CN116726190A - Aureostatin analogs and conjugate conjugates thereof with cell-binding molecules - Google Patents

Abstract

The present application provides analogs of auristatin, particularly monomethyl auristatin F (MMAF) analogs, as cytotoxic molecules that form conjugated conjugates with cell-binding molecules, and the preparation and therapeutic uses of these cytotoxic molecules and conjugates to prevent or delay the growth and/or proliferation of abnormal cells.

Description

Aureostatin analogs and conjugate conjugates thereof with cell-binding molecules

The present application is a divisional application of application number 201580080907.6, entitled "Australian statin analog and conjugate thereof with a cell-binding molecule", having application number 2015, 6 and 20.

Technical Field

The present application describes a class of derivatives of auristatin cytotoxic small molecules, in particular methyl auristatin F (MMAF), conjugated to cell binding molecules and methods of using them to block cell growth and proliferation.

Technical Field

Chemotherapy is one of the traditional primary means of treating cancer. Treatment of tumors with chemotherapeutic agents is based on the ability of the agent to distinguish between cancer cells and normal cells and to preferentially kill cancer cells. Lack of selectivity and systemic toxicity to humans are major drawbacks of chemotherapeutic agents.

In order to improve the anticancer effect of the drug, the activity of the drug can be improved, so that the therapeutic effect can be achieved with lower dosage without adverse reaction, or the selectivity of the drug to tumors can be improved, and further, the better therapeutic effect can be ensured with higher dosage.

The former have made much effort to find new toxic small molecule drugs with higher activity, including isolation of toxic small molecules from natural products such as plants, marine organisms, tissue cells of microorganisms. In 1984, pettit and his group isolated from sea mollusc tail-biting sea hares a class of toxic polypeptides named dolastatin. In the next few years, the structure of dolastatin 10, one of the most active compounds, was determined (Pettit, et al J.am.chem.Soc.1987, 109, 6883-6885;Pettit,et al.J.Am.Chem.Soc.1989, 111:5463-5465). Dolastatin 10 has the properties of inhibiting tubulin polymerization and potent anti-cell proliferation, its IC ₅₀ On the picomolar scale. Dolastatin 10 rapidly enters human clinical evaluations. However, during the first clinical trial phase, the maximum tolerated dose (0.4 mg/m ² ) Far lower than other anticancer drugs such as doxorubicin (60-70 mg/m) ² ) There is also no significant clinical benefit seen in secondary clinical trials against various types of cancers such as melanoma, renal carcinoma, sarcoma, breast cancer and prostate cancer (Pettit et al, anticancer Drug des, 1998,13:243-277; poncet et al, curr.pharm.des.1999,5:139-162; ali et al, anticancer Res.1998, 18:1021-1026；Kalemkarian et al.，Pharmaco1.1999，43：507-515；Turner et a1.，Prostate 1998，34：175-181；Pitot，et a1.，Clin.Cancer Res.1999，5：525-531；Vaishampayan et a1.Clin.Cancer Res.2000，6：4205-4208)。

The concept of antibody-coupled drugs (ADCs), a means of increasing the selectivity of toxic small molecules for cancer cells, arose in the eighties of the twentieth century. Many antibodies preferentially bind cell surface antigens, specific small molecule markers, etc., and thus can be used as a vehicle to selectively transport anticancer drugs into tumor cells. An important attribute of the payload drug on an ADC is its efficient activity. Early ADCs utilized small molecule anticancer drugs that had been marketed, one of the reasons for their failure was the lack of sufficiently high in vitro activity of these drugs. Although dolastatin 10 itself does not show effective clinical therapeutic implications, its unusually high activity still makes it an alternative to ADC-loaded small molecules.

Intensive structure-activity studies have been conducted for dolastatin 10. It was found that dolastatin has a C-terminal dolaphene unit substituted with phenethylamine and an N-terminal tertiary amine substituted with a primary or secondary amine, which is still active (Pettit et al, anticancer Drug des, 1998,13:243-277;Doronina et al.Bioconjugate Chem.2006,17:114-124). Methyl Auristatin E (MMAE) and methyl auristatin F (MMAF) were subsequently prepared as small load molecules of ADC drugs.

In 2011, the FDA approved the market for novel anticancer drugs brentuximab vedotin (trade name adecris) for the treatment of recurrent Hodgkin's Lymphoma (HL) and recurrent intersystem degenerative large cell lymphoma (sALCL). Brentuximab vedotin it comprises the chimeric monoclonal antibody brentuximab (cAC, targeting the cell membrane protein CD 30), cathepsin protease hydrolysis linker (valine-citrulline), benzyl carbamate spacer and 3-5 MMAE units. This approval provides an example of the use of ADC drugs in cancer therapy and verifies the feasibility of an analog of auristatin (e.g., MMAE) as a small molecule of the ADC drug payload.

MMAF has a poorer in vitro activity than MMAE, but its methyl ester derivative MMAF-OMe is approximately 100-fold more active than MMAE on most cell lines. The charge at the C-terminus of MMAF is detrimental to its transport across the cell membrane, thus reducing its activity. But its neutral derivative MMAF-OMe is able to diffuse into cancer cells where it is hydrolyzed by esterases in the plasma, reconverted to MMAF, killing cancer cells (Doronina et al bioconjugate chem.2006, 17:114-124). Another advantage of using MMAF-OMe is that once it is hydrolyzed, the hydrolysis product MMAF is more hydrophilic and less capable of transmembrane transport, can become trapped inside cancer cells, thus resulting in reduced systemic toxicity.

To increase the activity of tubulysins as ADC drug loading we modified the C-terminal gamma amino acids tubutyrosine (Tut) and tubuphenylalanine (Tup) (PCT/IB 2012/053554, wo 2014009774). The results show that the introduction of epoxy or vinyl groups on Tut and Tup can significantly increase the therapeutic window of the tubulysin-like ADC. (Med Chem. #44,249) ^th ACS National Meeting, dever, CO, mar.22-26,2015; AACR national meeting, #4532,Philadelphia,Apr 18 to 22,2015). Using the same strategy, we modified the structure of the C-and N-terminal amino acid residues based on MMAF or MMAF-OMe, for use as ADC drug payload small molecules. These modified MMAF compounds showed good activity against a variety of cancer cells, could be linked to antibodies, and did not have significant aggregation problems. The invention discloses these modified compounds and methods of conjugated coupling of small molecules as supported molecules to cell binding molecules.

Summary of the invention

In one aspect, the structure of the drug-linker-ligand conjugate of the invention may be expressed as formula (I):

G-Lm-Dn(I)

and pharmaceutically acceptable salts and solvates thereof;

wherein G is a cell surface binding molecule (ligand); l is a cleavable or non-cleavable linker; n is 1-20; m is 1-10; d is a small molecule drug having the structural formula (II):

Wherein- - -represents that the small molecule drug and the linker are independently linked; r is R ¹ ，R ² ，R ³ And R is ⁴ Is independent C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Heterocyclic, carbocyclyl, cycloalkyl, alkylcycloalkyl or heterocycloalkyl, C ₃ -C ₈ Aryl, aralkyl, heteroalkylcycloalkyl or alkanoyl groups of (a); 2R groups, e.g. R ¹ R ² ，R ¹ R ³ ，R ² R ³ ，R ³ R ⁴ And R is ⁵ R ⁶ A 3-7 membered carbocyclic, cyclic, heterocyclic or heterocyclic ring structure; in addition, R ² ，R ³ And R is ⁴ May be H, and R ¹ Can default;

Y ¹ is N or CH;

R ⁵ and R is ⁶ Each independently is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Alkyl carbocyclyl or C ₁ -C ₈ An alkyl heterocyclic group; r is R ⁵ And R is ⁶ Can be combined to form a cycloalkyl, heterocycloalkyl, carbocycle or heterocyclic ring structure;

R ⁷ is H or C ₁ -C ₈ An alkyl group;

R ⁸ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ⁹ is H, OH, C ₁ -C ₈ Alkyl, C ₃ -C ₈ Carbocycles or O- (C) ₁ -C ₈ An alkyl group);

R ¹⁰ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ¹¹ is H or C ₁ -C ₈ An alkyl group;

R ¹² is aryl, alkylaryl, alkyl or C ₃ -C ₈ A heterocycle;

Y ² selected from: wherein R is ¹³ ，R ¹⁴ And R is ¹⁵ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle; x is CH ₂ ，O，S，NH，NR ¹⁶ S (O) or S (O) ₂ Wherein R is ¹⁶ Is H or C ₁ -C ₈ An alkyl group;

z is H, C ₁ -C ₈ Alkyl, -O- (C) ₁ -C ₈ Alkyl), -halogen, -NO ₂ ，-CN，-COOH，-C(O)OR ¹⁷ ，-C(O)NHR ¹⁸ ，-C(O)NR ¹⁸ R ¹⁹ ，-CH ₂ R ²⁰ ，-CH ₂ OP(O)(OR ²¹ ) ₂ Wherein R is ¹⁷ ，R ¹⁸ ，R ¹⁹ ，R ²⁰ And R is ²¹ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ Alkyl heterocycles.

On the other hand, the linker L has the structure-Ww- (Aa) r-Vv- (wherein: -W-is a stretch unit linking the target binding unit (T) to an amino acid unit (Aa) and to V when Aa is absent, and W may comprise each independently a self-pinning spacer, a short peptide unit, a hydrazone, disulfide, thioether, ester or amide bond; w is 0 or 1; aa are each independently a natural or unnatural amino acid unit; r is an integer from 0 to 12; (Aa) _r Represents a natural or unnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit;

V is a spacer unit, each independently is H, O, NH, S, C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl or carbocyclyl, C ₃ -C ₈ Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylcycloalkyl, heteroalkylcycloalkyl or alkylcarbonyl, or 1 to 4 amino acid units, or (CH) ₂ CH ₂ O) _r Wherein r is an integer from 0 to 12; v is 0,1 or 2.

In another aspect, G is any of a variety of currently known or known cell surface binding molecules, such as a peptide and a non-peptide. In general, the cell-binding molecule G 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, a binding agent that mimics an antibody, an ankyrin repeat protein, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule (transferrin), a binding polypeptide, protein or antibody, a small molecule that binds to albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid. A preferred binding molecule G is a monoclonal antibody.

In another aspect, the compounds of formula (I) or pharmaceutically acceptable salts and solvates are used for the treatment of cancer, autoimmune diseases or infectious diseases.

Description of the drawings:

FIG. 1 Synthesis of Dap unit (2R, 3R, 4S) -dolaproine) in MMAF.

FIG. 2 Synthesis of the Dil unit ((3R, 4S, 5S) -dolasicoleucine) in MMAF.

FIG. 3 synthesis of an MMAF-OMe analog containing maleimide linker.

FIG. 4 synthesis of an MMAF-OMe analog containing maleimide linker.

FIG. 5 synthesis of an MMAF-OMe analog containing maleimide linker.

FIG. 6 Synthesis of an MMAF-OMe analog containing maleimide linker.

FIG. 7 synthesis of MMAF-OMe analogs containing maleimide linkers.

Disclosure of Invention

1. Definition of the definition

"alkyl" refers to a class of linear or cyclic straight or branched aliphatic hydrocarbons containing from 1 to 8 carbon atoms. Branched means that there are one or more lower alkyl groups, such as methyl, ethyl or propyl groups, attached to a linear alkyl group. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3-dimethylpentyl, 2,3, 4-trioctyl Methylpentyl, 3-methylhexyl, 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. C (C) ₁ -C ₈ Alkyl groups may be unsubstituted or substituted with one or more of the following, but not limited to: c (C) ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, aryl, -C (O) R ', -OC (O) R ', -C (O) OR ', -C (O) NH ₂ ，-C(O)NHR'，-C(O)N(R') ₂ ，-NHC(O)R'，-S(O) ₂ R ', -S (O) R', -OH, halogen, -N ₃ ，-NH ₂ ，-NH(R')，-N(R') ₂ -CN; wherein R' means each independently C ₁ -C ₈ Alkyl or aryl; halogen means-F, -Cl, -Br, or-I, preference is given to-F and-Cl.

"heteroalkyl" refers to C having 1 to 4 carbon atoms independently replaced by O, S and N atoms ₂ ～C ₈ An alkyl group.

"carbocycle" refers to a saturated or unsaturated monocyclic structure containing 3 to 8 carbon atoms or a bicyclic structure containing 7 to 13 carbon atoms. Monocyclic carbocycles have 3 to 6 ring members, typically 5 to 6. Bicyclic carbocycles having 7 to 12 ring members [4,5 ]]、[5,5]、[5,6]Or [6,6 ]]The ring structure, or having 9 to 10 ring-forming atoms, forming [5,6 ]]Or [6,6 ]]A ring structure. "C ₃ -C ₈ Carbocycle "refers to a saturated or unsaturated, non-aromatic cyclic compound containing 3,4,5,6,7,8 carbon atoms. C (C) ₃ ～C ₈ Carbocycles may be unsubstituted or substituted with one or more of the following, but not limited to: c (C) ₁ -C ₈ Alkyl, C ₁ -C ₈ Alkoxy, aryl, acyl, acyloxy, ester, -C (O) NH ₂ ，-C(O)NHR’，-C(O)N(R’) ₂ ，-NHC(O)R’，-S(O) ₂ R ', -S (O) R', -OH, halogen, -N ₃ ，-NH ₂ ，-NHR’，-N(R’) ₂ -CN; wherein R' means each independently C ₁ -C ₈ Alkyl or aryl. Typical C ₃ -C ₈ Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cycloHexyl, cyclohexenyl, 1, 3-cyclohexadienyl, 1, 4-cyclohexadienyl, cycloheptyl, 1, 3-cycloheptadienyl, 1,3, 5-cycloheptatrienyl, cyclooctyl and cyclooctadienyl.

"alkenyl" refers to an aliphatic hydrocarbon having one carbon-carbon double bond and from 2 to 8 carbon atoms, either straight or branched. Examples of alkenyl groups include, but are not limited to: vinyl, allyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, hexenyl, heptenyl, octenyl.

"alkynyl" refers to an aliphatic hydrocarbon containing one carbon-carbon triple bond and from 2 to 8 carbon atoms in either a straight or branched chain. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, 2-n-butynyl, 3-methylbutynyl, n-pentynyl, hexynyl, heptynyl, octynyl.

"aromatic hydrocarbon" or "aryl" refers to an aromatic or heteroaromatic group containing one or more ring systems consisting of 3 to 14, preferably 6 to 10 carbon atoms. "heteroaryl" means that the aromatic group has one or more, preferably 1, 2, 3 or 4 carbon atoms replaced by O, N, S, se and P, preferably O, S and N. "aromatic hydrocarbon" or "aryl" also refers to an aromatic group in which one or more hydrogen atoms are independently replaced by: alkyl, halogen, OR, SR, NRR ', n=nr, n=r, NRR', NO ₂ ，SOR，SO ₂ R，SO ₃ R，OSO ₃ R, PRR ', PORR ', P (OR) (OR '), P (O) (OR) (OR ') OR OP (O) (OR) (OR '). Wherein R and R' each independently represent H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aralkyl or carbonyl groups or a pharmaceutical salt.

"heterocycle" refers to a class of aromatic or non-aromatic rings containing 3 to 14 carbons wherein 1 to 4 carbon atoms in the ring are each independently substituted with a heteroatom such as O, N, S, se and P. Preferred heteroatoms are O, N and S. For a description of the relevant heterocycles, see also The Handbook of Chemistry and Physics,78 ^th Edition, CRC Press Inc. 1997-1998, pages 225 to 226. Preferred non-aromatic heterocyclic groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl,piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydropyranyl, azepanyl, and fused rings thereof with phenyl.

"heteroaryl" or "aromatic heterocyclic" refers to an aromatic hetero mono-, bi-or polycyclic structure containing 5 to 14, preferably 5 to 10 atoms. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, benzofuranyl, furanyl, 1,2, 4-thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolinyl, isobenzofuranyl, pyrazolyl, benzothienyl, carbazolyl, benzimidazolyl, isoxazolyl, pyridine nitroxide, and fused rings thereof with phenyl.

The terms "alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heterocycle", "heteroaryl" and the like also refer to "alkylene", "cycloalkylene", "alkenylene", "alkynylene", "arylene", "heteroarylene" and the like, which are generated by the loss of two hydrogen atoms from the corresponding hydrocarbon.

"aralkyl" means one of the groups bound to a carbon atom, e.g. terminal carbon or sp ³ Carbon, an acyclic alkyl group in which a hydrogen atom attached thereto is substituted with an aryl radical. Typical aralkyl groups include, but are not limited to, benzyl, 2-phenethyl, 2-phenylethynyl, naphthylmethyl, 2-naphthylethyl, 2-naphthylethynyl, naphtholbenzyl, 2-naphtholbenzophenethyl and the like.

"heteroaralkyl" means one of the groups bound to a carbon atom, e.g. terminal carbon or sp ³ Carbon, a non-cyclic alkyl group in which the attached hydrogen atom is replaced by a heteroaryl radical. Typical heteroaralkyl groups include, but are not limited to, 2-phenylimidazolylmethyl, 2-furanethyl, and the like.

"hydroxy protecting groups" include, but are not limited to, methoxy ether, 2-methoxyethoxy ether, -tetrahydropyran ether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, t-butyldimethylsilyl ether, triphenylmethylsilyl ether, acetate, substituted acetate, trimethylacetate, benzoate, mesylate, and p-toluenesulfonate.

"leaving group" refers to a functional group that may be substituted with additional other functional groups. These groups can be found in various literature in the art including, but not limited to, halogen (chlorine, bromine and iodine), methanesulfonate (OMs), p-toluenesulfonate (OTs) and trifluoromethanesulfonate (OTf).

The following abbreviations have the meanings:

boc, t-butoxycarbonyl; broP, brominated tris (dimethylamino) phosphine hexafluorophosphoric acid; CDI, carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIBAL-H, diisobutylaluminum hydride; DIPEA, diisopropylethylamine; DEPC, diethyl pyrocarbonate; DMA, N-dimethylacetamide; DMAP, p-dimethylaminopyridine; DMF, N-dimethylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDC,1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; ESI-MS, electrospray mass spectrometry; HATU,2- (7-oxybenzotriazol) -N, N' -tetramethylurea hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; NHS, N-hydroxysuccinimide; MMP, 4-methylmorpholine; PAB, p-aminophenyl; PBS, phosphate buffer (pH 7.0-7.5); PEG, polyethylene glycol; SEC, size exclusion chromatography; TCEP, tris (2-carboxyethyl) phosphine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; val, valine.

"pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that, when applied to an animal or human, do not produce deleterious, allergic or other untoward effects.

"pharmaceutically acceptable salts" refer to a class of derivatives of the compounds disclosed, which are acid or base addition salts produced by modification of the parent compound. Pharmaceutically acceptable salts include conventional non-toxic salts derived from non-toxic inorganic or organic acids, as well as quaternary ammonium salts. Such salt-forming inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like, and organic acids such as 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 addition salts include ammonium salts derived from tromethamine, meglumine, pyrroloethanol, etc., and metal salts of sodium, potassium, calcium, zinc, magnesium, etc.

"pharmaceutically acceptable solvate" or "solvate" refers to a complex of one or more solvent molecules in combination with a disclosed compound. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

"pharmaceutically acceptable excipients" include all carriers, diluents, adjuvants or excipients 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. The addition of such media and agents to pharmaceutically active substances is known in the art. All conventional media and agents, unless they are incompatible with the pharmaceutically active ingredient, are contemplated for use in the pharmaceutical compositions. Auxiliary active ingredients may also be added to obtain a suitable pharmaceutical combination.

2. Drug-linker-ligand conjugate

The drug-linker-ligand conjugate of the invention may be partially degraded after delivery into cells to release the drug or drug with a linker residue. The drug (or drug-linker residue) would then kill the tumor cells as a cytotoxin. The small molecule drugs are dolastatin/auristatin series compounds which interfere with microtubule dynamics, GTP hydrolysis, and cell nucleus and cell division, and have anticancer and antifungal activities.

In one aspect, the drug-linker-ligand conjugate of the invention has the structural formula (I)

G-Lm-Dn(I)

And pharmaceutically acceptable salts and solvates thereof;

wherein G is a cell surface binding molecule (ligand); l is a cleavable or non-cleavable linker; n is 1-20; m is 1-10; d is a small molecule drug having the structural formula (II):

wherein-represents the individual connection of the small molecule drug and the linker; r is R ¹ ，R ² ，R ³ And R is ⁴ Is independent C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Heterocyclic, carbocyclyl, cycloalkyl, alkylcycloalkyl or heterocycloalkyl, C ₃ -C ₈ Aryl, aralkyl, heteroalkylcycloalkyl or alkanoyl groups of (a); 2R groups, e.g. R ¹ R ² ，R ¹ R ³ ，R ² R ³ ，R ³ R ⁴ And R is ⁵ R ⁶ A ring structure which may be a 3-7 membered carbocyclic, cyclic, heterocyclic or heterocyclic ring; in addition, R ² ，R ³ And R is ⁴ May be H, and R ¹ Can default;

Y ¹ is N or CH;

R ⁵ and R is ⁶ Each independently is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Alkyl carbocyclyl or C ₁ -C ₈ An alkyl heterocyclic group; r is R ⁵ And R is ⁶ Can be combined to form a cycloalkyl, heterocycloalkyl, carbocycle or heterocyclic ring structure;

R ⁷ is H or C ₁ -C ₈ An alkyl group;

R ⁸ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ A heterocyclic ring,C ₃ -C ₈ carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ⁹ is H, OH, C ₁ -C ₈ Alkyl, C ₃ -C ₈ Carbocycles or O- (C) ₁ -C ₈ An alkyl group);

R ¹⁰ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ¹¹ is H or C ₁ -C ₈ An alkyl group;

R ¹² is aryl, alkylaryl, alkyl or C ₃ -C ₈ A heterocycle;

Y ² selected from: wherein R is ¹³ ，R ¹⁴ And R is ¹⁵ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle; x is CH ₂ ，O，S，NH，NR ¹⁶ S (O) or S (O) ₂ Wherein R is ¹⁶ Is H or C ₁ -C ₈ An alkyl group;

z is H, C ₁ -C ₈ Alkyl, -O- (C) ₁ -C ₈ Alkyl), -halogen, -NO ₂ ，-CN，-COOH，-C(O)OR ¹⁷ ，-C(O)NHR ¹⁸ ，-C(O)NR ¹⁸ R ¹⁹ ，-CH ₂ R ²⁰ ，-CH ₂ OP(O)(OR ²¹ ) ₂ Wherein R is ¹⁷ ，R ¹⁸ ，R ¹⁹ ，R ²⁰ And R is ²¹ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ Alkyl heterocycles.

The linker L may be composed of one or more linker units. The linker unit comprises 6-Maleimidocaproyl (MC), maleimidopropionyl (MP), p-aminobenzyloxycarbonyl (PAB), 4-mercaptopentanoate (SPP), 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (MCC), (4-acetyl) aminobenzoate (SIAB), 4-mercapto-butyrate (SPDB), 4-mercapto-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), ethyleneoxy (-CH) ₂ CH ₂ One or more repeating units of O-) (EO or PEO), or a peptide unit such as valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af). Other linker units can be found in the literature that combine by chemical bonding, such as to form olefins, alkenyl olefins, alkynyl olefins, ethers, polyethylene glycols, esters, amines, imines, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxyamides, polyurethanes, amino acids, acyloxyamines, hydroxamic acids, and many other chemical structures. Furthermore, it is particularly emphasized that the structure of the linker units may be saturated or unsaturated, may be free radicals, or may form divalent cyclic structures with each other within the linker, including cycloalkanes, cyclic ethers, cyclic amines, aralkenes, heteroaralkenes.

On the other hand, the linker L has the structure-Ww- (Aa) r-Vv- (wherein: w-is a stretch unit, the target binding unit (T, defined below) is linked to an amino acid unit (Aa), and when Aa is absent, to V, andand W may comprise each independently a self-pinning spacer, a short peptide unit, a hydrazone, disulfide, thioether, ester or amide bond; w is 0 or 1; aa are each independently a natural or unnatural amino acid unit; r is an integer from 0 to 12; (Aa) _r Represents a natural or unnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit;

v is a spacer unit, each independently is H, O, NH, S, C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl or carbocyclyl, C ₃ -C ₈ Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylcycloalkyl, heteroalkylcycloalkyl or alkylcarbonyl, or 1 to 4 amino acid units, or (CH) ₂ CH ₂ O) _r Wherein r is an integer from 0 to 12; v is 0,1 or 2.

On the other hand, the linker-small molecule drug (L) in the conjugate (I) _m -D _n ) The structural formula of (C) is shown as (III):

and pharmaceutically acceptable salts and solvates thereof;

wherein L is a cleavable or non-cleavable linker;

Y ¹ is N or CH; r is R ² Is H or C ₁ -C ₈ An alkyl group;

R ³ and R is ⁴ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocyclic group; r is R ³ And R is ⁴ Can be combined to form cyclic rings, heterocycloalkyl, carbocyclic or heterocyclic ring structuresConstructing a structure;

R ⁵ and R is ⁶ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocyclic group; r is R ³ And R is ⁴ Can be combined to form a cyclic structure of a cycloalkane, heterocycloalkyl, carbocycle or heterocycle;

R ⁷ is H or C ₁ -C ₈ An alkyl group;

R ⁸ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ⁹ is H, OH, C ₁ -C ₈ Alkyl, C ₃ -C ₈ Carbocycles or O- (C) ₁ -C ₈ An alkyl group);

R ¹⁰ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ¹¹ is H or C ₁ -C ₈ An alkyl group;

R ¹² is aryl, alkylaryl, alkyl or C ₃ -C ₈ A heterocycle;

Y ² selected from: wherein R is ¹³ ,R ¹⁴ And R is ¹⁵ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle; x is CH ₂ ，O，S，NH，NR ¹⁶ S (O) or S (O) ₂ Wherein R is ¹⁶ Is H or C ₁ -C ₈ An alkyl group;

z is H, C ₁ -C ₈ Alkyl, -O- (C) ₁ -C ₈ Alkyl), -halogen, -NO ₂ ，-CN，-COOH，-C(O)OR ¹⁷ ，-C(O)NHR ¹⁸ ，C(O)NR ¹⁸ R ¹⁹ ，-CH ₂ R ²⁰ ，-CH ₂ OP(O)(OR ²¹ ) ₂ Wherein R is ¹⁷ ，R ¹⁸ ，R ¹⁹ ，R ²⁰ And R is ²¹ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ Alkyl heterocycles.

Linker-small molecule drug (L) having structure as formula (III) _m -D _n ) Examples of (a) include, but are not limited to, the following:

wherein M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ Or a pharmaceutically acceptable salt; r is R ¹ ，R ² And R is ³ Is as defined above; t is a target binding unit, independently selected from L, R ¹ ，-O-，-S-，-SS-，-NH-，＝N-，＝NNH-，-NH(R ¹ )，-OR ¹ ，-C(＝O)-，-C(＝O)R ¹ ，-COOR ¹ -，-C(O)NH-，-C(＝O)NHR ¹ -，-SR ¹ -，-S(＝O)R ¹ -，-P(＝O)(OR ¹ ) ₂ -，-P(＝O)(NHR ¹ ) ₂ -，-CH ₂ OP(＝O)(OR ¹ ) ₂ -，-CH ₂ NHP(＝O)(NHR ¹ ) ₂ -，-CH ₂ NHP(＝O)(NHR ¹ ) ₂ -，-CH ₂ NHP(＝O)(NHR ¹ )(NHR ² )-，-SO ₂ R ¹ -,(CH ₂ CH ₂ O) _r -，-(CH(CH ₃ )CH ₂ O) _r -，C ₁ -C ₈ Alkyl, or heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocycle or carbocycle, C ₃ -C ₈ Aryl, cycloalkyl, alkylcycloalkyl, in cycloalkyl, heteroaryl, heteroalkylcycloalkyl, alkylcarbonyl or 1-10 amino acid units, wherein r is an integer from 0 to 12; l, R ¹ And R is ² Is defined as in formulae (I) and (II).

T may also consist of one or more linker units. The linker unit comprises 6-Maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit or v)c) Alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl (PAB), 4-mercaptopentanoyl (SPP), 4- (N-maleimidomethyl) -cyclohexane-1 acyl (MCC), (4-acetyl) aminobenzoyl (SIAB), 4-mercapto-butyryl (SPDB), 4-mercapto-2-hydroxysulfonyl-butyryl (2-sulfoo-SPDB), ethyleneoxy (-CH) ₂ CH ₂ One or more repeating units of O- > (EO or PEO), oxime (aldoxime or ketoxime), 1,2, 3-triazole (produced by click chemistry), acetal (produced by reaction of sugar or the like). Other linker units can be found in the literature, in part as exemplified below.

Examples of linker units:

(MC, 6-Maleimidohexanoyl); (ME, maleimide ethyl);(MP, maleimide propionyl);

(PAB, p-aminobenzyloxycarbonyl analog); />

(carbobenzyl polypeptide analogs);

(valine-citrulline);(MCC, 4- (N-maleimidomethyl) -cyclohexane-1 acyl);((4-acetyl) aminobenzoyl);(2-sulfoo-SPDB, 4-mercapto-2-hydroxysulfonyl-butyryl);(oxime);(1, 2, 3-triazole);(acetals); (hydrazone);(stable peptide chain);((2-dithio) ethoxycarbonyl); wherein X is ¹ And Y ¹ Independently selected from O, S, NH or CH ₂ ；R ¹ And R is ² The definition is as before.

Q is a cell binding unit or a functional group that can be readily attached to a cell binding molecule. Functional groups include, but are not limited to, disulfide substitutes, maleimido groups, haloacetyl groups, hydrazines, aldehydes, ketones, azides, amino groups, alkoxyamino groups, or N-hydroxysuccinimide esters.

On the other hand, the linker-small molecule drug of formula (III) has the following preferred structure:

wherein Q, T, R ¹ ，R ² And R is ³ Is as defined above; r is R ²² Is H, OPO ₃ M ₂ ，OSO ₃ M，OCH ₂ PO ₃ M，OH，NO ₂ ，F，Cl，Br，I，SR ¹³ ，SSR ¹³ ，NH ₂ ，NHR ¹³ ，NR ¹³ R ¹⁴ OR ¹³ The method comprises the steps of carrying out a first treatment on the surface of the M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ ；R ¹³ ，R ¹⁴ ，R ¹⁵ ，R ¹⁷ ，R ¹⁸ And R is ¹⁹ Is as defined above. In addition, R ¹⁸ And R is ¹⁹ Can be combined to form a cycloalkane, heterocycloalkyl, carbocyclic or heterocyclic ring structure, an aromatic ring, or a heteroaromatic ring structure.

Preferred structures comprising the conjugate of the antibody and the structure of formula (III) include, but are not limited to:

and pharmaceutically acceptable salts and solvates thereof, wherein the mAb is an antibody and n is 1-30.

On the other hand, the linker-small molecule drug (L) on the conjugate (I) _m -D _n ) Has a structure as shown in formula (IV):

and pharmaceutically acceptable salts and solvates thereof, wherein R ¹ ，R ² ，R ³ ，R ⁴ ，R ⁵ ，R ⁶ ，R ⁷ ，R ⁸ ，R ⁹ ，R ¹⁰ ，R ¹¹ ，R ¹² ，Y ¹ ，Y ² Z and L are as defined for formula (III).

Linker-small molecule drug (L) of formula (IV) _m -D _n ) Examples of (a) include, but are not limited to, the following:

and pharmaceutically acceptable salts and solvates thereof. Wherein R is ¹ ，R ² T, Q and M are as defined for formulae (IIIa to IIIff).

On the other hand, the linker-small molecule drug represented by formula (IV) has the following preferred structure:

and pharmaceutically acceptable salts and solvates thereof. Wherein Q, T, R ¹ ，R ² ，R ³ ，R ¹⁷ ，R ¹⁸ ，R ¹⁹ And R is ²² Is defined as above. In addition, R ¹⁷ And R is ¹⁸ May be default.

Preferred structures comprising the conjugate of the antibody and the structure of formula (IV) include, but are not limited to:

and pharmaceutically acceptable salts and solvates thereof. Wherein mAb is an antibody, and n is 1-30.

On the other hand, linker-small molecule drug (L) on conjugate (I) _m -D _n ) Has a structure as shown in formula (V):

and pharmaceutically acceptable salts and solvates thereof, wherein R ¹ ，R ² ，R ³ ，R ⁴ ，R ⁵ ，R ⁶ ，R ⁷ ，R ⁸ ，R ⁹ ，R ¹⁰ ，R ¹¹ ，R ¹² ，Y ¹ ，Y ² Z and L are as defined for formula (III).

Linker-small molecule drug (L) of formula (V) _m -D _n ) Examples of (a) include, but are not limited to, the following:

wherein R is ¹ ，R ² T, Q and M are defined in accordance with formulae (IIIa to IIIff).

On the other hand, the linker-small molecule drug represented by formula (V) has the following preferred structure:

and pharmaceutically acceptable salts and solvates thereof. Wherein Q, T, R ¹ ，R ² ，R ³ ，R ¹⁷ ，R ¹⁸ ，R ¹⁹ And R is ²² Is defined as above. In addition, R ¹⁷ And R is ¹⁸ May be default.

Preferred conjugate conjugates comprising an antibody and a structure of formula (V) include, but are not limited to:

and pharmaceutically acceptable salts and solvates thereof. Wherein mAb is an antibody, and n is 1-30.

The cytotoxic small molecule drug (dolastatin/auristatin) in the present invention can be prepared by the method described in the following literature: U.S. Pat. nos. 5,635,483; U.S. Pat. nos. 5,780,588; pettit et al, j.am.chem.soc.1989,111:5463-5465; pettit et al, J.chem.Soc.Perkin Trans.1996,15:859-863; shioiri et al, tetrahedron Lett.1993,49 (9): 1913-1924. Scheme 1 illustrates a process for preparing the Dap unit (2R, 3R, 4S) -dolaprine. Scheme 2 illustrates a method for preparing the Dil unit ((3R, 4S, 5S) -dolaseucine). In general, polypeptide drugs can be prepared by forming peptide bonds between two or more amino acids or modified amino acids, or peptide fragments. Such peptide bonds can be formed according to standard liquid or solid phase synthesis methods well known in the art of peptide chemistry. Schemes 3-7 illustrate methods of synthesis of MMAF analogs and drug-linker compounds.

3. Cell binding molecules (ligands)

The cell binding molecules and modified cell binding molecules on the conjugates of the invention may be any known or unknown molecule that binds to, complexes with, or reacts with a portion of the sites of cells that may be of therapeutic interest or that have been biologically modified to be of therapeutic interest.

Cell binding molecules include, but are not limited to, large molecular weight proteins, such as whole antibodies (polyclonal, monoclonal, dimeric, multimeric, multispecific (e.g., bispecific antibodies)), single chain antibodies, antibody fragments such as Fab, fab ', F (ab') ₂ Fv (Parham, J.Immunol.1983, 131:2895-2902), fragments produced from Fab expression libraries, anti-idiotype antibodies (anti-Id), CDR's, and epitope-binding fragments of any of the above antibodies, which immunospecifically bind to an antigen of a cancer cell, a viral antigen, a microbial antigen, or a protein produced by the immune system that is capable of recognizing, binding to a particular specific antigen, or of producing a desired biological activity (Miller et al, J.Immunol.2003, 170:4854-4861), interferon (e.g., type I, II, III); a polypeptide; lymphokines such as IL-2, IL-3, IL-4, IL-6, GM-CSF, interferon gamma (IFN-gamma); hormones such as insulin, TRH (thyroid stimulating hormone releasing hormone), MSH (melanocyte stimulating hormone), steroid hormones such as androgens and estrogens; growth factors and colony stimulating factors such as Epidermal Growth Factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), transforming Growth Factors (TGF), such as TGF alpha, TGF beta, insulin and insulin-like growth factors (IGF-I and IGF-II), G-CSF, M-CSF and GM CSF (Burgess, immunol. Today,1984, 5:155-158); vaccinia Virus Growth Factor (VGF); fibroblast Growth Factor (FGF); small molecular weight proteins, peptides and peptide hormones, such as bombesin, 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); vitamins such as folic acid; apolipoproteins and glycoproteins, such as transferrin (O' keefa et al, j.biol. Chem.1985, 932-937); a sugar-binding protein or lipoprotein, such as lectin; cell camp A carrier molecule for the culture; and small molecule inhibitors such as Prostate Specific Membrane Antigen (PSMA) inhibitors and small molecule Tyrosine Kinase Inhibitors (TKI), non-peptide or any other cell-binding molecules or substances such as biopolymers (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, bioconj. Chem.2008, 19:1309-12); liposomes (Medinai et al, curr. Phar. Res.2004, 10:2981-9); viral capsids (Flenniken et al, viruses nanotechnol 2009, 327:71-93). In general, monoclonal antibodies are preferred cell surface binding agents, if appropriate. Antibodies may be derived from murine, human, humanized, chimeric, or from other species.

The production of antibodies for use in the present invention includes in vivo or in vitro processes or a combination thereof. Methods for preparing polyclonal antibodies are well known in the art, for example as described in U.S. Pat. No.4,493,795. Monoclonal antibodies are typically produced by fusing myeloma cells with spleen cells from mice immunized with the desired antigen and Milstein, nature,1975, 256:495-497). Detailed methods are described in antibody laboratory manuals, antibodies-A Laboratory Manual, harlow and Lane, eds., cold Spring Harbor Laboratory Press, new York (1988). Specifically, monoclonal antibodies are obtained by immunizing mice, rats, hamsters, or other mammals with the antigen of interest, which is derived from a target cell, an intact virus, inactivated intact virus, viral proteins, and the like. Spleen cells are typically fused with myeloma cells using polyethylene glycol (PEG) 6000, selected based on their sensitivity to HAT (hypoxanthine-aminopterin-thymidine). Hybridomas producing monoclonal antibodies of the present invention are determined by their ability to immunoreact with a particular receptor or inhibit the activity of a target cell receptor.

Monoclonal antibodies for use in the present invention can also be produced using a medium containing cells comprising a hybridoma capable of secreting an antibody molecule having the appropriate antigen specificity and nutrients. The hybridoma secretes the antibody molecule into the culture medium under certain culture conditions and for a period of time. The medium containing the antibodies was then collected. The antibody molecules are further isolated and purified by various techniques, such as protein a affinity chromatography, anion, cation, hydrophobicity, or size exclusion chromatography, centrifugation, differential solubilization, or other standard techniques for purifying proteins.

Media for antibody production are well known and commercially available in the art, including synthetic media. A typical synthetic medium is Dulbecco's minimal medium (DMEM; dulbecco et al, virol.1959, 8:396) supplemented with 4.5 g/l glucose, 20mM glutamine, 20% fetal bovine serum and an anti-foaming agent, such as a polyoxyethylene polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be made by fusion techniques other than fusion techniques, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection of tumor viruses, such as Epstein Barr virus (EBV, also known as human herpesvirus 4 (HHV-4)) or Kaposi sarcoma-associated herpesvirus (KSHV) (U.S. Pat. Nos. 4,341,761;4,399,121;4,427,783;4,444,887;4,451,570;4,466,917;4,472,500;4,491,632;4,493,890). Monoclonal antibodies can also be produced by peptides comprising a carboxy-terminal or anti-receptor peptide (Niman et al, proc. Natl. Acad. Sci.1983,80:4949-4953;Geysen et al, proc. Natl. Acad. Sci.1985,82:178-182;Lei et al.Biochemistry 1995,34 (20): 6675-6688). Typically, an anti-receptor peptide or peptide analog is used alone or conjugated to an immunogenic carrier to prepare an anti-receptor peptide monoclonal antibody as an immunogen.

There are many other methods by which monoclonal antibodies of the invention can be produced as binding molecules. Particularly useful are methods of making fully human antibodies. One method is phage display technology, which uses affinity enrichment to select a series of human antibodies that specifically bind to an antigen. Phage display has been fully described in the literature, and construction and screening of phage display libraries is well known in the art, e.g., literature Dente et al, gene 1994,148 (1): 7-13; little et al, biotechnol adv.1994,12 (3): 539-55; clackson et al, nature 1991,352:264-628; huse et al, science 1989, 246:1275-1281.

Monoclonal antibodies obtained by hybridoma technology from animals derived from another non-human species, such as mice, may be humanized to avoid the production of anti-murine antibodies. The most common methods for humanization of antibodies are complementarity determining region grafting and surface substitution. These methods are widely described in the following documents: U.S. Pat. nos. 5,859,205 and 6,797,492; liu et al, immunol Rev.2008,222:9-27; almagro et al, front biosci.2008,13:1619-33; lazar et al mol. Immunol.2007,44 (8): 1986-98; li et al, proc.Natl. Acad.Sci.2006,103 (10): 3557-62. Fully human antibodies can also be prepared by immunizing transgenic mice, rabbits, monkeys, or other mammals carrying the heavy and light chains of the majority of human immunoglobulins with an immunogen. Examples of such mice are: xeno mice (Abgenix/Amgen), huMAb mice (UltiMab platform, medarex/BMS), veloci mice (Regeneron), kymab (Kymouse technology), omniRat ^TM and OmniMouse ^TM Platforms (Open Monoclonal Technology, inc), U.S. Pat. nos. 6,596,541;6,207,418;6,150,584;6,111,166;6,075,181;5,922,545;5,661,016;5,545,806; also seen in 5,436,149 and 5,569,825. In human therapy, the mouse variable and human constant regions may also be fused to construct "chimeric antibodies" that are significantly less immunogenic than the human monoclonal murine antibody. (Kipriyanov et al, molBiotechnol.2004,26:39-60; houdine, curr. Opin. Biotechnol.2002, 13:625-9). In addition, site-directed mutagenesis in the variable regions of antibodies can produce an antibody with higher affinity and specificity for its antigen (Brannigan et al, nat. Rev. Mol. Cell biol.2002,3:964-70; adams et al, J. Immunol. Methods1999, 231:249-60), and by exchanging the constant regions of monoclonal antibodies, its ability to mediate binding and cytotoxic effector functions can be enhanced.

Immunospecific antibodies against malignant cell antigens may also be obtained commercially or by any other method, for example, chemical synthesis or recombinant expression techniques. The nucleotide sequence of an antibody that is immunospecific for a malignant cell antigen can be obtained commercially, for example from the GenBank database or similar databases, or from literature publications, or by conventional cloning and sequencing.

In addition to antibodies, peptides or proteins that bind/block/target or otherwise interact with target cell epitopes or corresponding receptors may also be used as binding molecules. These peptides or proteins may be any random peptide or protein having an affinity for an epitope or corresponding receptor, which is not necessarily a member of the immunoglobulin family. These peptides can be isolated by techniques similar to phage display antibodies (Szardings, J. Recept. Signal transfer Res.2003,23 (4): 307-49). The use of peptides in such random peptide libraries is analogous to antibodies and antibody fragments. The binding molecules of the peptides or proteins may be bound or linked to macromolecules or materials such as albumin, polymers, liposomes, nanoparticles, but are not limited thereto, as long as the peptides or proteins retain their antigen binding specificity after linking.

Examples of antibodies on antibody drug conjugates for the treatment of cancer, autoimmune diseases and infectious diseases include, but are not limited to, 3F8 (anti-CD 2), abagonomab (anti-CA-125), abciximab (anti-CD 41 (integrin. AlphSub>A. IIb), adaliumab (anti-TNF-. AlphSub>A.), adecaumumab (anti-EpCAM, CD 326), afelimomab (anti-TNF-. AlphSub>A.), afutuzumab (anti-CD 20), alacizumab (anti-VEGFR 2), ALD518 (anti-IL-6), altuzumab (Campath, mabcamapath, anti-CD 52), altumomab (anti-CEA), anatumab (anti-TAG-72), anruzumab (IMA-638, anti-IL-13), apolizumab (anti-HLA-DR), artumomab (anti-CEA), aselizumab (anti-L-selectin (CD 62), actmurSub>A, roactmurSub>A, anti-IL-6 receptor), atoolimiumab (anti-Rhesus factor), bapineuzumab (anti-betSub>A-amylase), basaliximab (Simulect, anti-CD 25), bavituximab (anti-phosphatidylserine), becumomab (lymphosan, anti-CD 22), belimumab (BenlystSub>A, lymphoStat-B, anti-BAFF), benralizumab (anti-CD 125), bertilimiumab (anti-CCL 11 (eosinophil chemokine-1)), benilesomab (scintiuni, anti-CEA related antigen), bevacizumab (Avastin, anti-VEGF-Sub>A), biciromab (fibriint, anti-fibrin II betSub>A chain), bicuzumab (anti-CD 4) 4 v 6), blinatemomab (BiTE, anti-CD 19), brentuximab (cAC 10, anti-CD 30 TNFRSF 8), briakiumab (anti-IL-12, IL-23), canakiumab (Illar, anti-IL-1), cantuzumab (C242, anti-Canag), carrotuzumab, catuxomab (Removab, anti-EpCAM, anti-CD 3), CC49 (anti-TAG-72), cedellizumab (anti-CD 4), certolizumab (Cimzia, anti-TNF-alpha), cetuximab (Erbitux, IMC-225, anti-epidermal growth factor receptor), citatuzumab (anti-EpCAM), cixutuzumab (anti-IGF-1), clenoximab (anti-CD 4), clituzumab (anti-MUC 1), contuzumab (anti-IL-R2), anti-CR 61, anti-influenza Datuzumab (anti-CD 40), anti-CD 25 (IL-2 receptor alpha chain)), daatumumab (anti-CD 38 (cyclic adenosine diphosphate ribohydrolase), denosuumab (Prolia, anti-RANKL), detumomomab (anti-B cell lymphoma cells), dorlimomab, dorlixizumab, ecromeximab (anti-GD 3 ganglioside), eculizumab (Soliris, anti-C5), edobacomab (anti-endothelial aflatoxin), edrecolomab (Panorex, MAb17-1A, anti-EpCAM), efalizumab (Raptiva, anti-LFA-1 (CD 11A)), efurumab (Mygradab, anti-Hsp 90), elotuzumab (anti-SLAMF 7), elsillimomab (anti-IL-6), enlimomab (anti-ICAM-1 (CD 54)), epitumab (anti-epicenter), ratuzumab (anti-CD 22), lizumab (anti-GB 2 (CD 18)) Ertumaxomab (Rexoma, anti-HER 2/neu, CD 3), etaracizumab (Abegrin, anti-integrin. Alpha. -V.beta.3), exbivirus mab (anti-hepatitis B virus surface antigen), fanolesemia ab (Neutrosum, anti-CD 15), farolimomab (anti-interferon receptor), farletuzumab (anti-folate receptor 1), felvizumab (anti-respiratory syncytial virus), fezakinumab (anti-IL-22), figituzumab (anti-IGF-1 receptor), fontolizumab (anti-IFN-. Gamma.), formiriumab (anti-rabies glycoprotein), fresolimumab (anti-TGF-. Beta.), galiximab (anti-CD 80), gantenerumab (anti-beta-amyloid), gavi momab (anti-CD 147 (Bain)), gemtuzumab (anti-CD 33), and Giuzumab (anti-CR 9), anti-GPNMB), golimumab (Simmoni, anti-tumor necrosis factor-alpha), gomiliximab (anti-CD 23 (IgE receptor)), ibalizumab (anti-CD 4), iblitumomab (anti-CD 20), igoviomab (Inindiacas-125, anti-CA-125), imleromab (pentetate-Yingximab, anti-myocardial myoglobin), intliximab (Infliximab, anti-tumor necrosis factor-alpha), intulimumab (anti-CD 51), inolimiomab (anti-tumor) CD25 (IL-2 receptor alpha chain)), inotuzumab (anti-CD 22), iplilimumab (anti-CD 152), iratumumab (anti-CD 30 (TNFRSF 8)), kelimimab (anti-CD 4), labetuzumab (CEA-CIDE, anti-CEA), lebrikizumab (anti-IL-13), lemalesomab (anti-NCA-90 (granulocyte antigen)), lerdelomab (anti-TGF-beta 2), lerotuzumab (anti-TRAIL-R2), libivirus b (anti-hepatitis B surface antigen), linatumab (anti-CD 33), lucatumumab (anti-CD 40), lumiliximab (anti-CD 23 (IgE receptor), mapatuzumab (anti-TRAIL-R1), maximab (anti-T cell receptor), nemamma (anti-growth factor receptor), mepolemab (bosama), IL-5, anti-IL-5), lerdelomab (anti-TGF-beta 2), lerdelomab (anti-R2), lefumagumab (anti-CD 3, oshadow-2), oshadow factor (anti-tumor factor (anti-CD 3), oshadow factor (anti-tumor 2), oshadow (anti-tumor 2 (anti-CD 3, oshadow 2), omamma (anti-tumor 2 (anti-CD 23 (anti-Ig receptor), makamaumab (anti-tumor 2), nemamumab (anti-tumor 2), mazoma (anti-R1), mazoma (anti-tumor 2 (anti-tumor 1), mazoma (anti-tumor 2) and (anti-tumor 5 (anti-tumor 2) Oftuzumab (Arzerra, anti-CD 20), olamateumab (anti-PDGF-Rα), omalizumab (Xolair, anti-IgE Fc region), oportuzumab (anti-EpCAM), oregoumab (ovarex, anti-CA-125), otelixizumab (anti-CD 3), pagibaxximab (anti-lipoteichoic acid), palivizumab (Synagis, abbosyngitis, anti-respiratory syncytial virus), panitumumab (Vectibix, ABX-EGF, anti-epidermal growth factor receptor), panobacumab (anti-Pseudomonas aeruginosa), pascalizumab (anti-IL-4), pemtumomab (Thermolyn anti-MUC 1), pertuzumab (Omnitarg, 2C4, anti-HER 2/neu), pexelizumab (anti-C5), pintum omab (anti-adenocarcinoma antigen), priliximab (anti-CD 4), pritumumab (anti-vimentin), pro 140 (anti-CCR 5), racotumomoma B (1E 10, anti- (N-glycosyl neuraminic acid (NeuGc, NGNA) -ganglioside GM 3), rafivirumab (anti-rabies glycoprotein), ramucicrumab (anti-VEGFR 2), ranibizumab (Lepidizumab, anti-vascular endothelial growth factor A), raxibacumab (anti-anthrax protective antigen), regambirumab (anti-cytomegaloglycoprotein B), resilizumab (anti-IL-5), rilotumamab (anti-Cytomegalovirus B) HGF), rituximab (Rituximab, anti-CD 20), robatumumab (anti-IGF-1 receptor), rontallizumab (anti-IFN- α), rovalicumab (leukarrimab, anti-CD 11, CD 18), ruplizumab (Antova), anti-CD 154 (CD 40L)), satumomab (anti-TAG-72), sevirus (anti-cytomegalovirus), sibutramab (anti-FAP), simalimab (anti-IFN- α), sibamab (anti-IL-6), sibuzumab (anti-CD 2), (smart) MI95 (anti-CD 33), solanezumab (anti- βamyoid), sonepuzumab (anti-1-phospho-sphingosine), sontuzumab (antiepelin), stabub (anti-myostatin), suremab (anti-TAG-72), sevizumab (anti-shadow), sevisuab (anti-human CTLA (anti-CD 40), anti-shadow (anti-tumor), anti-factor (anti-CD 40), sibutramine (anti-shadow), sibutramine (anti-rna) 2, anti-shadow (anti-tumor) and anti-tumor factor (anti-CD 4), shadow (anti-tumor factor (anti-tumor) and anti-tumor (anti-tumor) 2, anti-tumor (anti-tumor) and anti-tumor) 2, anti-tumor (anti-tumor) and anti-tumor (anti-tumor) rna (anti-tumor) 35) and anti-tumor, tucotuzumab (anti-EpCAM), tuvirumab (anti-hepatitis B virus), urtoxazumab (anti-Escherichia coli), ustekinumab (Stelara, anti-IL-12, IL-23), vapaliximab (anti-AOC 3 (VAP-1)), vedolizumab (anti-Engineα4β7), veltuzumab (anti-CD 20), vepalimomab (anti-AOC 3 (VAP-1), visilizumab (Nuvin, anti-CD 3), vitaxin (anti-vascular integrin avb 3), volociximab (anti-integrin α5β1), votumumab (HumaSPECT, tumor antigen CTA 16.88), zalutamab (HumaxEGFr, anti-growth factor receptor), zaniumab (HUoliumab-CD 4, anti-CD 4), ziracimab (anti-CD 147 (anti-CD 5), uvaluzumab (anti-CD 5) AlefaceptAbataceptRilonacept (Arcalyst), 14F7 (anti-irp-2, iron regulatory protein 2), 14G2a (anti-GD 2 ganglioside), J591 antibody (anti-PSMA, welchenal medical college), 225.28S (anti-HMW-MAA, high molecular weight melanoma-associated antigen), sorin Radiofarmaci S.R.L., COL-1 (anti-CEACAM 3, CGM 1), CYT-356 #Prostate cancer), HNK20 (OraVax Inc respiratory syncytial virus), immuRAIT (Immunomedics), lym-1 (anti-HLA-DR 10), MAK-195F (anti-TNFA, TNFSF 2), medi-500 (T10B 9, anti-CD 3, trαβ complex (T cell receptor a/β)), ring Scan (anti-TAG 72), avicidin (anti-EpCAM (epithelial cell adhesion molecule), anti-TACSTD 1 (tumor associated calcium signaling 1), anti-GA 733-2 (gastrointestinal tumor associated Protein 2), anti-EGP-2 (epithelial glycoprotein 2), anti-KSA, KS1/4 antigen, M4S (tumor antigen 17-1a, CD 326), lymphoCide (Immunomedics), smart ID10 (Protein Design laboratory), oncolym (Techniclone Inc), allomune (Biotransplant), anti-vascular endothelial growth factor (gene tex), CEAcide (Immunomedics), IMC-1C11 (ImClone/gift), and ceximab (image/gift).

Other antibodies that may be used as binding molecules include, but are not limited to, antibodies to the following antigens: aminopeptidase N (CD 13), annexin A1, B7-H3 (CD 276, various cancers), CA125 (ovarian cancer), CA15-3 (various cancers), CA19-9 (various cancers), L6 (various cancers), lewis Y (various cancers), lewis X (various cancers), alpha fetoprotein (various cancers), CA242 (colorectal cancer), placental alkaline phosphatase (various cancers), prostate specific antigen (prostate cancer), prostatic acid phosphatase (prostate cancer), epidermal growth factor (various cancers), CD2 (hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, gastric cancer, lung cancer, breast 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 (hodgkin's lymphoma), CD33 (leukemia, autoimmune disease), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, merck cell carcinoma, liquid tumor, multiple myeloma), CD66E (various cancers), CD70 (metastatic renal cell carcinoma and non-hodgkin's lymphoma), and, CD74 (multiple myeloma), CD80 (lymphoma), CD98 (various cancers), mucin (various cancers), CD21 (solid tumors), CD227 (breast cancer, ovarian cancer), CD262 (non-small cell lung cancer and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (large intestine cancer, stomach cancer), CEACAM5 (carcinoembryonic antigen, CEA, CD 66E) (breast, colorectal and lung cancer), DLL4 (delta-class-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD 184, heme tumor, solid tumor), endoglin (CD 105, solid tumor), epCAM (epithelial cell adhesion molecule, bladder cancer, head cancer, neck cancer, colon cancer, lymphoma, prostate cancer, ovarian cancer), ERBB2 (epidermal growth factor receptor 2, lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune disease), FOLR (folate receptor, ovarian cancer), gangliosides of GD2 (various cancers), G-28 (melanoma), GD3 idiotypes (various cancers), heat shock proteins (various cancers), HER1 (lung cancer, stomach cancer), HER2 (breast cancer, H lung cancer, ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotrophin (various cancers), IGF1R (insulin-like growth factor 1 receptor), solid tumors, hematological tumors), interleukin-2 receptors (interleukin 2 receptors, T cell leukemia and lymphoma), IL-6 (interleukin 6 receptors, multiple myeloma, rheumatoid arthritis, castleman's disease, IL6 dependent tumors), integrins (αVβ3, α5β1, α6β4, α11β3, α5β5, αVβ5, various cancers), MAGE-1 (various cancers), MAGE-2 (various cancers), MAGE-3 (various cancers), MAGE-4 (various cancers), anti-transferrin receptor (various cancers), p97 (melanoma), MS4A1 (non-Hodgkin B cell lymphoma, leukemia), MUC1 or MUC1-KLH (breast cancer), ovarian cancer, cervical cancer, bronchial and gastrointestinal cancers), MUC16 (CA 125, ovarian cancer), CEA (colorectal cancer), GP100 (melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (small cell lung cancer, NHL), nucleolin (Neu oncogene product, various cancers), p21 (various cancers), anti (N-glycosylneuraminic acid) antibody binding sites (breast cancer, malignant melanoma), PLAP-like testis alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate cancer), PSA (prostate cancer), ROBO4, TAG72 (tumor-associated glycoprotein 72, aml, gastric cancer, colon cancer, ovarian cancer), T cell transmembrane protein (various tumors), tie (CD 202B), TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, various cancers), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, lymphoma, NHL, other cancers, RA and SLE), TPBG (trophoblastic glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis-inducing ligand receptor 1, lymphoma, NHL, colon cancer, lung cancer), VCAM-1 (CD 106, melanoma), vascular endothelial growth factor a (VEGF), vascular endothelial growth factor 2 (CD 309) (various cancers). Other tumor-associated antigens that antibodies can recognize can be found in the review literature: gerber et al, mAbs2009,1 (3): 247-253; novellino et al, cancer immunol. Immunother.2005,54 (3): 187-207; franke et al, cancer biother. Radiopharm.2000,15:459-76. Examples of such antigens are: CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11B, CD11c, CD12w, CD14, CD15, CD16, CD w17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49B, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD L, CD62 5663, CD53, CD54, CD55, CD58, CD59 CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186, CD195, CD202 (a, B), CD209, CD235a, CD271, CD303, CD304, annexin A1, nucleolin, endothelial factor (CD 105), ROBO4, aminopeptidase N, DLL, VEGFR-2 (CD 309), CXCR4 (9 CD 184), tie2, B7-H3, WT1, MUC1, LMP2, HPV E6E 7, EGFRvIII, HER-2/neu, idiotype antigen, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, melanA/MART1, RAS mutant, gp100, p53 mutant, protease 3 (PR 1), HIV BCR-AB1, tyrosinase, survivin, hTERT, sarcoma translocation breakpoint, ephA2, PAP, ML-IAP, AFP, epCAM, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, rhoC, TRP-2, GD3, fucose GM1, mesothelin, PSCA, MAGE A1, sLe (a), CYP1B1, PLAC1, GM3, BORIS, tn, globoH, ETV-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, tie2, page4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, fos-related antibody 1.

In a specific embodiment, the conjugates of the invention are useful for treating cancer. Such cancers include, but are not limited to, adrenocortical carcinoma, anal carcinoma, bladder carcinoma, brain tumors (adult, brain stem glioma, childhood cerebellar astrocytoma, ependymoma, medulloblastoma, astrocytoma, primary extraneuro-embryonic and pineal tumor, ocular pathway and hypothalamic glioma), breast carcinoma, carcinoid, gastrointestinal tract, primary carcinoma, cervical carcinoma, colon carcinoma, endometrial carcinoma, esophageal carcinoma, extrahepatic cholangiocarcinoma, ewing's family tumor (PNET), extracranial germ cell tumor, ocular tumor, gallbladder carcinoma, intraocular melanoma, gastric carcinoma (stomach), extragonadal germ cell tumor, gestational trophoblastoma; head and neck cancer, hypopharynx cancer, islet Cell cancer, kidney cancer (renal Cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy Cell), lip cancer, oral cancer, liver cancer, lung cancer (non-small Cell, lymphoma (AIDS), central nervous system, skin T-cells, hodgkin's disease, non-Hodgkin's disease, malignant mesothelioma, melanoma, merck Cell carcinoma, occult primary, multiple myeloma metastatic squamous cervical cancer, and other plasma Cell tumors, mycosis fungoides, myelodysplastic syndrome, myeloproliferative diseases, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer (epithelium, gemm Cell Tumor, low malignancy), pancreatic cancer (exocrine glands, islet Cell cancer), sinus and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasmacytoma, prostate cancer rhabdomyosarcoma, rectal cancer, renal cancer (renal Cell carcinoma), ureter (renal pelvis transitional Cell), salivary gland cancer, sezary syndrome, skin cancer (cutaneous T-Cell lymphoma, kaposi's sarcoma, melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma (malignancy), thyroid cancer, urinary tract cancer, uterine cancer (sarcoma), childhood, unusual cancer vaginal cancer, vulval cancer, nephroblastoma.

In another embodiment, the medicament of the invention may be used in a pharmaceutical combination or method to treat or prevent autoimmune diseases. Autoimmune diseases include, but are not limited to, achlorhina autoimmune chronic hepatitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoence encephalitis, addison's disease, agaropectinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM, TBM nephritis, anti-phospholipid syndrome, anti-synthetase syndrome, arthritis, anaphylaxis, allergic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune multiple endocrine glandular syndrome type I, II, and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, balo disease/Balo concentric sclerosis, bechets syndrome, berger's disease, bickertaff encephalitis, blue syndrome, bullous Pemphigoid, castleman disease, trypanosomiasis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic lyme disease, chronic obstructive pulmonary disease, allergic Stlaus syndrome, cicatricia Pemphigoid, celiac disease, cogan syndrome, condensed collectinopathy, complement component 2 deficiency, craniarteritis, CREST syndrome, crohn's disease (a idiopathic inflammatory bowel disease), kuh-sence Euphoria, cutaneous leukocytoclasticangiitis, idiopathic Dou Genshi disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse cutaneous systemic sclerosis, deretsler syndrome, discoid lupus erythematosus, eczema, endometriosis, adnexitis-related arthritis, eosinophilic fasciitis, epidermolysis bullosa, erythema nodosum, cryoglobulinemia, egypt syndrome, fibrodysplasia ossificans progressive fibrosis, myofibrositis, fibromylgia, aveolitis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, pneumonic nephritis syndrome, graves disease, guillain Barre syndrome, hashimoto's encephalitis, hashimoto thyroiditis, hemolytic anemia, herpes gestation, henochSchonleinpurpura, hidradenitissuppurativa, huntin syndrome (see antiphospholic syndrome), hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (see immune thrombocytopenic purpura), igA nephropathy (Berger's disease), inclusion body myositis, inflammatory demyelinating polyneopathies, interstitial cystitis, irritable bowel syndrome, juvenile idiopathic arthritis, rheumatoid arthritis, kawasaki's disease, lanbert-Eatonic syndrome, white blood cell disruption vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), lou Gehrig's disease (amyotrophic lateral sclerosis lupoid also), hepatitis, lupus erythematosus, ma Jide syndrome, microscopic polyangiitis, miller Fisher syndrome, mixed connective tissue disease, scleroderma, muchaHabermann disease, poly-Wiles syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromeyelitisia, neurominonia, ocular cicatricial pemphigoid, ocular clonus-myoclonus syndrome, ORD thyroiditis, recurrent rheumatism, pandas (streptococcal related childhood autoimmune neuropsychiatric disease), paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria, parry-Romberg syndrome, pasmonnage tenna syndrome, platykurtic ciliary inflammation, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous inflammation, poe ms syndrome, polyarteritis nodosa, polymyalgia rheumatica, Myositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyodermangrenosum, pure erythrocyte aplastic anemia, lasmarson's encephalitis, raynaud's phenomenon, recurrent polychondritis, lyote's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid, sarcoidosis, schizophrenia, schmitt syndrome, schnithler's syndrome, scleritis, scleroderma, sjogren's syndromeSjogren's syndrome, spondyloarthropathies, mucoid syndrome, stele's disease, stiff person's syndrome, subacute bacterial endocarditis, susac's syndrome, sweet's syndrome, chorea minor, sympathogenic ophthalmia, takayasu's arteritis, temporal arteritis (giant cell arteritis), tolosa Hunt's syndrome, myelitis, ulcerative colitis (an idiopathic inflammatory bowel disease), undifferentiated connective tissue disease, undifferentiated spinoarthropathies, vasculitis, vitiligo, wegener granulomatosis, wilson's syndrome, wiskott Aldrich syndrome.

In another specific embodiment, the binding molecules on the conjugates for use in the treatment or prevention of autoimmune diseases include, but are not limited to, anti-elastin antibodies; an anti-epithelial antibody Abys; an anti-basement membrane type IV collagen antibody; an antinuclear antibody; anti-ds-DNA; anti-single stranded DNA, anti-cardiolipin antibodies IgM, igG antibodies; anti-abdominal cavity; anti-phospholipid antibody IGK, igG antibody; an anti-Sm antibody; an anti-mitochondrial antibody; thyroid microsomal antibodies, T cell antibodies; an antibody; thyroglobulin antibody, anti-Scl-70 antibody; anti Jo; anti-u.sub.1rnp; la/SSB resistance; an anti-SSA antibody; anti-SSB; anti-perital cell antibodies; antihistamine; anti-RNP; C-ANCA; P-ANCA; anti-sticking points; and an anti-fibrin antibody, an anti-GBM antibody, an anti-ganglioside; an anti-desmogein 3 antibody; an anti-P62 antibody; an anti-SP 100 antibody; anti-mitochondrial antibody (M2); a rheumatoid factor antibody; an anti-MCV antibody; an anti-topoisomerase antibody; anti-neutrophil cytoplasmic (cANCA) antibodies.

In certain preferred embodiments, the binding molecules on the conjugates of the invention may bind to a receptor or receptor complex expressed on activated lymphocytes associated with autoimmune disease. These receptors or receptor complexes include immunoglobulin gene superfamily members (e.g., CD2, CD3, CD4, CD8, CD19, CD22, CD28, 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, apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (type C, type S, or type I), or complement regulatory proteins.

In another embodiment, the binding molecule is a humanized or human monoclonal antibody that is immunospecific for a viral or microbial antigen. "viral antigens" include, but are not limited to, any viral peptide, polypeptide protein capable of eliciting an immune response, such as HIV gp120, HIV nef, RSV F glycoprotein, influenza neuraminidase, influenza hemagglutinin, HTLV, herpes simplex virus glycoprotein (e.g., gB, gC, gD and gE), and hepatitis b surface antigen. "microbial antigens" include, but are not limited to, any microbial peptide, polypeptide, protein, carbohydrate, polysaccharide or lipid molecule capable of eliciting an immune response, such as bacterial, fungal, pathogenic protozoan or yeast polypeptides (including lipopolysaccharide and capsular polysaccharide). Examples of antibodies that may be used for viral or microbial infection diseases include, but are not limited to, palivizumab, a humanized anti-respiratory syncytial virus monoclonal antibody that treats RSV; pro542, a CD4 fusion antibody for the treatment of HIV infection; ostavir, which is a humanized antibody for the treatment of hepatitis b virus; protvir, a humanized IgG sub.1 antibody, is used to treat cytomegalovirus and is also an anti-endotoxin antibody.

The drug conjugates of the invention are useful in the treatment of infectious diseases. Such infectious diseases include, but are not limited to, acinetobacter infection, actinomycosis, african sleeping disease (african trypanosomiasis), aids (acquired immunodeficiency syndrome), amoeba disease, intangium disease, anthrax, alcovenia haemolytica, argentina hemorrhagic fever, ascariasis, aspergillosis, astrovirus infection, babesia, bacillus cereus infection, bacterial pneumonia, bacterial vaginitis, bacteroides infection, pouch ciliate disease, bayer Lin Huichong infection, BK virus infection, black hair sarcoidosis, human blastocyst infection, budding disease, bovinia hemorrhagic fever, bordetella infection, botulism (and infant botulism) barre syndrome, brucellosis infection, brucellosis, caliciviris, curculi infection, curculia infection, campylobacter (norovirus and saprobiosis); thrush), cat's disease, cellulitis, chargs disease (trypanosomiasis americana) pneumochlamydia infection, cholera, chromob infectious clostridium infection, coccidiosis, colorado tick fever, cold (acute viral nasopharyngitis; acute rhinitis), creutzfeldt-Jakob disease, skin larva migration, cyclosporin disease, cysticercosis, cytomegalovirus infection, dengue fever, diabrosis, diphtheria, schizotaeniasis, maidendra nematodiasis, ebola hemorrhagic fever, echinococcosis, enterobiasis, enterococcidiosis, enterovirus infection, epidemic typhus, infectious erythema (fifth disease), endometritis, fasciolopsis, fatal familial insomnia, filariasis, clostridium perfringens food poisoning, free living amoeba infection, jetty-strahlung-smith syndrome, giardiasis, jaundice, palatinose, gonorrhea, granuloma (Du Nuofan), group a streptococcal infection, group B streptococcal infection, haemophilus influenzae infection, hand-foot disease and oral disease (HFMD), hantavirus lung syndrome, hel kidney syndrome hemorrhagic fever, hepatitis a, hepatitis B, hepatitis c, hepatitis d, hepatitis e, herpes simplex, histoplasmosis, hook worm infection, human bocavirus infection, human ehrlichiosis, human granulocytic leukemia human monocyte, ehrlichiosis, human papilloma virus infection, human parainfluenza virus infection, epstein-barr virus infectious mononucleosis (monofil), influenza, isospora, kawasaki disease, keratitis, kuru, lassa fever, legionellosis, dewing soldier's disease, leprosy, leptospirosis, listeriosis, lyme disease, lymphocytic choriomeningitis, malaria, marburg hemorrhagic fever, measles, melenoid (whitpore disease), meningitis, microsporides, molluscum contagiosum, mumps, mouse typhus (endemic typhus), mycoplasma pneumonia, podophy, myiasis, neonatal conjunctivitis (neonatal ophthalmitis), mutant cortical-striatal-spinal degenerative diseases (vCJD, nvCJD), discoid filariasis (south america mycosis), and schistosomiasis, pasteurellosis, cephalic pediculosis (head lice), pediculosis (body lice), pubic lice (pubic lice, crab lice), pelvic inflammatory disease, pertussis, pneumococcal infection, pneumococcal pneumonia, poliomyelitis, prasuvorexa infection, primary amebic meningoepitis, progressive multifocal leukopathy, psittacosis, Q-fever, rabies, mouse bite fever, respiratory syncytial virus infection, rhinosporosis, rhinovirus infection, rickettsial infection, rickettsia, rift fever, rocky mountain spot fever, rotavirus infection, sham severe acute respiratory syndrome, sc sore, schistosomiasis, septicemia, shigella disease (bacillary dysentery), herpes zoster (shingles), smallpox (smallpox), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, granulomatoid disease, syphilis, taeniasis, tetanus (Lockjaw), tinea capitis (Barber's itch), tinea capitis (scalp tinea), tinea cruris (tinea corporis), tinea cruris (tinea cruris), tinea manus (tinea manus), tinea pedis (tinea unguium), tinea versicolor (tinea versicolor), moth (eyeball larva), toxoplasmosis, trichinosis, trichomoniasis, tuberculosis, rabbit fever, venezuela equine encephalitis, venezuela hemorrhagic fever, viral pneumonia, west Nile fever, white epidemic, yersinia pseudotuberculosis infection, yersinia disease, yellow fever, zygomycosis.

The binding molecules of the invention, preferably antibodies, may be against pathogenic strains including, but not limited to, acinetobacter baumannii, actinobacillus israeli, actinobacillus goides and Actinobacillus propionicus, trypanosoma brucei, HIV (human immunodeficiency Virus), america dysentery, leptospermum, bacillus anthracis, vibrio haemolyticus, huning Virus, ascaris, rhizoviridae, babesia, bacillus cereus, various bacteria, bacteroides, escherichia coli, abelmoschus, BK virus, manodulation bacteria, blakeslea, mikrootheca dermatitis, ma Chapu virus, bos, botulinum, saba, brucella, burkholderia cepacia and other Burkholderia species, mycobacterium ulcerans, callicarpae, campylobacter, candida albicans and other candida species, bartonella, streptococcus parathyroides and staphylococci, trypanosoma, xenoptera, varicella Zoster Virus (VZV), chlamydia trachomatis, vibrio cholerae, pei Shi phoma, chinese branch fluke, clostridium difficile, coccidioides, coulosa swine fever virus, rhinovirus, coronavirus, CJD prion protein, cryptococcus krigineus, cryptosporidium, brazilian hook worm; polysacharin, cyclosporin, taenias, cytomegalovirus, dengue viruses (den-1, den-2, den-3 and den-4), flaviviruses, friable binuclear amoeba, corynebacterium diphtheriae, candidiasis, taenias, echinococcus, ebola, elymus, enterococcus, enterovirus, rickettsia, pal Wo Bingdu B19, human herpesvirus 6 and human herpesvirus 7, fascioma, liver flukes and giant flukes, FFI prions, the finarioidea superfamily, clostridium perfringens; other clostridia, geotrichum candidum, GSS prions, giardia enterica, burkholderia, echinococci and echinococci, neisseria gonorrhoeae, klebsiella granulosa, streptococcus pyogenes, streptococcus agalactiae, haemophilus influenzae, enteroviruses, mainly coxsackie virus a and enterovirus 71, sin nomore virus, helicobacter pylori, escherichia coli O157: h7, bunyaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus type 1, herpes simplex virus type 2, histoplasmosis capsulata, duodenal adenocarcinoma and black neck gray ant, haemophilus influenzae, human Bokala virus, ehrlichia, hemophilus phagocytes, human metapneumovirus, ehrlichia, human papilloma virus, human parainfluenza virus, etstaenia and Emtheca breve, epstein-Barr virus, orthomyxoviridae, bessezia and other coccidium, gold-gerbera, klebsiella, kluyveromyces, kuru prions, laevigata, legionella pneumophila, leishmania, mycobacterium leptospiri and Mycobacterium, leptospira, listeria monocytogenes, borrelia burgdorferi and other borrelia species, ban Shi filaria and malaysia, lymphocytic choriomeningitis virus (LCMV), plasmodium, marburg virus, measles virus, pseudopseudopseudomeningitis, neisseria hyphae, ectopic myositis, microsporidia, mollusc infectious virus (MCV), mumps virus, typhoid rickettsia, mycoplasma pneumoniae, innumerable bacteria (actinomyces) and fungi (eupodophy), parasitic diptera larva, chlamydia trachomatis and gonorrhoeae, vCJD prions, nocardia and nocardia species, schistosome torsion, blastogenesis, merozoites and other merozoites, pasteella, head lice, body lice, yin lice, bordetella pertussis, yersinia pestis, streptococcus pneumoniae, jirooveii pneumococcus, poliovirus, prevotella, gray-tolerant, JC-virus, chlamydia psittaci, bennett rickettsia, rabies virus, candida, spirochete, respiratory syncytial virus, nasosporium, rhinovirus, rickettsia, riflesh-fever virus, rickettsia, rotavirus, rubella virus, salmonella, SARS coronavirus, scabies, schistosoma, shigella, varicella zoster virus, major smallpox virus or smallpox virus, sporozoites, staphylococcus aureus, streptococcus pyogenes, toxoid, trephine, treponema pallidum, ribbon, clostridium tetani, trichophyton, epidermomyces floccosum, trichophyton rubrum and Trichophyton mentagrophytes, trichophyton rubrum, trichophyton, malachillea, canine or toxoplasma, toxoplasma, trichinella, trichomonas vaginalis, trichomonas, mycobacterium tuberculosis, francisella, venezuela equine encephalitis, vibrio cholerae, guanlitupe virus, west Nile virus, trichosporon fuscum, yersinia pseudotuberculosis, yersinia enterocolitica, yellow fever virus, mucor (Mucor ycis) and arboobject mildew (Entomophthia-mycsis), pseudomonas aeruginosa, campylobacter (Vibrio) fetuses, aeromonas hydrophila, edwardsiella tarda, yersinia pestis, shigella dysenteriae, shigella sonnei, salmonella typhimurium, treponema, leucospira, leucopia icterus, leucopia angusta, pneumocystis californicus, brucella abortus, brucella, rickettsia praecox, rickettsia tsutsugamushi, chlamydia; pathogenic fungi (aspergillus fumigatus, candida albicans, histoplasma capsulatum); protozoa (Endomonas histolytica, trichomonas mandshurica, torula gambia, trypanosoma rotundifolia, leishmania leishmania, leishmania thermophila, leishmania brasiliensis, pneumosporoal pneumonia, plasmodium vivax, plasmodium falciparum); or Helminiths (Schistosoma japonicum, schistosoma mansoni, schistosoma japonicum and ancylostomiasis).

Other antibodies on conjugates useful in the treatment of viral diseases in the present invention include, but are not limited to, antibodies against the following pathogenic viral antigens: poxviruses, herpesviruses, adenoviruses, papillomaviruses, enteroviruses, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial viruses, rubella, arboviruses, rhabdoviruses, salmonella viruses, non-a/non-b hepatitis viruses, rhinoviruses, coronaviruses, roscovariates, oncological viruses (e.g., HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), kaposi's sarcoma-associated herpesviruses (kaposi sarcoma), epstein barr virus (nasopharyngeal carcinoma, burkitt lymphoma, primary central nervous system lymphoma), MCPyV (merker cell cancer), SV40 (simian virus 40), HCV (hepatocellular carcinoma), lv-1 (adult T-cell leukemia/lymphoma)), viruses caused by immune disorders (e.g., human immunodeficiency virus (aids)); central nervous system viruses (e.g., JCV (progressive multifocal leukoencephalopathy), meV (subacute sclerotic panencephalitis), LCV (lymphocytic choriomeningitis), arboviral encephalitis, orthomyxoviridae (possible) (epidemic type a encephalitis), RV (rabies), cadipra virus, herpes virus meningitis, lambda-hunter syndrome type II; polioviruses (poliomyelitis, post polio syndrome), HTLV-I (tropical spastic lower limb paresis)); cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)); cardiovascular viruses (e.g., CBV (pericarditis, myocarditis)); respiratory system/acute viral nasopharyngitis/viral pneumonia (epstein-barr virus (EBV infection/infectious mononucleosis), cytomegalovirus, SARS coronavirus (severe acute respiratory syndrome) orthomyxoviridae: influenza virus a/B/C (influenza/avian influenza), paramyxovirus: human parainfluenza virus (parainfluenza), RSV (human respiratory syncytial virus), hMPV); digestive system viruses (MuV (mumps), cytomegalovirus (cytomegalovirus esophagitis), adenovirus (adenovirus infection), rotavirus, norovirus, astrovirus, coronavirus, HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus)); genitourinary virus (e.g., BK virus, muV (mumps)).

The invention also includes pharmaceutical compositions consisting of the foregoing conjugates and a pharmaceutically acceptable carrier for use in the treatment of cancer and autoimmune disorders. Methods of treating cancer and autoimmune diseases may be performed in vitro, in vivo, or in vitro. Examples of in vitro uses include treating a cell culture with it to kill all cells except for variants that do not express a target antigen; or kill variants expressing unwanted antigens. Examples of in vitro uses include treatment of Hematopoietic Stem Cells (HSCs) to kill diseased or malignant cells prior to transplantation (HSCT). For example, tumor cells or lymphocytes are removed from bone marrow prior to autologous transplantation in cancer therapy or in the treatment of autoimmune diseases, or T cells and other lymphocytes are removed from allogeneic bone marrow or tissue prior to transplantation in order to prevent graft versus host disease. Such clinical in vitro treatments may be performed as follows: bone marrow is harvested from a patient or other individual and then incubated at about 37 ℃ for about 30 minutes to about 48 hours in a serum-containing medium to which the conjugate of the invention is added at a concentration ranging from about 1pM to 0.1mM. In particular the drug concentration and incubation time should be determined by a skilled clinician. After incubation, the bone marrow cells are washed with serum-containing medium and administered to the patient by intravenous injection according to known methods. If the patient receives additional therapy (e.g., ablative chemotherapy or systemic radiation therapy) between bone marrow harvesting and reinfusion of the therapeutic cells, the treated bone marrow cells should be stored in liquid nitrogen using standard medical equipment.

In clinical in vivo use, the conjugate will be supplied as a solution or as a lyophilized solid, which can be redissolved in sterile water for injection. Examples of conjugate administration regimens are as follows: the conjugate was given by intravenous injection once a week for 8 weeks. The bolus dose is administered in 50-500mL physiological saline to which human serum albumin (e.g., 0.5 to 1mL of a concentrated solution of human serum albumin, 100 mg/mL) may be added. Intravenous doses will be about 50 μg to 20mg/kg (body weight) per day, or weekly, bi-weekly, tri-weekly, or monthly (10 μg to 200mg/kg dose per injection). The patient can receive the second treatment course 4 to 20 weeks after the treatment. The specific clinical regimen regarding route of administration, excipients, diluents, dosages, numbers, etc., can be determined by a skilled clinician.

Examples of medical conditions that may be treated according to in vivo or in vitro methods of killing selected cell populations include malignant tumors of any type of cancer, autoimmune diseases, graft rejection and infections (viral, bacterial or parasitic).

The dosage of conjugate required to achieve the desired biological effect will vary with a variety of factors including the chemical nature, potency and bioavailability of the conjugate, the type of disease, the race of the patient, the disease state of development of the patient, and the route of administration; all factors determine the dosage, mode of administration and regimen desired.

In general, the conjugates of the invention are formulated for injection in an aqueous physiological buffer solution containing 0.1 to 10% w/v of the conjugate. Typical dosages range from 1 μg/kg to 1g/kg (body weight), 1 time per day, 1 time per week, 2 times per week, 3 times per week or once a month. The preferred dosage range is 0.01mg/kg to 20mg/kg body weight per day. The preferred dosage of the drug to be administered may depend on, for example, the type and extent of progression of the disease or disorder, the general health of the particular patient, the relative biological efficacy of the compound selected, the formulation of the drug, the route of administration (intravenous, intramuscular, or otherwise), the pharmacokinetic properties of the drug-prescribed delivery route, as well as the rate of administration (bolus or continuous infusion) and the dosing regimen (number of repetitions over a given period of time).

The conjugates of the present invention can also be administered in unit dosage form, wherein the term "unit dose" refers to a single dose that can be administered to a patient and can be easily handled and packaged while the active conjugate itself or a pharmaceutically acceptable composition as described below, maintains a physically and chemically stable dose. Typical total daily dosages range from 0.01 to 100mg/kg body weight. As a general guideline, a unit dose in humans ranges from 1mg to 3000mg per day or week, or 2 weeks, 3 weeks, or each month. The preferred unit dosage range is from 1 to 500mg, one to four times a day, more preferably from 10mg to 500mg, once a day. Conjugates provided herein may be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dose compositions may be tablets, simple capsules or soft gel capsules, for oral administration; or powder, nasal drops or aerosol, by intranasal administration; or an ointment, cream, lotion, gel or spray, by dermal or transdermal patch.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention is further illustrated by the following examples, the contents of which are not intended to limit the scope of the invention. In the examples, cell lines were maintained under the conditions specified by the American Type Culture Collection (ATCC) or the German type culture Collection (DSMZ), unless specified. Cell culture reagents were from Invitrogen corporation, except as specifically noted. All anhydrous reagents were obtained commercially and stored in nitrogen filled sealed bottles. Other reagents and solvents were purchased at the highest standard and used without further treatment. NMR data were obtained at Bruker 500MHz with chemical shifts in ppm and tetramethylsilane as reference (0 ppm) and coupling constants (J) in Hz. High resolution mass spectral data were obtained on a Waters XevoQTof mass spectrometer (by Waters Acquity UPLC high performance liquid chromatography).

Example 1 preparation of Compound 2

Boc-L-proline (10.0 g,46.4 mmol) was dissolved in 50mL tetrahydrofuran and cooled to 0deg.C, to which BH was added ₃ Tetrahydrofuran (1.0M, 46.4 mL). The reaction was stirred at 0℃for 1.5 hours, then poured into ice water, and extracted with ethyl acetate. The organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give compound 2 (8.5 g, yield 91%) as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ3.94(dd,J＝4.9,2.7Hz,2H),3.60(ddd,J＝18.7,11.9,9.3Hz,2H),3.49–3.37(m,1H),3.34–3.23(m,1H),2.06–1.91(m,1H),1.89–1.69(m,2H),1.65–1.51(m,1H),1.49–1.40(m,9H)。

Example 2 preparation of Compound 3

To a solution of compound 2 (13.0 g,64.6 mmol) in dimethyl sulfoxide (90 mL) was added triethylamine (40 mL) and stirring was continued for 15 min. The mixture was cooled in an ice bath and sulfur trioxide-pyri-dine was added in portions over 40 minutesPyridine complex (35.98 g,226 mmol). The reaction was warmed to room temperature and stirred for 2.5 hours. After ice (250 g) was added, the mixture was extracted with dichloromethane (150 mL. Times.3). The organic phase was washed with 50% citric acid solution (150 mL), water (150 mL), saturated sodium bicarbonate solution (150 mL) and brine (150 mL), and dried over anhydrous sodium sulfate. Filtration and distillation under reduced pressure gave aldehyde 3 (10.4 g, 81% yield) as a thick oil, which was used without further purification. ¹ H NMR(500MHz,CDCl ₃ )δ9.45(s,1H),4.04(s,1H),3.53(dd,J＝14.4,8.0Hz,2H),2.00–1.82(m,4H),1.44(d,J＝22.6Hz,9H)。

Example 3 preparation of Compound 5

N ₂ A solution of n-butyllithium in n-hexane (21.6 mL,2.2M,47.43 mmol) was added dropwise to 4-methyl-5-phenyloxazolidin-2-one (8.0 g,45.17 mmol) in tetrahydrofuran (100 mL) at-78deg.C under protection. The solution was stirred at-78 ℃ for 1 hour, then propionyl chloride (4.4 ml,50.59 mmol) was slowly added. The reaction mixture was naturally warmed to-50 ℃, stirred for 2 hours, and then quenched by addition of saturated ammonium chloride solution (100 mL). The organic solvent was distilled off under reduced pressure, and the residual solution was extracted with ethyl acetate (3X 100 mL). The organic layer was washed with saturated sodium bicarbonate solution (100 mL) and brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (20% ethyl acetate/n-hexane) to give compound 5 as a thick oil (10.5 g,98% yield). ¹ H NMR(500MHz,CDCl ₃ )δ7.45–7.34(m,3H),7.30(d,J＝7.0Hz,2H),5.67(d,J＝7.3Hz,1H),4.82–4.70(m,1H),2.97(dd,J＝19.0,7.4Hz,2H),1.19(t,J＝7.4Hz,3H),0.90(d,J＝6.6Hz,3H)。

EXAMPLE 4 preparation of Compound 6

To compound 5 (9.40 g,40.4 mmol) dichloro at 0deg.CEt is added to a solution of methane (60 mL) ₃ N (6.45 mL,46.64 mmol) followed by 1M solution of dibutyl boron trifluoro methane sulfonate in dichloromethane (42 mL,42 mmol). The mixture was stirred at 0deg.C for 45 min, cooled to-70deg.C, then a solution of aldehyde 3 (4.58 g,22.97 mmol) in dichloromethane (40 mL) was slowly added thereto over 30 min. The reaction was stirred at-70℃for 2 hours, 0℃for 1 hour, and at room temperature for 15 minutes, then quenched with phosphate buffer solution (pH 7,38 mL). Methanol-30% hydrogen peroxide (2:1 v/v,100 mL) was added below 10deg.C, after stirring for 20 min, water (100 mL) was added and the mixture concentrated in vacuo. To the concentrate was added more water (200 mL) and extracted with ethyl acetate (3X 100 mL). The organic layer was treated with 1N KHSO ₄ (100 mL), sodium bicarbonate solution (100 mL) and brine (100 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was purified by flash column chromatography (10% -50% ethyl acetate/n-hexane) to give 6 (7.10 g,71% yield) as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ7.39(dt,J＝23.4,7.1Hz,3H),7.30(d,J＝7.5Hz,2H),5.67(d,J＝7.1Hz,1H),4.84–4.67(m,1H),4.08–3.93(m,3H),3.92–3.84(m,1H),3.50(d,J＝9.0Hz,1H),3.24(d,J＝6.7Hz,1H),2.15(s,1H),1.89(dd,J＝22.4,14.8Hz,3H),1.48(d,J＝21.5Hz,9H),1.33(d,J＝6.9Hz,3H),0.88(d,J＝6.4Hz,3H)。

EXAMPLE 5 preparation of Compound 7

To compound 6 (5.1 g,11.9 mmol) and molecular sieves under nitrogen 5g) Dichloroethane (30 mL) was added to the mixture. Stirring was carried out at room temperature for 20 minutes and cooled to 0℃and then proton sponge (6.62 g,30.9 mmol) was added followed by trioxynium tetrafluoroborate (4.40 g,29.7 mmol). Stirring was continued at 0℃for 2 hours and at room temperature for 48 hours. The reaction mixture was filtered, the filtrate was concentrated and purified by column chromatography (20-70% ethyl acetate/n-hexane) to give compound 7 as a white solid(1.80 g,35% yield). ¹ H NMR(500MHz,CDCl ₃ )δ7.46–7.27(m,5H),5.65(s,1H),4.69(s,1H),3.92(s,1H),3.83(s,1H),3.48(s,3H),3.17(s,2H),2.02–1.68(m,5H),1.48(d,J＝22.3Hz,9H),1.32(t,J＝6.0Hz,3H),0.91–0.84(m,3H)。/>

EXAMPLE 6 preparation of Compound 8

To a solution of compound 7 (1.80 g,4.03 mmol) in tetrahydrofuran (30 mL) and water (7.5 mL) was added 30% hydrogen peroxide (1.44 mL,14.4 mmol) followed by lithium hydroxide (0.27 g,6.45 mmol) in water (5 mL) at 0deg.C for 5 min. After stirring at 0deg.C for 3 hours, 1N sodium sulfite (15.7 mL) was added, the mixture was warmed to room temperature, and stirred overnight. Tetrahydrofuran was distilled off under reduced pressure, and the aqueous phase was washed with methylene chloride (3X 50 mL) to remove oxazolidinone. The aqueous phase was acidified to pH 3 with 1N hydrochloric acid and extracted with ethyl acetate (3X 50 mL). The organic layer was washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound 8 as a colorless oil (1.15 g,98% yield). ¹ H NMR(500MHz,CDCl ₃ )δ3.99–3.74(m,2H),3.44(d,J＝2.6Hz,3H),3.23(s,1H),2.60–2.45(m,1H),1.92(tt,J＝56.0,31.5Hz,3H),1.79–1.69(m,1H),1.58–1.39(m,9H),1.30–1.24(m,3H)。

EXAMPLE 7 preparation of Compound 11

To an ice-cold solution of compound 9 (4.55 g,19.67 mmol) in tetrahydrofuran (20 mL) was added 1,1' -carbonyldiimidazole (3.51 g,21.63 mmol). After the evolution of gas ceased, the resulting mixture was stirred at room temperature for 3.5 hours. A solution of freshly prepared isopropyl magnesium bromide in tetrahydrofuran (123 mmol,30 mL) was added dropwise to pre-chilled (0 ℃) ethyl hydrogen malonate (6.50 g,49.2 mmol) keeping the internal temperature below 5 ℃. After stirring at room temperature for 1.5 hours, the magnesium enolate solution wasCooled in an ice-water bath, followed by the addition of a solution of the imidazolide at 0 ℃ over 1 hour via a double ended needle. The resulting mixture was stirred at 0 ℃ for 30 minutes, then at room temperature for 64 hours. The reaction was quenched by the addition of 10% aqueous citric acid (5 mL) and acidified to pH 3 with another 10% aqueous citric acid (110 mL). Extraction with ethyl acetate (150 mL. Times.3), washing of the organic extract with water (50 mL), saturated aqueous sodium bicarbonate (50 mL) and saturated aqueous sodium chloride (50 mL), and washing with Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The concentrate was purified by silica gel column chromatography using ethyl acetate/n-hexane (1:4) as an eluent to give compound 11 (5.50 g, yield 93%). ¹ H NMR(500MHz,CDCl ₃ )δ5.04(d,J＝7.8Hz,1H),4.20(p,J＝7.0Hz,3H),3.52(t,J＝10.7Hz,2H),1.96(d,J＝3.7Hz,1H),1.69(s,2H),1.44(s,9H),1.28(dd,J＝7.1,2.9Hz,3H),0.98(t,J＝6.9Hz,3H),0.92–0.86(m,3H)。

EXAMPLE 8 preparation of Compound 12a

To a solution of Compound 11 (5.90 g,19.83 mmol) in ethanol (6 mL) at-60℃was added sodium borohydride (3.77 g,99.2 mmol) in one portion. The reaction mixture was stirred below-55 ℃ for 5.5 hours, then quenched with 10% aqueous citric acid (100 mL) and the resulting solution was acidified to pH 2 with 10% aqueous citric acid and then extracted with ethyl acetate (100 mL x 3). The organic extract was washed with saturated aqueous sodium chloride (100 mL), dried over sodium sulfate and concentrated under reduced pressure. The concentrate was purified by column chromatography (10-50% ethyl acetate/n-hexane) to give pure diastereomer 12a (2.20 g,37% yield) and a mixture of 12a and 12b (2.0 g,34% yield, about 9:1 ratio). ¹ H NMR(500MHz,CDCl ₃ )δ4.41(d,J＝9.3Hz,1H),4.17(tt,J＝7.1,3.6Hz,2H),4.00(t,J＝6.9Hz,1H),3.55(dd,J＝11.7,9.3Hz,1H),2.56–2.51(m,2H),2.44(dd,J＝16.4,9.0Hz,1H),1.79(d,J＝3.8Hz,1H),1.60–1.53(m,1H),1.43(s,9H),1.27(dd,J＝9.3,5.0Hz,3H),1.03–0.91(m,7H)。

EXAMPLE 9 preparation of Compound 13

To a solution of compound 12a (2.20 g,7.20 mmol) in ethanol (22 mL) was added 1N aqueous sodium hydroxide (7.57 mL,7.57 mmol). The mixture was stirred at 0 ℃ for 30 minutes, then at room temperature for 2 hours. The resulting solution was acidified to pH 4 by the addition of 1N hydrochloric acid and then extracted with ethyl acetate (50 mL. Times.3). The organic extract was washed with a 1N aqueous potassium hydrogen sulfate solution (50 mL) and a saturated aqueous sodium chloride solution (50 mL), dried over sodium sulfate, and concentrated under reduced pressure to give compound 13 (1.90 g,95% yield). ¹ H NMR(500MHz,CDCl ₃ )δ4.50(d,J＝8.7Hz,1H),4.07(d,J＝5.5Hz,1H),3.59(d,J＝8.3Hz,1H),2.56–2.45(m,2H),1.76–1.65(m,1H),1.56(d,J＝7.1Hz,1H),1.45(s,9H),1.26(t,J＝7.1Hz,3H),0.93(dd,J＝14.4,7.1Hz,6H)。

EXAMPLE 10 preparation of Compound 14

To a solution of compound 13 (1.90 g,6.9 mmol) in tetrahydrofuran (40 mL) was added sodium hydride (60% oil suspension, 1.93g,48.3 mmol) at 0deg.C. After stirring for 1 hour, methyl iodide (6.6 mL,103.5 mmol) was added. Stirring was continued at 0deg.C for 40 hours, then saturated aqueous sodium bicarbonate (50 mL) was added followed by water (100 mL). The mixture was washed with diethyl ether (50 mL. Times.2), and the aqueous layer was acidified to pH 3 with 1N aqueous potassium hydrogen sulfate, followed by extraction with ethyl acetate (50 mL. Times.3). The combined organic extracts were washed with 5% aqueous sodium thiosulfate (50 mL) and saturated aqueous sodium chloride (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound 14 (1.00 g,48% yield). ¹ H NMR(500MHz,CDCl ₃ )δ3.95(d,J＝75.4Hz,2H),3.42(d,J＝4.4Hz,3H),2.71(s,3H),2.62(s,1H),2.56–2.47(m,2H),1.79(s,1H),1.47(s,1H),1.45(d,J＝3.3Hz,9H),1.13–1.05(m,1H),0.96(d,J＝6.7Hz,3H),0.89(td,J＝7.2,2.5Hz,3H)。

General reaction procedure for removal of Boc group with trifluoroacetic acid:

to a solution of N-Boc amino acid (1.0 mmol) in dichloromethane (2.5 mL) was added trifluoroacetic acid (1.0 mL). After stirring at room temperature for 1-3 hours, the reaction mixture was concentrated under reduced pressure. After adding toluene to the mixture for three times, the mixture was used without further purification.

Example 11 preparation of Compound 15

To a solution of Compound 8 (100 mg,0.347 mmol) and L-phenylalanine methyl ester hydrochloride (107.8 mg,0.500 mmol) in DMF (5 mL) was added diethyl cyanophosphonate (75.6. Mu.L, 0.451 mmol) followed by Et at 0deg.C ₃ N (131. Mu.L, 0.94 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight.

The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give 15 (130 mg,83% yield) as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ7.28(dd,J＝7.9,6.5Hz,2H),7.23(t,J＝7.3Hz,1H),7.16(s,2H),4.81(s,1H),3.98–3.56(m,5H),3.50(s,1H),3.37(d,J＝2.9Hz,3H),3.17(dd,J＝13.9,5.4Hz,2H),3.04(dd,J＝14.0,7.7Hz,1H),2.34(s,1H),1.81–1.69(m,2H),1.65(s,3H),1.51–1.40(m,9H),1.16(d,J＝7.0Hz,3H)。

EXAMPLE 12 preparation of Compound 16

To a solution of the deprotected product of compound 15 (0.29 mmol) and compound 14 (96.6 mg,0.318 mmol) in DMF (5 mL) was added diethyl cyanophosphate (58. Mu.L, 0.347 mmol) followed by Et at 0deg.C ₃ N (109. Mu.L, 0.78 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate and concentrated under reduced pressure. The concentrate was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give 16 (150 mg,81% yield) as a white solid. LC-MS (ESI) m/z C ₃₄ H ₅₅ N ₃ O ₈ [M+H] ⁺ Calculated 634.40, found 634.40.

EXAMPLE 13 preparation of Compound 17

To a solution of the deprotected product of compound 16 (0.118 mmol) and Boc-Val-OH (51.8 mg,0.236 mmol) in dichloromethane (5 mL) was added bromotris (dimethylamino) -phosphonium hexafluorophosphate (BroP, 70.1mg,0.184 mmol) followed by diisopropylethylamine (70. Mu.L, 0.425 mmol) at 0deg.C. The mixture was stirred at 0℃for 30 minutes under protection from light, then at room temperature for 2 days, diluted with ethyl acetate (80 mL), washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), and washed with Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (20-100% ethyl acetate/n-hexane) to give 17 (67 mg,77% yield) as a white solid. LC-MS (ESI) m/z C ₃₉ H ₆₄ N ₄ O ₉ [M+H] ⁺ Calculated 733.47, found 733.46.

EXAMPLE 14 preparation of the Compound Boc-N-Me-Val-OH

To a solution of Boc-L-Val-OH (2.00 g,9.2 mmol) and methyl iodide (5.74 mL,92 mmol) in anhydrous tetrahydrofuran (40 mL) was added sodium hydride (3.68 g,92 mmol) at 0deg.C. Mixing the reactionThe mixture was stirred at 0℃for 1.5 hours, then warmed to room temperature and stirred for 24 hours. The reaction mixture was quenched with ice water (50 mL), water (100 mL) was added, and the reaction mixture was washed with ethyl acetate (50 mL. Times.3), acidified to pH3 with aqueous solution, and extracted with ethyl acetate (50 mL. Times.3). The combined organic phases were dried over sodium sulfate, filtered and concentrated to give Boc-N-Me-Val-OH as a white solid (2.00 g,94% yield). ¹ H NMR(500MHz,CDCl ₃ )δ4.10(d,J＝10.0Hz,1H),2.87(s,3H),2.37–2.13(m,1H),1.44(d,J＝26.7Hz,9H),1.02(d,J＝6.5Hz,3H),0.90(t,J＝8.6Hz,3H)。

EXAMPLE 15 preparation of Compound 18

To a solution of the deprotected product of compound 17 (0.091 mmol) and Boc-N-Me-Val-OH (127 mg,0.548 mmol) in DMF (5 mL) was added diethyl cyanophosphonate (18.2. Mu.L, 0.114 mmol) and 4-methylmorpholine (59. Mu.L, 0.548 mmol) at 0deg.C. The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by chromatography (20-100% ethyl acetate/n-hexane) to give 18 (30 mg,39% yield) as a white solid. LC-MS (ESI) m/z C ₄₅ H ₇₅ N ₅ O ₁₀ [M+H] ⁺ Calculated 846.55, found 846.56.

EXAMPLE 16 preparation of Compound 19

To a solution of the deprotected product of compound 18 (0.071 mmol) and 4-maleimide butyric acid (26 mg,0.142 mmol) in dichloromethane (5 mL) at 0deg.C was added BroP (81 mg,0.213 mmol) followed by diisopropylethylamine 42 μl,0.256 mmol). The mixture is mixedStirred at 0℃for 30 minutes in the absence of light, and at room temperature for 2 days. The reaction mixture was diluted with ethyl acetate (40 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (50% ethyl acetate/n-hexane to 10% methanol/ethyl acetate) to give white solid 19 (19 mg,29% yield). LC-MS (ESI) m/z C ₄₈ H ₇₄ N ₆ O ₁₁ [M+H] ⁺ Calculated 911.54, found 911.53.

EXAMPLE 17 preparation of Compound 20

To a solution of compound 8 (58 mg,0.201 mmol) and (4R) -methyl-4-amino-2-methyl-5-phenylpentanoate (64 mg,0.29 mmol) in DMF (5 mL) was added diethyl cyanophosphonate (43.9. Mu.L, 0.262 mmol) followed by Et at 0deg.C ₃ N (76. Mu.L, 0.544 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give 20 (83 mg,84% yield) as a white solid. LC-MS (ESI) m/z C ₂₇ H ₄₂ N ₂ O ₆ [M+H] ⁺ Calculated 491.30, found 491.29.

EXAMPLE 18 preparation of Compound 21

To a solution of the deprotected product of compound 20 (0.17 mmol) and compound 14 (57 mg,0.187 mmol) in DMF (5 mL) at 0deg.C was added diethyl cyanophosphate (35 μL,0.204 mmol) and Et ₃ N (64. Mu.L, 0.459 mmol). The reaction mixture is reacted in the presence ofStirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give 21 (100 mg,87% yield) as a white solid. LC-MS (ESI) m/z C ₃₇ H ₆₁ N ₃ O ₈ [M+H] ⁺ Calculated 676.45, found 676.43.

EXAMPLE 19 preparation of Compound 22

To a solution of the deprotected product of compound 21 (0.124 mmol) and Boc-Val-OH (77 mg,0.354 mmol) in dichloromethane (5 mL) at 0deg.C was added BroP (68.5 mg,0.176 mmol) followed by diisopropylethylamine (96 μL,0.584 mmol). The mixture was stirred at 0℃for 30 min under protection from light, then at room temperature for 24 h. TLC analysis indicated that starting material was still present. More Boc-Val-OH (77 mg), broP (68.5 mg) and diisopropylethylamine (96. Mu.L) were added and the reaction stirred for an additional 24 hours, then diluted with ethyl acetate (80 mL), washed with 1N aqueous potassium hydroxide sulfate (40 mL), water (40 mL), saturated aqueous sodium bicarbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (20-100% ethyl acetate/n-hexane) to give 22 (65 mg,67% yield) as a white solid. LC-MS (ESI) m/z C ₄₂ H ₇₀ N ₄ O ₉ [M+H] ⁺ Calculated 775.51, found 775.49.

EXAMPLE 20 preparation of Compound 23

Deprotection product of Compound 22 (0.084 mmol) and Boc-N-Me-Val-OH (116 mg,0.503 mm)To a solution of ol) in DMF (5 mL) was added diethyl cyanophosphonate (16.8. Mu.L, 0.105 mmol) and 4-methylmorpholine (54. Mu.L, 0.503 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (80 mL), which was washed with 1N aqueous potassium hydrogen sulfate (40 mL), water (40 mL), saturated aqueous sodium hydrogen carbonate (40 mL) and saturated aqueous sodium chloride (40 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (20-100% ethyl acetate/n-hexane) to give 23 (60 mg,80% yield) as a white solid. LC-MS (ESI) m/z C ₄₈ H ₈₁ N ₅ O ₁₀ [M+H] ⁺ Calculated 888.60, found 888.60.

EXAMPLE 21 preparation of Compound 24

To a solution of the deprotected product of compound 23 (0.034 mmol) and 4-maleimidobutyric acid (17.7 mg,0.097 mmol) in dichloromethane (5 mL) at 0deg.C was added BroP (18.7 mg,0.0483 mmol) and diisopropylethylamine (26 μL,0.159 mmol). The mixture was stirred at 0℃for 30 minutes in the absence of light and then at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (40 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (50% ethyl acetate/n-hexane to 10% methanol/ethyl acetate) to give 24 (23 mg,70% yield) as a white solid. LC-MS (ESI) m/z C ₅₁ H ₈₀ N ₆ O ₁₁ [M+H] ⁺ Calculated 953.59, found 953.63.

EXAMPLE 22 preparation of Compound 25

A mixture of L-phenylalanine methyl ester hydrochloride (7.90 g,36.6 mmol), di-tert-butyl dicarbonate (12.9 g), methylene chloride (100 mL) and saturated aqueous sodium bicarbonate (80 mL) was stirred at room temperature for 2 hours. The organic phase was separated and the aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated to give a colorless oil (9.76 g,95% yield). The crude product was used directly in the next step.

EXAMPLE 23 preparation of Compound 26

A solution of Boc-L-phenylalanine methyl ester (9.76 g,35.0 mmol) in methylene chloride (60 mL) was cooled to-65℃and DIBAL-H (1.0M in n-hexane, 70 mL) was slowly added over 1 hour. After stirring for 2 hours, the reaction was quenched with methanol (20 mL) and warmed to room temperature. 1N hydrochloric acid (120 mL) was then added and the organic phase was separated. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with brine, dried over sodium sulfate, filtered and concentrated to give a colorless oil (7.0 g,80% yield). The crude product was used directly in the next step.

EXAMPLE 24 preparation of Compound 27

Aldehyde (7.0 g,28 mmol) and methyl (triphenylphosphine) acetate (11.7 g,35 mmol) were stirred in dichloromethane (100 mL) at room temperature for 2 hours. After removal of the solvent, the concentrate was purified by column chromatography (85% ethyl acetate/n-hexane) to give 27 (6.9 g,81% yield) as a white solid. ¹ H NMR(500MHz,CDCl ₃ )δ7.32–7.28(m,2H),7.24(dt,J＝4.2,1.8Hz,1H),7.18–7.14(m,2H),6.91(dd,J＝15.7,4.9Hz,1H),5.86(dd,J＝15.7,1.3Hz,1H),4.60(s,1H),4.58(s,1H),3.72(s,3H),2.89(d,J＝6.4Hz,2H),1.39(s,9H)。

EXAMPLE 25 preparation of Compound 28

Et is added to a solution of amine 27 (0.194 mmol) in DMF (2 mL) at 0deg.C ₃ N (54.1. Mu.L, 0.388 mmol) followed by a solution of compound 8 (37.2 mg,0.129 mmol) in DMF (1 mL) and diethyl cyanophosphonate (29.0. Mu.L, 0.194 mmol). The reaction mixture was stirred at 0 ℃ for 30 minutes, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (30-50% ethyl acetate/n-hexane) to give compound 28 (58.2 mg,95% yield) as a colorless oil. LC-MS (ESI) m/z C ₂₆ H ₃₈ N ₂ O ₆ [M+H] ⁺ Calculated 475.27, found 475.26.

EXAMPLE 26 preparation of Compound 29

Et is added to a solution of deprotected product of compound 28 (0.123 mmol) in DMF (2 mL) at 0deg.C ₃ N (51.4. Mu.L, 0.369 mmol) followed by a solution of compound 14 (37.3 mg,0.123 mmol) in DMF (1 mL) and diethyl cyanophosphonate (22.1. Mu.L, 0.148 mmol). The reaction mixture was stirred at 0 ℃ for 30 minutes, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (70-90% ethyl acetate/n-hexane) to give compound 29 (81.0 mg,99% yield) as a pale yellow oil. LC-MS (ESI) m/z C ₃₆ H ₅₇ N ₃ O ₈ [M+H] ⁺ Calculated 660.41, found 660.41.

EXAMPLE 27 preparation of Compound 30

To a solution of the deprotected product of compound 29 (0.123 mmol) and Boc-Val-OH (80.2 mg,0.369 mmol) in dichloromethane (5 mL) was added BroP (71.6 mg,0.185 mmol) followed by diisopropylethylamine (107.4. Mu.L, 0.615 mmol) at 0deg.C. The mixture was stirred at 0℃for 30 minutes in the absence of light, and then at room temperature for 24 hours. TLC analysis showed the starting material still present. More Boc-Val-OH (80.2 mg), broP (71.6 mg) and diisopropylethylamine (107.4. Mu.L) were added, the reaction stirred for an additional 24 hours, then diluted with ethyl acetate (50 mL), washed with 1N aqueous potassium hydroxide sodium sulfate (20 mL), water (20 mL), saturated aqueous sodium bicarbonate (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The concentrate was purified by column chromatography to give white foam 30 (86.3 mg,92% yield). LC-MS (ESI) m/z C ₄₁ H ₆₆ N ₄ O ₉ [M+H] ⁺ Calculated 759.48, found 759.48.

EXAMPLE 28 preparation of Compound 31

To a solution of the deprotected product of compound 30 (0.107 mmol) in DMF (2 mL) was added 4-methylmorpholine (70.6. Mu.L, 0.642 mmol) followed by a solution of Boc-N-Me-Val-OH (148.5 mg, 0.640 mmol) in DMF (1 mL) and diethyl cyanophosphonate (32.0. Mu.L, 0.214 mmol) at 0deg.C. The reaction mixture was stirred at 0 ℃ for 30 minutes, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (75% ethyl acetate/n-hexane) to give compound 31 (57.9 mg,62% yield) as a colorless oil. LC-MS (ESI) m/zC ₄₇ H ₇₇ N ₅ O ₁₀ [M+H] ⁺ Calculated 872.57, found 872.57.

EXAMPLE 29 preparation of Compound 32

To a solution of compound 31 (0.0316 mmol) and 4-maleimide butyric acid (17.4 mg,0.0949 mmol) in dichloromethane (3 mL) at 0deg.C was added BroP (18.4 mg,0.0474 mmol) followed by diisopropylethylamine (27.6 μL,0.158 mmol). The mixture was stirred at 0℃for 30 minutes in the absence of light, and then at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (50 mL), washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL) and saturated aqueous sodium chloride (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (pure ethyl acetate to 10% methanol/ethyl acetate) to give 32 (30.0 mg,99% yield) as a white solid. LC-MS (ESI) m/z C ₅₀ H ₇₆ N ₆ O ₁₁ [M+H] ⁺ Calculated 938.17, found 938.17.

EXAMPLE 30 preparation of Compound 33

Preparation of (S, E) -4-amino-2-methyl-5-phenylpent-2-enoic acid methyl ester the procedure described in example 24 was followed starting from L-Phe-OMe.

To a solution of compound 8 (32 mg,0.112 mmol) and methyl (S, E) -4-amino-2-methyl-5-phenylpent-2-enoate (34 mg,0.157 mmol) in DMF (5 mL) was added diethyl cyanophosphonate (23. Mu.L, 0.146 mmol) and Et at 0deg.C ₃ N (42. Mu.L, 0.302 mmol). The reaction mixture was stirred at 0 ℃ for 2 hours, then warmed to room temperature and stirred overnight. The reaction mixture was diluted with ethyl acetate (50 mL), which was washed with 1N aqueous potassium hydrogen sulfate (20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated aqueous sodium chloride (20 mL), and the organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography (15-75% ethyl acetate/n-hexane) to give 33 (54 mg, 98%) as a white solidYield). LC-MS (ESI) m/z C ₂₇ H ₄₀ N ₂ O ₆ [M+H] ⁺ Calculated 489.29, found 489.28.

EXAMPLE 28 preparation of Compound 31

Compound 30 (0.107 mol) was dissolved in DMF (2 mL), 4-methylmorpholine (70.6. Mu.L, 0.642 mmol) was added at 0deg.C followed by a solution of Boc-N-Me-Val-OH (148.5 mg, 0.640 mmol) in DMF (1 mL) and diethyl cyanophosphate (32.0. Mu.L, 0.214 mmol) was added. The reaction was carried out at 0℃for 30min, then warmed to room temperature and stirred overnight. The reaction solution was diluted with ethyl acetate (50 mL), potassium hydrogen sulfate (1 n,20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by a silica gel column (75% ethyl acetate/n-hexane) to give compound 31 (57.9 mg, 62%) as a colorless oil. LC-MS (ESI) m/z C ₄₇ H ₇₇ N ₅ O ₁₀ [M+H] ⁺ Calculated 872.57, found 872.57.

EXAMPLE 29 preparation of Compound 32

Compound 31 (0.0316 mmol) and maleimide butyric acid (17.4 mg,0.049 mmol) were dissolved in DCM (3 mL), broP (18.4 mg,0.0474 mmol) and DIPEA (27.6. Mu.L, 0.158 mmol)) were added at 0deg.C, the reaction was carried out in the dark at 0deg.C for 30min, then warmed to room temperature, reacted for 16 h, the reaction solution was diluted with ethyl acetate (50 mL), potassium bisulfate (1N, 20 mL), water (20 mL), saturated sodium chloride (20 mL) washed, dried over anhydrous sodium sulfate, concentrated in vacuo, silica gel column purified (ethyl acetate/methanol=1 to 0.1) to give compound 32 (30.0 mg, 99%) as a white solid LC-MS (ESI) m/z C) ₅₀ H ₇₆ N ₆ O ₁₁ [M+H] ⁺ Calculated 938.17, found 938.17.

EXAMPLE 30 preparation of Compound 33

(S, Z) methyl 4-amino-2-methyl-5-phenyl-2-pentenoate was prepared from L-Phe-OMe in the same manner as in example 24.

Compound 8 (32 mg,0.112 mmol) and (S, Z) 4-amino-2-methyl-5-phenylpentanoate (34 mg,0.157 mmol) were dissolved in DMF (5 mL). To this was added diethyl cyanophosphate (23 μl,0.146 mmol) at 0deg.C, followed by triethylamine (42 μl,0.302 mmol). The reaction was carried out at 0℃for 2 hours, then warmed to room temperature, reacted for 16 hours, the reaction solution was diluted with ethyl acetate (50 mL), potassium hydrogen sulfate (1N, 20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated sodium chloride (20 mL) were washed, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by a silica gel column (15-75% ethyl acetate/n-hexane) to give compound 33 (54 mg, 98%) as a white solid. LC-MS (ESI) m/zC ₂₇ H ₄₀ N ₂ O ₆ [M+H] ⁺ Calculated 489.29, found 489.28.

EXAMPLE 31 preparation of Compound 34

Compound 33 (0.112 mmol) and compound 14 (37.6 mg,0.124 mmol) were dissolved in DMF (5 mL). Diethyl cyanophosphate (21. Mu.L, 0.135 mmol) and triethylamine (42. Mu.L, 0.304 mmol) were added at 0deg.C. The reaction was carried out at 0℃for 2 hours, then warmed to room temperature, reacted for 16 hours, the reaction solution was diluted with ethyl acetate (50 mL), potassium hydrogen sulfate (1N, 20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated sodium chloride (20 mL) were washed, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by a silica gel column (15-75% ethyl acetate/n-hexane) to give compound 34 (61 mg, 80%) as a white solid. LC-MS (ESI) m/z C ₃₇ H ₅₉ N ₃ O ₈ [M+H] ⁺ Calculated 674.43, found 674.43.

Example 32 preparation of Compound 35

Compound 34 (0.091 mmol) and Boc-Val-OH (56 mg,0.258 mmol) were dissolved in DCM (5 mL), broP (50 mg,0.129 mmol) and DIPEA (70. Mu.L, 0.426 mmol) were added at 0deg.C, the reaction was carried out at 0deg.C in the absence of light for 30min, then warmed to room temperature and reacted for 16 hours. TLC showed that the starting material was not fully reacted, supplemented with Boc-Val-OH (56 mg), broP (50 mg) and DIPEA (70 μl), and the reaction was continued for 24 hours, the reaction solution was diluted with ethyl acetate (50 mL), washed with potassium bisulfate (1 n,20 mL), water (20 mL), saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified on a silica gel column (ethyl acetate/n-hexane=0.2 to 1) to give compound 35 (62 mg, 88%) as a white solid. LC-MS (ESI) m/z C ₄₂ H ₆₈ N ₄ O ₉ [M+H] ⁺ Calculated 773.50, found 773.50.

EXAMPLE 33 preparation of Compound 36

Compound 35 (0.0804 mol) and Boc-N-Me-Val-OH (111.4 mg, 0.480 mmol) were dissolved in DMF (5 mL), diethyl cyanophosphate (16.0. Mu.L, 0.100 mmol) and 4-methylmorpholine (52. Mu.L, 0.482 mmol) were added at 0deg.C, and after 2 hours the reaction was allowed to warm to room temperature and reacted for 16 hours. The reaction solution was diluted with ethyl acetate (50 mL), potassium hydrogen sulfate (1 n,20 mL), water (20 mL), saturated aqueous sodium hydrogen carbonate (20 mL) and saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by a silica gel column (20% -100% ethyl acetate/n-hexane) to give compound 36 (56 mg, 78%) as a colorless oil. LC-MS (ESI) m/z C ₄₈ H ₇₉ N ₅ O ₁₀ [M+H] ⁺ Calculated 886.58, found 886.59.

EXAMPLE 34 preparation of Compound 37

Compound 36 (0.0316 mmol) and maleimide butyric acid (16.5 mg,0.090 mmol) were dissolved in DCM (5 mL), broP (17.4 mg,0.0450 mmol) and DIPEA (24. Mu.L, 0.149 mmol) were added at 0deg.C, the reaction was carried out at 0deg.C in the absence of light for 30min, then warmed to room temperature and reacted for 24 hours. The reaction solution was diluted with ethyl acetate (50 mLl), which was washed with potassium hydrogen sulfate (1 n,20 mL), water (20 mL), saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by a silica gel column (50% ethyl acetate/n-hexane-10% methanol/ethyl acetate) to give compound 37 (15 mg, 50%) as a white solid. LC-MS (ESI) m/z C ₅₁ H ₇₈ N ₆ O ₁₁ [M+H] ⁺ Calculated 951.57, found 951.58.

EXAMPLE 35 preparation of Compound 38

A (0.18 g,0.722 mmol) and b (0.31 g,0.866 mmol) were dissolved in DCM (3.6 mL), stirred at room temperature for 16 hours and concentrated, purified on a silica gel column (20% ethyl acetate/n-hexane) to give compound 38 as a white solid (0.19 g, 80%).

EXAMPLE 36 preparation of Compound 39

Compound 38 (0.19 g,0.57 mmol) was dissolved in DCM (6 mL), m-CPBA (75 wt%,0.22g,0.90 mmol) was added in portions under an ice-water bath, reacted for half an hour at 0deg.C, then warmed to room temperature, reacted for 16 hours, TLC showed complete starting material, reacted further warmed to 40deg.C, then cooled to room temperature, the precipitated white solid was filtered, and the filtrate was washed with saturated sodium hydrogen sulfite, saturated sodium bicarbonate and brine. After drying and concentration, purification was performed by silica gel column (10 to 50% ethyl acetate/n-hexane) to obtain a white solid (0.1 g, 50%).

Example 37 preparation of Compound 40

Compound 39 (28 mg,0.08 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.3 mL) was added at 0deg.C, the reaction was warmed to room temperature and stirred for 2 hours, diluted with dichloromethane (10 mL), saturated sodium bicarbonate (10 mL) and saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give compound 40 (16.9 mg, 84%) as a colorless oil, which was used directly in the next step without further purification. LC-MS (ESI) m/z C ₁₄ H ₁₉ NO ₃ [M+H] ⁺ Calculated 250.14, found 250.14.

EXAMPLE 38 preparation of Compound 41

Compound 8 (63 mg,0.219 mmol) was dissolved in methanol (2 mL), thionyl chloride (79.5. Mu.L, 1.09 mmol) was added dropwise at 0deg.C, then the reaction was stirred overnight at room temperature, the reaction mixture was concentrated and then azeotropically distilled with toluene to give compound 41 (44 mg) as a colorless oil, which was used directly in the next reaction without purification. LC-MS (ESI) m/z C ₁₀ H ₁₉ NO ₃ [M+H] ⁺ Calculated 202.14, found 202.14.

Example 39 preparation of Compound 42

Compound 41 (0.219 mmol) and compound 14 (73 mg,0.24 mmol) were dissolved in DMF (5 mL), DECP (45. Mu.L, 0.263 mmol) and triethylamine (152.3. Mu.L, 1.09 mmol) were added at 0deg.C, after stirring for 2 hours at 0deg.C, the reaction was stirred overnight at room temperature, diluted with ethyl acetate (50 mL), potassium bisulfate (1N, 20 mL), water (20 mL), saturated aqueous sodium bicarbonate (20 mL) and saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified on a silica gel column (15-75% ethyl acetate/n-hexane) to give compound 42 (61 mg, 80%) as a white solid. LC-MS (ESI) m/z C ₂₅ H ₄₆ N ₂ O ₇ [M+H] ⁺ Calculated 487.33, found 487.32.

EXAMPLE 40 preparation of Compound 43

Compound 42 (0.173 mmol) and Boc-Val-OH (107.1 mg,0.493 mmol) were dissolved in DCM (5 mL), broP (95.4 mg,0.246 mmol) and DIPEA (133.7. Mu.L, 0.813 mmol) were added at 0deg.C, the reaction was protected from light for 30min at 0deg.C, then warmed to room temperature, reacted for 48 h, the reaction was diluted with ethyl acetate (50 mL), washed with potassium bisulfate (1N, 20 mL), water (20 mL), saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified on a silica gel column (15-80% ethyl acetate/n-hexane) to give compound 43 (80 mg, 78%) as a white solid. LC-MS (ESI) m/z C ₃₀ H ₅₅ N ₃ O ₈ [M+H]Calculated + 586.40, found 586.37.

EXAMPLE 41 preparation of Compound 44

Compound 43 (72 mg,0.123 mmol) was dissolved in THF (2 mL), a solution of lithium hydroxide (26 mg,0.614 mmol) in water (1 mL) was added at 0deg.C, the reaction solution was warmed to 25deg.C, stirred overnight, THF was removed by low-pressure concentration, then water (50 mL) was added, the pH was adjusted to 3 with potassium hydrogen sulfate (1N), and extraction was performed with ethyl acetate (20 mL. Times.3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated at low pressure to give compound 44 (70 mg, 98%) as a white solid, which was used in the next step without further purification. LC-MS (ESI) m/z C ₂₉ H ₅₃ N ₃ O ₈ [M+H] ⁺ Calculated 572.38, found 572.38.

EXAMPLE 42 Compound 45

Compound 44 (27.7 mg,0.048 mmol) and compound 40 (16.9 mg,0.068 mmol) were dissolved in DMF (5 mL), diethyl cyanophosphate (10. Mu.L, 0.063 mmol) and triethylamine (18.2. Mu.L, 0.131 mmol) were added at 0deg.C, the reaction was allowed to warm to room temperature after 2 hours, the reaction was diluted with ethyl acetate (50 mL), potassium bisulfate (1N, 20 mL), water (20 mL), saturated aqueous sodium bicarbonate (20 mL) and saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified on a silica gel column (20% -100% ethyl acetate/n-hexane) to give compound 45 (10 mg, 26%) as a colorless oil. LC-MS (ESI) m/z C ₄₃ H ₇₀ N ₄ O ₁₀ [M+H] ⁺ Calculated 803.51, found 803.47.

EXAMPLE 43 preparation of Compound 46

Compound 45 (10 mg,0.0124 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (0.3 mL) was added at 0deg.C. The reaction solution was warmed to room temperature, stirred for 2 hours, diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate (10 mL) and saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give compound 46 (8 mg, 99%) as a colorless oil, which was used directly in the next step without further purification. LC-MS (ESI) m/z C ₃₈ H ₆₂ N ₄ O ₈ [M+H] ⁺ Calculated 703.46, found 703.44.

EXAMPLE 44 preparation of Compound 47

Compound 46 ((8 mg,0.0113 mmol) and Boc-N-Me-Val-OH (15.7 mg,0.0682 mmol) were dissolved in DMF (5 mL) and diethyl cyanophosphate (2. Mu.L, 0.0142 mmol) and 4-methylmorpholine (7.3. Mu.L, 0.0682 mmol) were added at 0deg.C, the reaction was allowed to proceed for 2 hours at 0deg.C, then allowed to react at room temperature for 16 hours, the reaction mixture was diluted with ethyl acetate (50 mL), potassium bisulfate (1N, 20 mL), water (20 mL), saturated aqueous sodium bicarbonate (20 mL) and saturated sodium chloride (20 mL)Washing, drying over anhydrous sodium sulfate, concentration in vacuo, and purification on a silica gel column (20% -100% ethyl acetate/n-hexane) afforded compound 47 (8 mg, 76%) as a colorless oil. LC-MS (ESI) m/z C ₄₉ H ₈₁ N ₅ O ₁₁ [M+H] ⁺ Calculated 916.59, found 916.60.

EXAMPLE 45 preparation of Compound 48

Compound 47 (8 mg,0.0087 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (0.3 mL) was added at 0deg.C. The reaction was warmed to room temperature, stirred for 2 hours, diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate (10 mL) and saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate and concentrated in vacuo to give compound 48 (7 mg, 99%) as a colorless oil which was used directly in the next step without further purification. LC-MS (ESI) m/z C ₄₄ H ₇₃ N ₅ O ₉ [M+H] ⁺ Calculated 816.54, found 816.53.

EXAMPLE 46 preparation of Compound 49

Compound 48 (0.0087 mmol) and maleimide butyric acid (4.6 mg,0.025 mmol) were dissolved in DCM (5 mL), broP (4.8 mg,0.0124 mmol) and DIPEA (6.7. Mu.L, 0.041 mmol) were added at 0deg.C, the reaction was carried out in the absence of light at 0deg.C for 30min, then warmed to room temperature for 24 h, the reaction was diluted with ethyl acetate (50 mL), washed with potassium bisulfate (1N, 20 mL), water (20 mL), saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and purified on a silica gel column (50% ethyl acetate/n-10% methanol/ethyl acetate) to give compound 49 (3.2 mg, 37%) as a white solid. LC-MS (ESI) m/z C ₅₂ H ₈₀ N ₆ O ₁₂ [M+H] ⁺ Calculated 981.58, found 981.57.

Example 47 preparation of ADC with Compounds 19,24,32,37 and 49

Monoclonal antibodies (e.g., her2 antibody herceptin, 5 mg/ml) were dissolved in a phosphate buffer solution containing 0.05mol/L sodium tetraborate, pH 7.8, and TCEP (2-10 times the equivalent of antibody) and compound 19,24,32,37 or 49 (4-10 times the equivalent of antibody) were added at 25 ℃. After stirring at 25℃for 2-24 hours, the reaction was quenched with an excess of cysteine (or n-ethylmaleimide, dehydroascorbic acid). The buffer solution was replaced with a NAP desalting column (Illustra Sephadex G-25DNA Grade,GE Healthcare) with a solution containing 0.05mol/L glycine, 0.01mol/L histidine, 1% sucrose, 0.01% Tween-20 and 0.1mol/L sodium dihydrogen phosphate (pH=6.0). Four hours of dialysis was performed with slide-a-lyzer dialysis cartridge (Thermo Scientific 20,000MWCO) in the same buffer. Each antibody in the purified conjugate was grafted with 3-4 cytotoxic molecules (LC-MS detection), 99% monomer (exclusion chromatography analysis), and <0.2% small molecule compound that did not form conjugate (dual column reversed phase hplc analysis), with a final protein concentration of 0.8-2.5 mg/mL (protein concentration and drug loading measured spectrophotometrically at 280nm and 254nm, respectively).

Example 47 in vitro cytotoxicity assays of conjugates 50, 51, 52, 53 and 54 of anti-Her 2 antibodies

Cell lines used in the cytotoxicity experiments included NCI-N87, which is a human gastric cancer cell line, BT-474, which is a human invasive ductal carcinoma cell line, and SKOV3, which is a human ovarian cancer cell line. For NCI-N87 and BT-474 cells, these cells were grown in RPMI-1640 medium with 10% FBS. For SKOV3 cells, the cells were in McCoy 5A medium with 10% fbs. In running the assay, cells (180 μl,6000 cells) were added to 96-well plates and incubated for 24 hours at 37 ℃ and 5% carbon dioxide, after which the cells were treated with different concentrations of compounds (50, 51, 52, 53 and 54) (20 μl) (total volume 0.2 mL). Control wells contained cells and medium, no test compound. After incubating the well plate for 120 hours at 37℃and 5% carbon dioxide, MTT (5 mg/mL, 20. Mu.L) was added thereto and incubated for 1.5 hours at 37 ℃. After careful removal of the medium, DMSO (180. Mu.L) was added, and the absorbance was measured at 490nm and 570nm with shaking for 15 minutes. The inhibition ratio was calculated according to the following formula:

inhibition% = [1- (assay value-blank value)/(control value-blank value) ] ×100%

TABLE 1 in vitro Activity of conjugate conjugated conjugate in vitro Activity of saturated anti-Her 2 naked antibody (shielding antigen) (anti-N-87 cells (Ag+))

Compounds 50, 51, 52 and 53 have similar windows of in vitro activity and specificity. Compound 54, which contains one epoxy group, performs better than the other 4 conjugate conjugates in both killing effect and specificity window against antigen-positive tumor cells.

Claims (23)

1. The conjugated conjugate is formed by an auristatin analogue serving as a cytotoxic molecule and a cell binding molecule, and has a structural general formula (I):

G-Lm-Dn(I)

and pharmaceutically acceptable salts and solvates thereof;

wherein G is a cell surface binding molecule (ligand); n is 1-20; m is 1-10; l is a cleavable or non-cleavable linker; d is a cytotoxic molecule (small molecule drug) having structural formula (II):

wherein- - -represents that the small molecule drug and the linker are independently linked; r is R ¹ ，R ² ，R ³ And R is ⁴ Is independent C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl orHeterocycloalkyl, C ₃ -C ₈ Aryl, aralkyl, heteroalkylcycloalkyl or alkanoyl groups of (a); 2R groups, e.g. R ¹ R ² ，R ¹ R ³ ，R ² R ³ ，R ³ R ⁴ And R is ⁵ R ⁶ A 3-7 membered carbocyclic, cyclic, heterocyclic or heterocyclic ring structure; in addition, R ² ，R ³ And R is ⁴ May be H, and R ¹ Can default;

Y ¹ is N or CH;

R ⁵ and R is ⁶ Each independently is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Alkyl carbocyclyl or C ₁ -C ₈ An alkyl heterocyclic group; r is R ⁵ And R is ⁶ Can be combined to form a cycloalkyl, heterocycloalkyl, carbocycle or heterocyclic ring structure;

R ⁷ is H or C ₁ -C ₈ An alkyl group;

R ⁸ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ⁹ is H, OH, C ₁ -C ₈ Alkyl, C ₃ -C ₈ Carbocycles or O- (C) ₁ -C ₈ An alkyl group);

R ¹⁰ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ¹¹ is H or C ₁ -C ₈ An alkyl group;

R ¹² is aryl, alkylaryl, alkyl or C ₃ -C ₈ A heterocycle;

Y ² selected from: wherein R is ¹³ ，R ¹⁴ And R is ¹⁵ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle; x is CH ₂ ，O，S，NH，NR ¹⁶ S (O) or S (O) ₂ Wherein R is ¹⁶ Is H or C ₁ -C ₈ An alkyl group;

z is H, C ₁ -C ₈ Alkyl, -O- (C) ₁ -C ₈ Alkyl), -halogen, -NO ₂ ，-CN，-COOH，-C(O)OR ¹⁷ ，-C(O)NHR ¹⁸ ，-C(O)NR ¹⁸ R ¹⁹ ，-CH ₂ R ²⁰ ，-CH ₂ OP(O)(OR ²¹ ) ₂ Wherein R is ¹⁷ ，R ¹⁸ ，R ¹⁹ ，R ²⁰ And R is ²¹ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ Alkyl heterocycles.

The structure of the linker L is-Ww-(Aa) r-Vv-, wherein: -W-is a stretch unit linking the target binding unit (T) to an amino acid unit (Aa) and to V when Aa is absent, and W may comprise each independently a self-pinning spacer, a short peptide unit, a hydrazone, disulfide, thioether, ester or amide bond; w is 0 or 1; aa are each independently a natural or unnatural amino acid unit; r is an integer from 0 to 12; (Aa) _r Represents a natural or unnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit;

v is a spacer unit, each independently is H, O, NH, S, C ₁ -C ₈ Alkyl or heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocyclyl or carbocyclyl, C ₃ -C ₈ Aryl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroarylcycloalkyl, heteroalkylcycloalkyl or alkylcarbonyl, or 1 to 4 amino acid units, or (CH) ₂ CH ₂ O) _r Wherein r is an integer from 0 to 12; v is 0,1 or 2.

The cell-binding molecule G 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, a binding agent that mimics an antibody, an ankyrin repeat, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient transport molecule (transferrin), a binding polypeptide, protein or antibody, a small molecule that binds to albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid.

2. According to claim 1, the linker-small molecule drug (L) of the general structural formula (III) _m -D _n )：

And pharmaceutically acceptable salts and solvates thereof;

wherein L is a cleavable or non-cleavable linker; l is provided with a functional group which can be connected with small molecular medicines through disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary or quaternary), imine, cycloheteroalkane, heteroarene, oxime, amide bond and the like;

Y ¹ is N or CH;

R ² is H or C ₁ -C ₈ An alkyl group;

R ³ and R is ⁴ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocyclic group; r is R ³ And R is ⁴ Can be combined to form a cyclic structure of a cycloalkane, heterocycloalkyl, carbocycle or heterocycle;

R ⁵ and R is ⁶ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocyclic group; r is R ³ And R is ⁴ Can be combined to form a cyclic structure of a cycloalkane, heterocycloalkyl, carbocycle or heterocycle;

R ⁷ is H or C ₁ -C ₈ An alkyl group;

R ⁸ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ⁹ is H, OH, C ₁ -C ₈ Alkyl, C ₃ -C ₈ Carbocycles or O- (C) ₁ -C ₈ An alkyl group);

R ¹⁰ is H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocycle, C ₃ -C ₈ Carbocycles, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle;

R ¹¹ is H or C ₁ -C ₈ An alkyl group;

R ¹² is aryl, alkylaryl, alkyl or C ₃ -C ₈ A heterocycle;

Y ² selected from: wherein R is ¹³ ,R ¹⁴ And R is ¹⁵ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ An alkyl heterocycle; x is CH ₂ ，O，S，NH，NR ¹⁶ S (O) or S (O) ₂ Wherein R is ¹⁶ Is H or C ₁ -C ₈ An alkyl group;

z is H, C ₁ -C ₈ Alkyl, -O- (C) ₁ -C ₈ Alkyl), -halogen, -NO ₂ ，-CN，-COOH，-C(O)OR ¹⁷ ，-C(O)NHR ¹⁸ ，C(O)NR ¹⁸ R ¹⁹ ，-CH ₂ R ²⁰ ，-CH ₂ OP(O)(OR ²¹ ) ₂ Wherein R is ¹⁷ ，R ¹⁸ ，R ¹⁹ ，R ²⁰ And R is ²¹ Are each independently H, C ₁ -C ₈ Alkyl, C ₁ -C ₈ Heteroalkyl, C ₂ -C ₈ Alkenyl, C ₂ -C ₈ Alkynyl, C ₃ -C ₈ Heterocyclyl, C ₃ -C ₈ Carbocyclyl, aryl, C ₁ -C ₈ Alkylaryl, C ₁ -C ₈ Carbocycle or C ₁ -C ₈ Alkyl heterocycles.

3. According to claim 2, the linker-small molecule drug (L _m -D _n )：

Wherein M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ Or a pharmaceutically acceptable salt; r is R ¹ ，R ² And R is ³ Is defined by claim 1;

t is independently selected from L, R ¹ ，-O-，-S-，-SS-，-NH-，＝N-，＝NNH-，-NH(R ¹ )，-OR ¹ ，-C(＝O)-，

-C(＝O)R ¹ ，-COOR ¹ -，-C(O)NH-，-C(＝O)NHR ¹ -，-SR ¹ -，-S(＝O)R ¹ -，-P(＝O)(OR ¹ ) ₂ -，

-P(＝O)(NHR ¹ ) ₂ -，-CH ₂ OP(＝O)(OR ¹ ) ₂ -，-CH ₂ NHP(＝O)(NHR ¹ ) ₂ -，-CH ₂ NHP(＝O)(NHR ¹ ) ₂ -，

-CH ₂ NHP(＝O)(NHR ¹ )(NHR ² )-，-SO ₂ R ¹ -,(CH ₂ CH ₂ O) _r -，-(CH(CH ₃ )CH ₂ O) _r -，C ₁ -C ₈ Alkyl, or heteroalkyl, C ₂ -C ₈ Alkenyl, alkynyl, heterocycle or carbocycle, C ₃ -C ₈ Aryl, cycloalkyl, alkylcycloalkyl, in cycloalkyl, heteroaryl, heteroalkylcycloalkyl, alkylcarbonyl or 1-10 amino acid units, wherein r is an integer from 0 to 12; l, R ¹ And R is ² Is as defined in claim 1.

Q is a cell binding unit or a functional group that can be readily attached to a cell binding molecule. Functional groups include disulfide substituents, maleimide groups, haloacetyl groups, hydrazine, aldehydes, ketones, azides, amino groups, alkoxyamino groups or N-hydroxysuccinimide esters.

4. According to claim 1, a linker-small molecule drug having the following preferred structure:

and pharmaceutically acceptable salts and solvates thereof;

wherein Q, T, R ¹ ，R ² And R is ³ Is as defined in claims 1 and 3; r is R ²² Is H, OPO ₃ M ₂ ，OSO ₃ M，OCH ₂ PO ₃ M，OH，NO ₂ ，F，Cl，Br，I，SR ¹³ ，SSR ¹³ ，NH ₂ ，NHR ¹³ ，NR ¹³ R ¹⁴ OR ¹³ The method comprises the steps of carrying out a first treatment on the surface of the M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ ；R ¹³ ，R ¹⁴ ，R ¹⁵ ，R ¹⁷ ，R ¹⁸ And R is ¹⁹ Is as defined in claim 1. In addition, R ¹⁸ And R is ¹⁹ Can be combined to form a cycloalkane, heterocycloalkyl, carbocyclic or heterocyclic ring structure, an aromatic ring, or a heteroaromatic ring structure.

5. Conjugated drugs according to claims 2,3 and 4, having the following preferred structures:

and pharmaceutically acceptable salts and solvates thereof, wherein the mAb is an antibody and n is 1-30.

6. According to claim 1, the linker L consists of one or more of the following linker units: 6-Maleimidohexanoyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe or af), p-aminobenzyloxycarbonyl Phenyl (PAB), 4-mercaptopentanoyl (SPP), 4- (N-maleimidomethyl) -cyclohexane-1 acyl (MCC), (4-acetyl) aminobenzoyl (SIAB), 4-mercapto-butyryl (SPDB), 4-mercapto-2-hydroxysulfonyl-butyryl (2-Sulfo-SPDB), ethyleneoxy (-CH) ₂ CH ₂ One or more repeating units of O- > (EO or PEO), oxime (aldoxime or ketoxime), 1,2, 3-triazole (produced by click chemistry), acetal (produced by reaction of sugar or the like). The structures are as follows:

(MC, 6-Maleimidohexanoyl); (ME, maleimide ethyl);(MP, maleimide propionyl);(PAB, p-aminobenzyloxycarbonyl analog, X ¹ And Y ¹ Independently selected from O, S, NH or CH ₂ )；(carbonyl benzyl polypeptide analogue, X) ¹ And Y ¹ Independently selected from O, S, NH or CH ₂ ，R ₁ And R is ₂ Is as defined above);

(valine-citrulline);(MCC, 4- (N-maleimidomethyl) -cyclohexane-1 acyl);((4-acetyl) aminobenzoyl);(2-sulfoo-SPDB, 4-mercapto-2-hydroxysulfonyl-butyryl);(oxime);(1, 2, 3-triazole);(acetals);(hydrazone);

((2-dithio) ethoxycarbonyl).

7. According to claim 1, a linker-small molecule drug having the structure of formula (IV):

And pharmaceutically acceptable salts and solvates thereof, wherein R ¹ ，R ² ，R ³ ，R ⁴ ，R ⁵ ，R ⁶ ，R ⁷ ，R ⁸ ，R ⁹ ，R ¹⁰ ，R ¹¹ ，R ¹² ，Y ¹ ，Y ² Z and L are as defined in claim 2.

8. Linker-small molecule drug (L) of formula (IV) _m -D _n )：

And pharmaceutically acceptable salts and solvates thereof. Wherein M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ ；R ¹ ，R ² ，R ³ T and Q are as defined in claims 2 and 3.

9. According to claim 7, the preferred linker-small molecule drug represented by structural formula (IV):

and pharmaceutically acceptable salts and solvates thereof. Wherein Q, T, R ¹ ，R ² ，R ³ ，R ¹⁷ ，R ¹⁸ ，R ¹⁹ And R is ²² Is as defined in claims 1,3 and 4. In addition, R ¹⁷ And R is ¹⁸ May be default.

10. According to claims 1 and 7, preferred conjugate conjugates comprising an antibody and a structure of general formula (IV):

and pharmaceutically acceptable salts and solvates thereof. Wherein mAb is an antibody, and n is 1-30.

11. According to claim 1, the linker-small molecule drug (L) having the structure of formula (V) _m -D _n )：

And pharmaceutically acceptable salts and solvates thereof, wherein R ¹ ，R ² ，R ³ ，R ⁴ ，R ⁵ ，R ⁶ ，R ⁷ ，R ⁸ ，R ⁹ ，R ¹⁰ ，R ¹¹ ，R ¹² ，Y ¹ ，Y ² Z and L are as defined in claim 2.

12. According to claim 11, the linker-small molecule drug (L _m -D _n )：

And pharmaceutically acceptable salts and solvates thereof. Wherein M is H, na, K, ca, mg, zn or N ⁺ R ¹ R ² R ³ ；R ¹ ，R ² ，R ³ T and Q are as defined in claims 2 and 3.

13. According to claim 11, a linker-small molecule drug having the following preferred structure:

and pharmaceutically acceptable salts and solvates thereof. Wherein Q, T, R ¹ ，R ² ，R ³ ，R ¹⁷ ，R ¹⁸ ，R ¹⁹ And R is ²² Is as defined in claims 1,3 and 4.In addition, R ¹⁹ May be default.

14. According to claims 1 and 11, a conjugate having the following structure:

and pharmaceutically acceptable salts and solvates thereof. Wherein mAb is an antibody, and n is 1-30.

15. According to claims 1,3,4,8,9, 12 and 13, the cell binding molecule is a molecule against the following antigens or receptors: CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11B, CD11C, CD12w, CD14, CD15, CD16, CD w17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49B, CD49C, CD51, CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79B, CD80, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD174, CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200B, CD209, CD221, CD227, CD235a, CD240, CD262, CD271, CD274, CD 276 (B7-H3), CD303, cd304, cd309, CD326, 4-1BB,5AC,5T4 (trophoblast glycoprotein, TPBG,5T4, WNT activation inhibitor 1 or WAIF 1), cancer antigen, AGS-5, AGS-22M6, activin receptor kinase 1, AFP, AKAP-4, ALK, alpha integrin, alpha V-beta 6, aminopeptidase N, beta amyloid, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, protective antigens for anthrax toxin, anti-transferrin receptor, AOC3 (VAP-1), B7-H3, bacillus anthracis, BAFF (B cell activating factor), B cell lymphoma cells, bcr-abl, bombesin, BORIS, C5, C242 antigen, CA125 (glycoantigen 125, MUC 16), CAIX (carbonic anhydrase 9), CALLA, canAg, canine IL31, myosin, CCL11 (C-C chemokine 11), CCR4 (C-C chemokine receptor type 4, CD 194), CCR5, CD3 epsilon, CEA (carcinoembryonic antigen), CEACAM3, CEACAM5, CFD, ch4D5, cholecystokinin 2 (CCK 2R), CLDN18 (claudin-18), lectin a, CRIPTO, CSF1R (colony stimulating factor 1 receptor, CD 115), CSF2 (colony stimulating factor 2 receptor), macrophage colony stimulating factor (GM-CSF), CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA tumor antigen, CXCR4 (CD 184), C-X-C chemokine receptor type 4, cyclic ADP-ribohydrolase, cyclin B1, cytomegalovirus, glycoprotein B, dabigaran, DLL4, DPP4 (dipeptidylpeptidase 4), DR5 (death receptor 5), shiga toxin-1 of Escherichia coli, shiga toxin-2, ED-B, EGFL7, EGFR, EGFRII, EGFRvIII, endoglin (CD 105), endothelin B receptor, endotoxin, epCAM (epithelial adhesion molecule), ephA2, episilin, ERBB2 (epidermal growth factor receptor 2), ERBB3, ERG (TMPRSS 2 ETS fusion gene), escherichia coli, ETV6-AML, FAP (fibroblast activation protein alpha), FCGR1, alpha fetoprotein, fibrin II, fibronectin additional domain name-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, fos-associated antigen 1, respiratory syncytial virus F protein, frizzled receptor, fucose GM1, gangliosides of GD2, G-28 (cell surface antigen glyvolipid), GD3 idiotypes, globoH, glypican 3, N-glycosylneuraminic acid, GM3, GM-CSF receptor alpha chain, growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2 (guanylate cyclase 2C), guanylate cyclase C (GC-C), enteroguanylate cyclase, guanylate cyclase C receptor, thermostable enterotoxin receptor (hSTAR), heat shock proteins, hemagglutin, hepatitis B surface antigen, hepatitis B virus, HER1 (HER 1), HER2, HER2/neu, HER3 (ERBB-3), igG4, HGF/SF (hepatocyte growth factor/spreading factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human spreading factor receptor kinase, HPV E6/E7, HSP90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-gamma, influenza virus Hemagglutinin, ig E Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, αIIBβ3, αVβ3, α4β7, α5β1, α6β4, α7β7, αIIβ3, α5β5, αVβ5), gamma interferon inducible protein, ITGA2, ITGB2, KIR2D, LCK, legumain, lewis-Y antigen, LFA-1 (lymphocyte function associated antigen 1, CD11 a), LHRH, LINGO-1, lipoteichoic acid, LIV1a, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3,MAGE A1,MAGE A3,MAGE 4,MART1,MCP-1, MIF (macrophage migration inhibitor, or Glycosylation Inhibitor (GIF)), MS4A1 (transmembrane four domain family member 1), MSLN (mesothelin), MUC1 (mucin 1, cell surface associated (MUC 1) or Polymorphic Epithelial Mucin (PEM)), MUC1-KLH, MUC16 (CA 125), MCP1 (monocyte chemotactic protein 1), MYCN, myelin associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), nectin-4 (ASG-22 ME), nerve growth factor, neuronal apoptosis regulator 1, nogo-A, notch receptor, nucleolin, neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, oxLDL (oxidized low density lipoprotein), OY-TES1, P21, P53 non-mutated, P97, PAP, anti (N-glycosylneuraminic acid) antibody binding sites, PAX3, PAX5, PCSK9, PDCD1 (PD-1, apoptosis protein 1, CD 279), PDGF-Rα (platelet-derived growth factor receptor α type), PDGF-Rβ, PDL-1, PLAC1, PLAP-testis-like alkaline phosphatase, platelet-derived growth factor receptor β, phospho-sodium cotransporter, PMEL 17, sialic acid, proteinase 3 (PR 1), prostate cancer, PS (phosphatidylserine), prostate cancer cells, pseudomonas aeruginosa, PSMA, PSA, prostate stem cell antigen, rabies glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD 240), rhesus factor, RANKL, rhoC, RAS mutation, RGS5, ROBO4, respiratory syncytial virus, RON, sarcoma translocation breakpoint, SART3, osteoprotegerin, SLAM7, selectin P, SDC1, systemic lupus erythematosus (a), somatodin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (prostate hexatransmembrane epithelial antigen 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor vascular endothelial marker 1), TENB2, tenascin C (TN-C), TGF-alpha (transforming growth factor alpha), TGF-beta 1, TGF-beta 2, tie (CD 202B), tie2, TIM-1 (CDX-014), TNF, TNF-alpha, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF13B, TPBG (trophoblastin), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor binding 1), TRAIL-R2 (death receptor 5, dr 5), tumor-associated calcium signaling receptor 2, tumor-specific glycosylation of muc1, TWEAK receptor, tumor-specific glycosylation-regulated receptor (glycoprotein gene 75), TRP-2, tyrosinase, VCAM-1 (CD 106), VEGF (vascular endothelial growth factor), VEGF-Sub>A, VEGF-2 (CD 309), VEGFR-1, VEGFR-2, VEGFR2, vimentin, WT1, XAGE1, or insulin growth factor or epidermal growth factor receptor expressing any cell.

16. According to claims 1,3,4,8,9, 12 and 13, the cell binding molecules are preferably intact antibodies (polyclonal and monoclonal); diabodies, triabodies, and antibody fragments (e.g., fab ', F (ab') ₂ Fv, fragments generated by Fab expression libraries); a monoclonal antibody; a single chain monoclonal antibody; a monoclonal antibody fragment capable of binding to a target cell; a chimeric antibody; a chimeric antibody fragment capable of binding to a target cell; a domain antibody; a domain antibody capable of binding to a target cell; a prototype antibody; a single chain prototype antibody; a prototype antibody fragment capable of binding to a target cell; a humanized antibody; a single chain humanized antibody; a humanized antibody fragment capable of binding to a target cell; an anti-idiotype antibody (anti-Id); lymphokines (e.g., IL-2, IL-3, IL-4, IL-6, IL-10); hormones (e.g., insulin, TRH (thyroid stimulating hormone releasing hormone), steroid hormones such as androgens and estrogens, MSH (melanocyte stimulating hormone)); interferons (e.g., IFN-gamma); vitamins such as folic acid; growth factors and colony stimulating factors (e.g., epidermal Growth Factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), transforming Growth Factors (TGF) such as TGF alpha and TGF beta, insulin and insulin-like growth factors (IGF-I and IGF-II), G-CSF, M-CSF and GM-CSF, vaccinia Virus Growth Factor (VGF), fibroblast Growth Factor (FGF) and platelet-derived growth factor); a transport molecule for cell nutrition; interleukins and cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6); peptides and peptide hormones, such as bombesin, gastrin releasing peptide; apolipoproteins and glycoproteins, such as transferrin; a sugar-binding protein or lipoprotein, such as lectin; small molecule inhibitors, such as Prostate Specific Membrane Antigen (PSMA) inhibitors and small molecule Tyrosine Kinase Inhibitors (TKIs); conjugated proteins that bind to target cells; macromolecules, dendrimers, nanoparticles, liposomes and viruses A cell binding molecule attached to the capsid, or a combination of the above.

17. Conjugate conjugates made using the cytotoxic molecules of claims 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13 and 14 for use in pharmaceutical compositions for targeted killing of abnormal cells, in particular tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, bone marrow cells, activated T cells and B cells, melanocytes, proliferative diseases, benign or malignant tumors, leukemias and lymphoid malignancies, neurogenic, glial, astrocytic, hypothalamic, glandular, macrophage, epithelial, stromal, blastula, angiogenic immune disorders, inflammatory, autoimmune diseases, destructive disorders, bone diseases, infectious diseases, viral diseases, liver diseases and neurodegenerative disorders; pancreatic or renal disease in a mammal.

18. A pharmaceutical composition for the treatment or prophylaxis of cancer, autoimmune diseases or infectious diseases comprising a therapeutically effective amount of a conjugate prepared according to claims 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13 and 14, or a pharmaceutically acceptable salt thereof and a carrier, diluent or excipient, or a combination thereof.

19. A pharmaceutical composition for the treatment or prophylaxis of cancer, autoimmune diseases or infectious diseases comprising a therapeutically effective amount of conjugated conjugates prepared according to claims 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13 and 14, simultaneously administered with other therapeutic agents such as chemotherapy, radiation therapy, immunotherapeutic agents, anti-autoimmune disease agents, anti-infective agents or other antibody drug conjugates, for the synergistic treatment or prophylaxis of cancer, autoimmune diseases or infectious diseases.

20. The tumor cell of claim 17 comprising a lymphoma cell, a myeloma cell, a renal cell, a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a colon cancer cell, a gastric cancer cell, a squamous cell carcinoma, a small cell lung cancer, a non-small cell lung cancer cell, a testicular cancer cell, or any other cell that grows and divides at an uncontrolled rate and is oncogenic.

21. Conjugate conjugates prepared according to claims 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13 and 14, having in vitro, in vivo and in vivo activity.

22. The process according to claim 1,2,3,4,7,8,9, 11, 12 and 13, wherein the linker L and T comprise a peptide unit of 1 to 20 natural or unnatural amino acids, or a p-aminobenzyl unit, or a 6-maleimidocaproyl unit, or a disulfide or thioether unit, or a hydrazone unit, or a triazole unit or an alkyl oxime unit, which can be hydrolyzed by proteases under certain conditions.

23. The co-therapeutic agent of claim 19 preferably selected from the group consisting of: abatacept (Orencia) AbirateroneAcetaminophen/hydrocodone, adalimumab, afatinib +.>AlbumabAlitretinoinado-Trastuzumab emtansine(Kadcyla ^TM ) Amphetamine mixed salt (amphetamine/racemic amphetamine, or Adderall XR), anastrozole +.>Aripiprazole, atazanavir, atezolizumab (MPDL 3280A), atorvastatin, axitinib->Belinostat(Beleodaq ^TM )，BevacizumabCabazitaxelCabozantinib(Cometriq ^TM ) Bexarotene->Blinatumomab(Blincyto ^TM )，BortezomibBoschatinib->Brentuximab vedotinBudesonide, budesonide/formoterol, buprenorphine, capecitabine, carfilzomib->Celecoxib，Ceritinib(LDK378/Zykadia)，CetuximabCiclosporin, cinacalcet, crizotinibDabigatran，DabrafenibDarbepoetin, darunavir, imatinibDasatinib->Denileukin diftitoxDenosumabDepakote，Dexlansoprazole，Dexmethylphenidate，Dinutuximab(Unituxin ^TM ) Doxycycline, duloxetine, emtricitabine/Rilpivirine/tenofovir disoproxil, emtricitabine/tenofovir/efavirenz, enoxaparin, enzalutamide>Epoetin, erlotinib +.>Esomeprazole, eszopiclone, etanercept, everolimus +.>Exemestane->Everolimus->Ezetimibe, ezetimibe/simvastatin, fenofibrate, feguisteine, fingolimod, fluticasone/salmeterol->Gefitinib>Glatiramer, goserelin (Zoladex), imatinib (Gleevec), ibritumomab tiuxetan Ibrutinib(Imbruvica ^TM )，IdelalisibInfliximab, insulin aspart, insulin distil, insulin glargine, insulin lispro, interferon beta 1a, interferon beta 1b, lapatinib->IpilimumabIpratropium bromide/salbutamol sulphate, lanreotide->lenaliomideLenretinib methanesulfonic acid (Lenvima ^TM ) Letrozole->Levothyroxine, lidocaine, linezolid, liraglutide, lidexamfetamine, MEDI4736 (Cergene, alekang Corp.), memantine, methylphenidate, metoprolol, modafinil, mometasone, nilotinib->NivolumabOfatumumabObinutuzumab(Gazyva ^TM )，Olaparib(Lynparza ^TM ) Olmesartan, olmesartan/hydrochlorothiazide, omalizumab, omega-3 fatty acid ethyl ester, oseltamivir, oxycodone, palbociclib->Palivizumab，PanitumumabPanobinostat->Pazopanib->PembrolizumabPemetrexed (Neotai), pertuzumab (Pertuzumab) ^TM ) Pneumococcal conjugate vaccine, pomalidomide->Pregabalin, quetiapine, rabeprazole, radium 223 chlorideRaloxifene, raltefravir, ramucicrumab +.>RanibizumabRituximabRivaroxaban, romidepsin +.>Rosuvastatin, ruxolitinib phosphate (Jakafi) ^TM ) Salbutamol, sevelamer, sildenafil, silumixmab (Sylvant) ^TM ) Sitagliptin, sitagliptin/metformin, solifenacin, sorafenibSunitinibTadalafil, tamoxifen, telaprevir, temsirolimus Testesterone gel, thalidomide (Immunprin, talidex), tiotopium, toremifene>TrametinibTrastuzumab，TretinoinUstekinumab, valsartan, van der Tani->VemurafenibVorinostatZivafliberceptZostavax and their analogs, derivatives, pharmaceutically acceptable salts, carriers, diluents or excipients, or combinations thereof. />