per antibody molecule.

The linker may be classified into dimethyl (phenyl) silyl (DMPS), SMDP, 4-succinimidyloxycarbonyl-methyl-alpha (2-pyridyldithio) toluene (SMPT), N-succinimidyl-4- (2-pyridylthio) valerate (SPP), N-succinimidyl-4- (2-pyridylthio) propionate (SPDP), N-succinimidyl-4- (2-pyridylthio) butyrate (SPDB), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimide- (polyethylene glycol) N-maleimide (SM (PEG) N), and the like. The antibody (> 5mg/mL) is diluted in a buffer (pH 6.5-7.5, 5mM PBS, 50mM NaCl, 1mM EDTA) and reacted with the linker for 2 hours, and the molar ratio of the linker to the antibody is 6-10 times or more. The reaction mixture was separated by Sephadex G25 gel chromatography, and the lower molecular weight molecules were removed. (the concentration of antibody was determined spectrophotometrically, the linker contained a pyridylthio group the extinction coefficient of the antibody at 280nm was 2067550M-1 cm-1. the 2-thiopyridine group released was determined after treatment of the modified antibody with excess dithiothreitol (20 fold more equivalent), the extinction coefficients at 343 and 280nm were 8080 and 5100M-1cm-1, respectively). Adding 1.2-1.5 equivalents of mitosis inhibitor molecules with sulfhydryl groups to the modified antibody. The reaction is carried out at room temperature for 5 to 18 hours. The reaction mixture was subjected to Sephadex G25 gel chromatography to remove the unbound drug or other low molecular weight substances. The concentration of the ligation product was then determined by measuring the absorbance at 280nm and 252 nm. The connection product is in a monomer form, and each antibody molecule is averagely connected with 3.2-4.5 drug molecules.

EXAMPLE 75 in vitro toxicity test

BJAB (Burkitt's lymphoma cells), BT-474 (breast cancer cells), Namalwa (human Burkitt's lymphoma cells), Ramos (human Burkitt's lymphoma cells), COLO 205 (human colon adenocarcinoma cells) and A375 (human malignant melanoma cells) were purchased from ATCC. The breast tumor cell line KPL-4 was awarded by doctor J.Kurebayashi (Kurebayashi, J.et al.Br J Cancer 1999; 79: 707-17). The cells were grown in RPMI 1640 medium containing 10% inactivated Fetal Bovine Serum (FBS) at 37 ℃ in a 6% CO2 incubator. The colony formation assay can be used as a method for toxicity assay detection, see literature (Franken, et al, Nature Protocols 1, 2315-2319 (2006)). The test cells were seeded in 6-well plates at 5000 cells per well and a gradient dilution of the drug (mitotic inhibitor or conjugate) from 1pM to 50nM was added and incubated for 72 hours. And replacing the old culture medium, continuously culturing the cells, and forming clones after 7-10 days. Cells were fixed and then stained with 0.2% crystal violet (diluted in 10% formalin or PBS) and cell clones counted. The number of untreated cells (medium only) can be determined by the number of clones formed in the well. Cell viability was calculated as the ratio of the number of colonies formed in wells of the drug treated and control (drug untreated).

TABLE 1 shows the structure of antimitotic drugs produced by solid phase synthesis and their ion peaks in mass spectra and their poisoning effect (IC) on Ramos cells (ATCC, human Burkitt lymphoma cells) in vitro tests₅₀) The value is obtained.

Table 2 shows the structural formulae of some antibody-antimitotic agent conjugates.

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Claims (20)

1. an antibody drug conjugate having the structural formula (I):

where T is a targeting or cell binding agent; l is a cleavable linker; represents a linkage linking L to an atom on the bracketed structure; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Each represents C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(2-8 carbon atoms) heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl (having 3 to 8 carbon atoms); or two R groups, e.g. R¹R²，R²R³，R³R⁴，R⁵R⁶，R¹²R¹³Two together may be C₃-C₇A carbocyclic group (containing 3-7 carbons), a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R³And R⁴May be H, and R²It is possible to delete the content of the content,

R⁵、R⁶、R⁸and R¹⁰Are each H or C₁～C₄An alkyl group or heteroalkyl group (containing 1 to 4 carbon atoms),

R⁷can be independently selected from H and R¹⁴or-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkane, or C₃-C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; x¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heteroCycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶、-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R1⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH₂OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each ring or heteroaromatic ring having from 4 to 10, preferably from 4 to 6, carbon atoms, and heteroaromatic rings in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atomsSubstituted, aryl Ar also refers to an aromatic or heteroaromatic ring in which one or more H atoms are substituted, the groups substituting for H atoms including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z1 is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)R¹⁸、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl,heteroalkyl group, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂～C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the group¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆An alkyl group or a heteroalkyl group,

the above-described conjugates of the present invention do not include the following structures:

wherein R is²⁴Is H or CH₃；R²⁵Is alkyl, fatty acyl or aromatic acyl; r²⁶Is H, C₁-C₅Alkyl radical, C₂-C₅Alkenyl, C (═ O) C₁-C₅Alkyl or C (═ O) C₂-C₅An alkenyl group; r²⁸Is H or C₁-C₅An alkyl group; r²⁷Is H OR 1-3 halogen, nitro, carboxylic ester OR its derivative, cyano, hydroxy, alkyl, haloalkyl, alkoxy, haloalkoxy, phenol protecting group, prodrug OR OR²⁹Wherein R is²⁹Is substituted aryl, C (O) R³⁰、P(O)(OR³⁰)₂Or SO₃R³⁰Wherein R is³⁰Is H, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl and aralkyl, or a metal cation; t is a targeting or cell binding body; l is a dissociable linker.

2. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (II):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Are respectively C₁～C₈Alkyl, heteroalkyl; c₂～C₈Heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl, or two R groups, e.g. R¹R²、R¹R³、R²R³、R³R⁴、R⁵R⁶；R¹²R¹³May be C₃～C₇(3-7 carbon) carbocyclic group, cycloalkyl group, heterocyclic group, heterocycloalkyl ring system group and the like; y is N or CH; furthermore, R¹、R³And R⁴May be H, and R²It is possible to delete the content of the content,

R⁵、R⁶、R⁸and R¹⁰Are respectively H, C₁～C₄An alkyl group or a heteroalkyl group,

R⁷is H, R¹⁴、-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl，C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkyl, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl or alkylcarbonyl, X¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶or-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH²OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each ring or heteroaromatic ring having from 4 to 10, preferably from 4 to 6, carbon atoms, heteroaromatic rings in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms, aryl Ar is an aromatic or heteroaromatic ring in which one or more H atoms are replaced, the H atom-replacing groups including R¹⁷、F、C1、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO²、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²Can be used forIs H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄-C₁₂(having 4 to 12 carbon atoms) glycoside or C₁-C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁-C₈Alkyl, carboxyalkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, or C₃-C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁-C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃-C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁-C₆Alkyl or heteroalkyl.

3. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (III):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Are respectively C₁～C₈Alkyl, heteroalkyl; c₂～C₈Heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl, or two R groups, e.g. R¹R²、R¹R³、R²R³、R³R⁴、R⁵R⁶；R¹²R¹³May be C₃～C₇(3-7 carbon) carbocyclic group, cycloalkyl group, heterocyclic group, heterocycloalkyl ring system group and the like; y is N or CH; furthermore, R¹、R²、R³And R⁴It may be a compound of formula (I) and (II),

R⁵、R⁶、R⁸and R¹⁰Are respectively H, C₁～C₄An alkyl group or a heteroalkyl group,

R⁷is R¹⁴，-R¹⁴C(O)X¹R¹⁵-or-R¹⁴X¹R¹⁵-，R¹⁴And R¹⁵Are respectively C₁-C₈Alkyl or heteroalkyl, C₂-C₈Alkylene, alkynylene, heterocyclyl, carbocyclyl, cycloalkyl, C₃-C₈Aryl, heterocyclylalkyl, heteroaralkyl, heteroalkylcycloalkyl or carbonylalkyl, X¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶、-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH₂OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁0 alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar denotes an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, eachThe aromatic or heteroaromatic ring having 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms, the heteroaromatic ring being an aromatic ring in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably by O, S or N atoms, the aromatic group Ar also being an aromatic or heteroaromatic ring in which one or more H atoms are replaced, the H atom-replacing groups comprising R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)R¹⁸、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂～C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the group¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆Alkyl or heteroalkyl.

4. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (IV):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is equal to 1 to 20; m is equal to 1 to 10, and,

the structure in brackets is an effective antimitotic agentWherein R is¹、R²、R³And R⁴Each represents C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(2-8 carbon atoms) heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl (having 3 to 8 carbon atoms); or two R groups, e.g. R¹R²，R²R³，R³R⁴，R⁵R⁶，R¹²R¹³Two together may be C₃-C₇A carbocyclic group (containing 3-7 carbons), a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R²、R³And R⁴It may be a compound of formula (I) and (II),

R⁵、R⁶、R⁸and R¹⁰Are each H or C₁～C₄An alkyl group or a heteroalkyl group (containing 1 to 4 carbon atoms).

R⁷Can be independently selected from H and R¹⁴or-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkane, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; x¹Is O, S, S-S, NH or NR¹⁴，

R⁹is-O-, -OR¹⁴-，-OC(＝O)R¹⁴-，-OC(＝O)NHR¹⁴-，-OC(＝O)R¹⁴SSR¹⁵-, OR-OP (═ O) (OR)¹⁴) O-, wherein R¹⁴And R¹⁵Are respectively C₁～C₈Alkyl or heteroalkyl, C₃～C₈Aryl, heteroaryl, heterocyclyl, carbocyclyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, carbonylalkyl or a pharmaceutical salt, and further R⁹It can also be a void that is left,

R¹¹is H,R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶、-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH₂OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁-C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each ring or heteroaromatic ring having from 4 to 10, preferably from 4 to 6, carbon atoms, heteroaromatic rings in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms, aryl Ar is an aromatic or heteroaromatic ring in which one or more H atoms are replaced, the H atom-replacing groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO₂、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、pR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When the group structure is as follows, R¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂～C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the group¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆Alkyl or heteroalkyl.

5. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (V):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Are respectively C₁～C₈Alkyl, heteroalkyl; c₂～C₈Heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl, or two R groups, e.g. R¹R²、R¹R³、R²R³、R³R⁴、R⁵R⁶；R¹²R¹³May be C₃～C₇(3-7 carbon) carbocyclic group, cycloalkyl group, heterocyclic group, heterocycloalkyl ring system group and the like; y is N or CH; furthermore, R¹、R²、R³And R⁴It may be a compound of formula (I) and (II),

R⁵、R⁶、R⁸and R¹⁰Are respectively H, C₁～C₄An alkyl group or a heteroalkyl group,

R⁷is H, R¹⁴、-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkyl, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl or alkylcarbonyl, X¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, -R¹⁴-、-R¹⁴C(＝O)R¹⁶-、-R¹⁴X²R¹⁶-or-R¹⁴C(＝O)X²-, wherein X²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclic radicalCarbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴-、C(O)NH₂、C(O)NHR¹⁴、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH²OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each ring or heteroaromatic ring having from 4 to 10, preferably from 4 to 6, carbon atoms, heteroaromatic rings in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms, aryl Ar is an aromatic or heteroaromatic ring in which one or more H atoms are replaced, the H atom-replacing groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO²、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄-C₁₂(having 4 to 12 carbon atoms) glycoside or C₁-C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁-C₈Alkyl, carboxyalkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, or C₃-C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁-C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃-C₈Aryl, alkyl carbonylsOr a pharmaceutically acceptable salt thereof; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁-C₆An alkyl group or a heteroalkyl group,

the above-described conjugates of the present invention do not include the following structures:

wherein R is²⁵Is alkyl, fatty acyl or aromatic acyl; r²⁶Is H, C₁～C₅Alkyl radical, C₂～C₅Alkenyl, C (═ O) C₁～C₅Alkyl or C (═ O) C₁～C₅An alkenyl group; r²⁷Is H OR 1-3 halogen, nitro, carboxylic ester OR its derivative, cyano, hydroxy, alkyl, haloalkyl, alkoxy, haloalkoxy, phenol protecting group, prodrug OR OR²⁹Wherein R is²⁹Is substituted aryl, C (O) R³⁰、P(O)(OR³⁰)₂Or SO₃R³⁰Wherein R is₃₀Is H, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl and aralkyl, or a metal cation; t is a targeting or cell binding body; l is a dissociable linker.

6. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (VI):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Are respectively C₁～C₈Alkyl, heteroalkyl; c₂～C₈Heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl, or two R groups, e.g. R¹R²、R¹R³、R²R³、R³R⁴、R⁵R⁶；R¹²R¹³May be C₃～C₇(3-7 carbon) carbocyclic group, cycloalkyl group, heterocyclic group, heterocycloalkyl ring system group and the like; y is N or CH; furthermore, R¹、R²、R³And R⁴It may be a compound of formula (I) and (II),

R⁵、R⁶、R⁸and R¹⁰Are respectively H, C₁～C₄An alkyl group or a heteroalkyl group,

R⁷is H, R¹⁴、-R¹⁴C(＝O)X¹R¹⁵or-R¹4X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkyl, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl or alkylcarbonyl, X¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -O-, -OR¹⁴、-OC(＝O)R¹⁴-、-OC(＝O)NHR¹⁴-、-OC(＝O)R¹⁴SSR¹⁵-、OP(＝O)(OR¹⁴) -OR OR¹⁴OP(＝O)(OR¹⁵) Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclic, carbonCyclic group, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶or-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-O-R¹⁴-、-S-R¹⁴-、-S(＝O)-R¹⁴-or-NHR¹⁴-；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-O-、-S-、-NH-、＝N-、＝NNH-、-NHR¹⁴-、--N(R¹⁴)-、-OR¹⁴-、-COR¹⁶-、-C(O)O-、-C(O)NH-、C(O)NR¹⁴-、-SR¹⁴-、-S(＝O)R¹⁴-、-NHR₁₄-、-P(＝O)(OR¹⁶)-、-OP(＝O)(OR¹⁶)₂-、-CH₂OP(＝O)(OR¹⁶)-、-C(O)OP(＝O)(OR¹⁶)-、-OP(＝O)(OR¹⁶) O-or-SO₂R¹⁶，R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each aromatic or heteroaromatic ring having 4 to 10 carbon atoms, preferably 4 to 6 carbon atoms, and heteroaromatic rings in which one or more carbon atoms are replaced by heteroatomsAromatic rings, preferably having only 1, 2 or 3 carbon atoms, substituted by O, N, Si, Se, P or S atoms, preferably by O, S, or N atoms, aryl Ar also meaning aromatic or heteroaromatic rings substituted by one or more H atoms, the H atom-substituting groups including R¹⁷、F、C1、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO²、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、PR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁-C₈Alkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃-C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄-C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄-C₁₂(having 4 to 12 carbon atoms) glycoside or C₁-C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁-C₈Alkyl, carboxyalkyl, heteroalkyl, C₂-C₈Alkenyl, alkynyl, heterocyclyl, or C₃-C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH, NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁-C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂-C₈Alkenyl, alkynyl, heterocyclyl, C₃-C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁-C₆Alkyl or heteroalkyl.

7. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (VII):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is equal to 1 to 10,

the structure in brackets is an effective antimitotic agent, where R¹、R²、R³And R⁴Each represents C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(2-8 carbon atoms) heterocycle, carbocycle, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈Aryl, aralkyl, heteroalkylcycloalkyl, alkylcarbonyl (having 3 to 8 carbon atoms); or two R groups, e.g. R¹R²，R²R³，R³R⁴，R⁵R⁶，R¹²R¹³Two together may be C₃～C₇A carbocyclic group (containing 3-7 carbons), a cycloalkyl group, a heterocyclic group, a heterocycloalkyl group, or the like; y is N or CH; furthermore, R¹、R²、R³And R⁴It may be a compound of formula (I) and (II),

R⁵、R⁶、R⁸and R¹⁰Are each H or C₁～C₄An alkyl group or a heteroalkyl group (containing 1 to 4 carbon atoms).

R⁷Can be independently selected from H and R¹⁴or-R¹⁴C(＝O)X¹R¹⁵or-R¹⁴X¹R¹⁵Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, cycloalkane, or C₃～C₈Aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; x¹Is O, S, S-S, NH or NR¹⁴，

R⁹Is H, -OH, -OR¹⁴、-OC(＝O)R¹⁴、-OC(＝O)NHR¹⁴、-OC(＝O)R¹⁴SSR¹⁵、OP(＝O)(OR¹⁴) OR OR¹⁴OP(＝O)(OR¹⁵)₂Wherein R is¹⁴And R¹⁵Are respectively C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl,

R¹¹is H, R¹⁴、-R¹⁴C(＝O)R¹⁶、-R¹⁴X²R¹⁶or-R¹⁴C(＝O)X²Wherein X is²is-O-, -S-, -NH-, -N (R)¹⁴)-、-N(R¹⁴)₂、-O-R¹⁴、-S-R¹⁴、-S(＝O)-R¹⁴or-NHR¹⁴；R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl; r¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹²is R¹⁴、-OH、-SH、-NH₂、＝NH、＝NNH₂、-NH(R¹⁴)、-OR¹⁴、-COR¹⁶、-COOR¹⁴、-C(O)NH₂、-C(O)NHR¹⁴、-C(O)N(R¹⁴)(R¹⁶)、-SR¹⁴、-S(＝O)R¹⁴、-P(＝O)(OR¹⁶)₂、-OP(＝O)(OR¹⁶)₂、-CH²OP(＝O)(OR¹⁶)₂or-SO₂R¹⁶，R¹⁴Is C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl, R¹⁶Is H, OH, R¹⁴Or 1 to 4 amino acid units,

R¹³is C₁～C₁₀Alkyl, heteroalkyl, alkyl acid, alkylamide, alkylamine, or aryl Ar; wherein Ar is an aromatic or heteroaromatic ring consisting of a single ring or multiple rings, each ring or heteroaromatic ring having from 4 to 10, preferably from 4 to 6, carbon atoms, heteroaromatic rings in which one or more carbon atoms are replaced by heteroatoms, preferably only 1, 2 or 3 carbon atoms are replaced by O, N, Si, Se, P or S atoms, preferably O, S, or N atoms, aryl Ar is an aromatic or heteroaromatic ring in which one or more H atoms are replaced, the H atom-replacing groups including R¹⁷、F、Cl、Br、I、OR¹⁶、SR¹⁶、NR¹⁶R¹⁷、N＝NR¹⁶、N＝R¹⁶、NR¹⁶R¹⁷、NO²、SOR¹⁶R¹⁷、SO₂R¹⁶、SO₃R¹⁶、OSO₃R¹⁶、pR¹⁶R¹⁷、POR¹⁶R¹⁷、PO₂R¹⁶R¹⁷、OP(O)(OR¹⁷)₂、OCH₂OP(O)(OR¹⁷)₂、OC(O)OP(O)(OR¹⁷)₂、PO(OR¹⁶)(OR¹⁷)、OP(O)(OR¹⁷)OP(O)(OR¹⁷)₂、OC(O)R¹⁷Or OC (O) NHR¹⁷Wherein R is¹⁶And R¹⁷Are respectively H, C₁～C₈Alkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, carbocyclyl, or C₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C₄～C₁₂A glycoside or a pharmaceutically acceptable salt thereof,

in addition, when R is¹⁰When not H, or when R¹³When it is a group¹²May be H:

where Z is¹Is H, CH₂OP(O)(OR¹⁸)₂、C(O)OP(O)(OR¹⁸)₂、PO(OR¹⁸)₂、C(O)R¹⁸、P(O)(OR¹⁸)OP(O)(OR¹⁸)₂、C(O)NHR¹⁸、SO₂(OR¹⁸)、C₄～C₁₂(having 4 to 12 carbon atoms) glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r¹⁹Is H, OH、NH₂、OSO₂(OR¹⁸)、XCH₂OP(O)(OR¹⁸)₂、XPO(OR¹⁸)₂、XC(O)OP(O)(OR¹⁸)₂、XC(O)R¹⁸、XC(O)NHR¹⁸、C₁～C₈Alkyl, carboxyalkyl, carboxylic acid derivatives, C₂～C₈Alkenyl, alkynyl, heterocyclyl, C₃～C₈Aryl, alkylcarbonyl or pharmaceutically acceptable salts; x is O, S, or NH; y is¹And Y²Are each a group consisting of N or CH,

or, when R is¹¹When it is the following group structure, R¹²May be H:

X²is O, S, N-R⁸；R⁸Is H, C₁～C₆An alkyl group or a heteroalkyl group,

the above-described conjugates of the present invention do not include the following structures:

wherein R is²⁴Is H or CH₃；R²⁵Is alkyl, fatty acyl or aromatic acyl; r²⁶Is H, C₁～C₅Alkyl radical, C₂～C₅Alkenyl, C (═ O) C₁～C₅Alkyl or C (═ O) C₂～C₅An alkenyl group; r²⁸Is H or C₁～C₅An alkyl group; t is a targeting or cell binding body; l is a dissociable linker.

8. The antibody drug conjugate according to claim 1, which has a structure represented by the structural formula (VIII):

t is a targeting or cell binding body; l is a cleavable linker attached to the molecule in parentheses; n is 1 to 20; m is 1-10; q is 1-5;

Z¹is H, CH₂OP(O)(OR¹⁸)₂，PO(OR¹⁸)₂，C(O)OP(O)(OR¹⁸)₂，P(O)(OR¹⁸)OP(O)(OR¹⁸)₂，C(O)R¹⁸，C(O)NHR¹⁸，SO₂(OR¹⁸)，C₄～C₁₂Glycoside or C₁～C₈Alkyl, carboxyalkyl, heterocyclyl; r¹⁸Is H, C₁～C₈Alkyl, carboxyalkyl, heteroalkyl, C₂～C₈Alkenyl, alkynyl, heterocyclyl, or C₃～C₈Aryl, alkylcarbonyl; r²⁴And R²⁴' is H or CH, respectively₃。

9. The linker L of the conjugate of claim 1, 2, 3, 4, 5, 6, 7 or 8 has the formula: -W^w-(Aa)_r-V_v-wherein-W-is an extension unit that links a target linker unit (T) to an amino acid unit (Aa), or W to a V unit when Aa is absent; w may independently comprise a self-immolative spacer unit, a peptidyl unit, a hydrazone, a disulfide bond, a thioether bond, an ester, or an amide bond; w may be 0 or 1; aa independently represents 1 natural or unnatural amino acid unit; r independently represents an integer of 0 to 12; (Aa)_rDenotes natural or non-natural amino acids, dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides or dodecapeptides,

v is a spacer unit which independently represents H, O, NH, S, C₁～C₈Alkyl or heteroalkyl of (a); c₂～C₈Alkenyl, alkynyl, heterocycle, carbocycle; c₃～C₈Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkylA group, an alkylsulfonyl group, an alkylcarbonyl group, or 1 to 4 amino acid units; v represents 0, 1 or 2.

10. The targeting or cell binding agent T of claim 1, 2, 3, 4, 5, 6, 7 or 8 may be selected from the group consisting of: antibodies or single chain antibodies, antibody fragments that can bind to target cells, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments that can bind to target cells, chimeric antibodies, chimeric antibody fragments that can bind to target cells, domain antibodies (including single domain antibodies), domain antibody fragments that can bind to target cells, mimetics (antibodies), DAPRins (designed ankyrin repeat), lymphokines, hormones, vitamins, growth factors, colony stimulating factors, nutrient transport molecules (such as transferrin), binding peptides, proteins, small molecules attached to albumin, high molecular polymers, artificial macromolecules, liposomes, nanoparticles, vesicles, viral capsids.

11. The antibody drug conjugate of claim 1, 2, 3, 4, 5, 6, 7 or 8, and pharmaceutically acceptable salts and solvates thereof having the formula IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIj, IIk, IIl, IIm, IIn, IIo, IIp, IIq, IIr, IIIa, IIIb, IIIc, IIId, IIIe, IIIf, IIIg, IIIh, IIIi, IIIj, IVa, IVb, IVc, IVd, IVf, Vg, Vvb, Vc, Vd, Ve, Vf, Vh, VIa, VIb, VIc, VIe, VIf, VIg, VIh, VIIi, VIIa, VIIb, VIIc, VIId, VIIe, VIIf, VIIg, VIIh, ii, VIIj, VIIk, VIIl, VIIm, VIIp, VIIt, VIIr, VIIk, VIIl, VIIk, and pharmaceutically acceptable salts and solvates thereof

Where T is a targeting or cell binding agent; aa is a natural or unnatural amino acid; ar is an aromatic group; n is 1 to 20; m is 1-10; q is 1-5; x ', Y ' and Z ' are each CH, O, S, NH, or NR²²；R²²、R²³And R²⁴Respectively represent C₁～C₈Alkyl (containing 1 to 8 carbon atoms) and heteroalkyl; c₂～C₈(containing 2-8 carbon atoms) alkene, alkyne, heterocycle, carbonCyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl; c₃～C₈(having 3 to 8 carbon atoms) aryl, aralkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl Or (OCH)₂CH₂)_n-; r 'and R' are each H or CH₃(ii) a Within the parentheses are the antimitotic drugs of the invention and within the square brackets the antimitotic drugs are linked to the cell binding molecule T via a linker.

12. The cytotoxic drug conjugate according to claims 1, 2, 3, 4, 5, 6, 7 or 8, and pharmaceutically acceptable salts and solvates thereof comprising an antimitotic drug and linker having the formula 232a, 232b, 232c, 232d, 232e, 232f, 232g, 232h, 232i, 232j, 232k, 2321, 232m, 232n, 232o, 232p, 232q, 232r, 232s, 232t, 221a, 221b, 233a, 233b, 233c, 233d, 222a, 308a, 308b, 309a, 309b, 290, 299a, 299b, 241, 247, 248, 206, 211, 346, 350, 354, 357, 361, 366, 371, and 376:

13. the cytotoxic drug conjugate according to claim 1, 2, 3, 4, 5, 6, 7 or 8 and pharmaceutically acceptable salts and solvates thereof have the structural formula as shown in mAb-TZ01, mAb-TZ02, mAb-TZ03, mAb-TZ04, mAb-TZ05, mAb-TZ06, mAb-TZ07, mAb-TZ08, mAb-TZ09, mAb-TZ10a, mAb-TZ10b, mAb-5TZ11, mAb-TZ12, mAb-TZ13, mAb-TZ14, mAb-TZ15, mAb-TZ16, mAb-TZ17, mAb-TZ18, mAb-TZ19, mAb-TZ20, mAb-TZ21, mAb-TZ 363672, mAb-TZ 21:

here mAb stands for antibody.

14. The target cells of the cytotoxic drug conjugate of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 comprising tumor cells, virally infected cells, microbially infected cells, parasitically infected cells, autoimmune cells, activated cells, bone marrow cells, activated T and B cells, or melanocytes, expressing 1 or more of the following antigens: apo2, ASLG659, BAFF-R, BMPR1B (bone morphogenetic protein), IGF-IR, CA125, CanAg, E16, EGFR, EphA2 receptor, ErbB2, interleukin-17, MUC 2, mucin 16(MUC 2), sodium-dependent phosphate transporter 2B (NaPi 22), NaPi 32 (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34, solute carrier family 2, type II sodium-dependent phosphate transporter 3B), VEGF, TF, MY 2, anti B2, EpfRH 2, FcRH2, C242, CD2, CD 3619, CD-CR 2, CD-derived CD-CR 2, CD-LREP-CR 2, CD-, FCRH1, glycoprotein NMB (GPNMB), GPNMB glycoprotein non-metastatic B (osteopectivin), HER2, HER2/neu, HER3, HLADOB (MHC class II antigen), integrin, IRTA2, LIV-1, Mesothelin (Mesothelin), MPF (MPF, MSLN, SMR, megakaryocyte enhancer, Mesothelin), GEDA, programmed death ligand 1(PDL1), Sema5b (FLJ10372, KIAA1445, Mm 15, Nectin-4, SEMA5B, 5EMAG, semaphorin 5b Hlog, Sema domain, seven thrombospondin repeats, cytoplasmic domain), PSCA, PSMA, STETRK 6, STEAP1 (prostate 1 six transmembrane antigen) and STEAP2(HGNC 8639, PCNC-1, PCAP-4, SLMP 42024, insulin 42024, and folate receptor for growth of epithelial cells.

15. According to claim 9, when a linker of the conjugate links two cytotoxic molecules, the linker has the following 3-bromo-maleimide group, or 3, 4-dibromomaleimide group structure:

where R is C₁～C₈Alkyl or C₂H₄(OC₂H₄)_nN is 1 to 20; x³Is H or Br.

16. According to claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, when two cytotoxic molecules of the conjugate are linked to a linker, the conjugate has the following structure:

where R is C₁～C₈Alkyl or C₂H₄(OC₂H₄)_nN is 1 to 20; d is a cytotoxic molecule/drug and T is a cell binding molecule.

17. According to claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, when two cytotoxic molecules of the conjugate are linked to a linker, the conjugate has the following structure:

where R is C₁～C₈Alkyl or C₂H₄(OC₂H₄)_nN is 1 to 20; d is a cytotoxic molecule/drug and T is a cell binding molecule.

18. The cytotoxic drug D of the conjugate according to claim 16 or 17 may also be selected from chemical drugs for cancer therapy, such as calicheamicin, microtubule polymer inhibitors (auristatins), maytansinoids (maytansinoids), dolastatins (dolastatins), CC-1065 analogs, doxorubicin, taxanes, Pyrrolobenzodiazepine dimers (pyrrobenzodiazepines), siRNA or mixtures thereof, and these conjugates may also be pharmaceutically acceptable salts, acids or derivatives of any of the above.

19. The conjugate of claim 1, 2, 3, 4, 5, 6, 7, 8, 11 or 13, or a pharmaceutically acceptable salt, carrier, diluent, excipient (adjuvant) or mixture thereof, in a therapeutically effective amount to form a pharmaceutical composition for the treatment or prevention of cancer, autoimmune disease or infectious disease.

20. The conjugate (conjugate) of claim 1, 2, 3, 4, 5, 6, 7, 8, 11 or 13 can be used in combination with other drugs (such as chemotherapeutic drugs, radiation therapy, immunological drugs, anti-autoimmune disorder drugs, anti-infective agents or other antibody-drug conjugates (conjugates)) in a therapeutically effective amount to constitute a pharmaceutical composition for treating or preventing cancer, autoimmune diseases or infectious diseases.

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