Maytansinoid derivatives having self-immolative peptide linkers and conjugates thereof

Abstract

Antibody-drug conjugates having a new class of linker moieties are provided. The present invention relates to a novel cell-binding agent-maytansinoid conjugate having a self-immolative peptide linker, more specifically, to a conjugate represented by the following formula (I) or a pharmaceutically acceptable salt thereof: TIFF2022137200000211.tif1981 [In the formula, CB is a cell binding agent; L2 is absent or a spacer; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Application No. 62 / 465,118, filed February 28, 2017, and U.S. Provisional Application No. 62 / 480,209, filed March 31, 2017. The entire contents of each of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have emerged as a powerful class of antitumor drugs with efficacy across a wide range of cancers. ADCs generally consist of three distinct components: a cell-binding agent, a linker, and a cytotoxic drug. The linker component of an ADC is a key element in developing targeted anticancer drugs with an optimal therapeutic window, i.e., high activity at low, nontoxic doses.

[0003] Therefore, there is a need for ADCs with new classes of linker components. Summary of the Invention

[0004] The present invention provides a cell-binding agent-cytotoxic drug conjugate represented by the formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, CB is a cell binding agent; L2 is absent or a spacer; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0005] The present invention also provides a compound of formula (II): [ka] or a pharmaceutically acceptable salt thereof [In the formula, L2' is absent or a spacer having a reactive moiety capable of forming a covalent bond with a cell-binding agent; A is a peptide containing 1 amino acid or 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0006] Compounds of formula (III): [ka] or a pharmaceutically acceptable salt thereof is also included in the present invention. [In the formula, A' is a peptide containing 1 amino acid or 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0007] The present invention also provides a compound of formula (IV): [ka] or a pharmaceutically acceptable salt thereof [In the formula, L3 is given by the following formula: [ka] Represented by; R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + , or -OH, halogen, SO3H or NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; k is an integer from 1 to 10; A is an amino acid or a peptide containing 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0008] The present invention is also directed to compositions (e.g., pharmaceutical compositions) comprising a conjugate (e.g., a conjugate of Formula (I)) or a compound (e.g., a compound of Formula (II), (III), or (IV)) and a carrier (a pharmaceutically acceptable carrier). The present invention also includes compositions (e.g., pharmaceutical compositions) comprising a conjugate (e.g., a conjugate of Formula (I)) or a compound (e.g., a compound of Formula (II), (III), or (IV)) described herein and a carrier (a pharmaceutically acceptable carrier), further comprising a second therapeutic agent. The compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human). The compositions are useful for treating cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), transplant rejection, lupus, myositis, infection, immune disorders such as AIDS, and the like in a mammal (e.g., a human). and are useful in treating conditions such as inflammatory diseases.

[0009] The present invention also includes a method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), comprising administering to said abnormal cell or said mammal a therapeutically effective amount of a conjugate (e.g., a conjugate of Formula (I)) or compound (e.g., a compound of Formula (II), (III), or (IV)), or a composition thereof, alone or in combination with a second therapeutic agent. [Brief explanation of the drawings]

[0010] [Figure 1] The Ab-sSPDB-DM4 conjugates tested and compared with the conjugates of the present invention are shown. [Figure 2] Peptide anilino maytansinoid ADCs that were tested and compared to the conjugates of the present invention are shown. [Figure 3] A synthetic scheme for preparing representative maytansinoid compounds of the invention having a self-immolative peptide linker is shown. [Figure 4] A synthetic scheme for preparing representative conjugates of the invention is shown. [Figure 5] A synthetic scheme for preparing representative maytansinoid compounds of the invention having a self-immolative peptide linker is shown. [Figure 6] A synthetic scheme for preparing S-methylated metabolites of the conjugates of the invention is shown. [Figure 7] The binding affinity of a representative conjugate of the invention for a target antigen on T47D cells is shown. [Figure 8] The in vitro cytotoxicity of representative conjugates of the invention against KB cells is shown. [Figure 9A] The bystander killing effect of representative conjugates of the present invention is shown. [Figure 9B] The bystander killing effect of representative conjugates of the present invention is shown. [Figure 9C] The bystander killing effect of representative conjugates of the present invention is shown. [Figure 9D] The bystander killing effect of representative conjugates of the present invention is shown. [Figure 10] The in vivo antitumor activity of a representative conjugate of the invention in mice bearing large 250 mm 2 EGFR receptor-positive xenografts is shown. [Figure 11A] The in vivo antitumor activity of a representative conjugate of the invention at a dose of 5 mg / kg (FIG. 11A) in mice bearing CanAg+HT-29 xenografts is shown. [Figure 11B] The in vivo antitumor activity of a representative conjugate of the invention at a dose of 2.5 mg / kg (FIG. 11B) in mice bearing CanAg+HT-29 xenografts is shown. [Figure 12] The in vivo antitumor activity of representative conjugates of the present invention in mice bearing NCI-H2110 folate receptor-positive xenografts is shown. Mice were administered conjugate 17c or 4b at a dose of 3 mg / kg. [Figure 13]The weight change in mice treated with a representative conjugate of the invention is shown compared to mice treated with conjugate 4b. [Figure 14] The in vitro cytotoxicity of representative conjugates of the invention against CA922 cells is shown compared to 1a. [Figure 15] The in vitro cytotoxicity of metabolites 25a, 25b and 25c is shown in comparison with three metabolites of the conjugate Ab-sSPDB-DM4. [Figure 16] The metabolite species and proposed cleavage sites detected from in vitro metabolic studies of conjugate 17c are shown. [Figure 17A] The in vivo antitumor activity of representative conjugates of the present invention is shown in mice bearing OV-90 ovarian xenografts expressing heterologous FRα. Mice were administered conjugate 17c or conjugate 4b at doses of 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg. [Figure 17B] The in vivo antitumor activity of representative conjugates of the present invention is shown in mice bearing OV-90 ovarian xenografts expressing heterologous FRα. Mice were administered conjugate 17c or conjugate 4b at doses of 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg. [Figure 18] The weight change in mice treated with representative conjugates of the invention with various peptide linkers is shown compared to mice treated with conjugate 1a. [Figure 19] Shown are UPLC traces of DTT and NEM treated cell lysates from COLO205 cells not exposed to any conjugate (top) and COLO205 cells exposed to conjugate 18c. [Figure 20] The SEC / MS spectrum of conjugate 18c is shown. [Figure 21] The SEC / MS spectrum of conjugate 16c is shown. [Figure 22] The SEC / MS spectrum of conjugate 17c is shown. [Figure 23] The SEC / MS spectrum of conjugate 26c is shown. [Figure 24A] Shown are the body weights (mean ± SD, g) of CD-1 mice administered 1200 μg / kg of huML66-GMBS-Ala3-Immol-DM (conjugates 16a, 16b, 16c, and 16d) or huML66-s-SPDB-DM4 (conjugate la). [Figure 24B] Shown is the change in body weight (mean ± SD, g) of CD-1 mice administered 1200 μg / kg of huME66-GMBS-Ala3-Immol-DM (conjugates 16a, 16b, 16c, and 16d) or huML66-s-SPDB-DM4 (conjugate la). [Figure 25A] Shown are the body weights (mean ± SD, g) of CD-1 mice administered 1000 or 1250 μg / kg Movl9vl.6-GMBS-lAladAlalAla-Immol-DM (conjugate 17c) or 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl (conjugate 4b). [Figure 25B] Shown is the weight change (mean ± SD, g) of CD-1 mice administered 1000 or 1250 μg / kg Movl9vl.6-GMBS-lAladAlalAla-Immol-DM (conjugate 17c) or 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl (conjugate 4b). [Figure 25C] Shown is the weight change (mean ± SD, g) of individual CD-1 mice administered 1000 or 1250 μg / kg Movl9vl.6-GMBS-lAladAlalAla-Immol-DM (conjugate 17c) or 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl (conjugate 4b). [Figure 25D]Shown are individual body weights (mean ± SD, g) of CD-1 mice administered 1000 or 1250 μg / kg Movl9vl.6-GMBS-lAladAlalAla-Immol-DM (conjugate 17c) or 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl (conjugate 4b). [Figure 26] The pharmacokinetic profiles of conjugate 17c and the M-SPDB-DM4 conjugate in mice are shown. [Figure 27] Normalized percent degradation for conjugate 26c and M9346A-C442-mal-SPDB-DM4 is shown versus time. DETAILED DESCRIPTION OF THE INVENTION

[0011] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

[0012] It is to be understood that any of the embodiments described herein, including those described in different aspects of the invention (e.g., compounds, compound-linker molecules, conjugates, compositions, methods of making and using) and in different parts of the specification (including embodiments described only in the Examples), can be combined with one or more other embodiments of the invention, unless expressly denied or inappropriate. Combinations of embodiments are not limited to specific combinations claimed by multiple dependent claims.

[0013] definition As used herein, the term "treating" or "treatment" includes reversing, alleviating, or halting the symptoms, clinical signs, and underlying pathology of a condition so as to improve or stabilize the subject's condition. As used herein, as is well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation, remission, or slowing of the progression of one or more symptoms or conditions associated with a condition, e.g., cancer, whether detectable or undetectable; reduction in disease magnitude; stabilization (i.e., not worsening) of the disease state; delay or slowing of disease progression; improvement or remission of the pathology; and relief (whether partial or total) of the condition, whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival time in the absence of treatment. Exemplary beneficial clinical results are described herein.

[0014] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and therefore its application includes instances where the circumstance occurs and instances where the circumstance does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and therefore its application includes structures where a non-hydrogen substituent is present and structures where a non-hydrogen substituent is not present.

[0015] Unless specifically stated as "unsubstituted," references herein to chemical moieties are understood to include substituted variants. For example, reference to an "alkyl" group or moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on a chemical moiety include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aryl or heteroaryl moieties.

[0016] As used herein, the term "cell binding agent" or "CBA" preferably refers to a specific CBAs refer to compounds that can bind to cells (e.g., on cell surface ligands) or to ligands associated with or in close proximity to cells. In certain embodiments, binding to cells or ligands on or near cells is specific. CBAs can include peptides and non-peptides.

[0017] As used herein, "alkyl" refers to a saturated straight- or branched-chain monovalent hydrocarbon radical. In preferred embodiments, a straight- or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C2 in a straight chain). 30 , branched chain C3 to C 30), more preferably 20 or less. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-l-propyl, -CHCH(CH)2), 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl-3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-l-butyl, 2-methyl-l-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbon atoms of the hydrocarbon backbone. In certain embodiments, a straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C6 in a straight chain). 30 , branched chain C3 to C 30 In a preferred embodiment, the chain is 10 carbons or less (C1 to C 10 ) atoms in its backbone. In other embodiments, the chain has six or fewer carbon (C1-C6) atoms in its backbone. In other embodiments, the chain has three or fewer carbon (C1-C3) atoms in its backbone.

[0018] As used herein, "alkylene" refers to a saturated straight- or branched-chain divalent hydrocarbon radical. In preferred embodiments, a straight- or branched-chain alkylene has 30 or fewer carbon atoms in its backbone (e.g., C1-C in a straight chain). 30 , branched chain C3 to C 30), more preferably 20 or fewer. Furthermore, the term "alkylene," as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkylene" and "substituted alkylene," the latter of which refers to an alkylene moiety having substituents replacing a hydrogen on one or more carbon atoms of the hydrocarbon backbone. In certain embodiments, a straight-chain or branched-chain alkylene has 30 or fewer carbon atoms in its backbone (e.g., C1-C in a straight chain). 30 , branched chain C3 to C 30 In a preferred embodiment, the chain is 10 carbons or less (C1 to C 10 ) atoms in its backbone. In other embodiments, the chain has six or fewer carbon (C1-C6) atoms in its backbone. In other embodiments, the chain has three or fewer carbon (C1-C3) atoms in its backbone.

[0019] "Alkenyl" refers to a straight- or branched-chain monovalent hydrocarbon radical of 2 to 20 carbon atoms having at least one site of unsaturation, i.e., a carbon-carbon double bond, where alkenyl radicals include radicals having "cis" and "trans" configurations, or alternatively, "E" and "Z" configurations. Examples include, but are not limited to, ethylenyl or vinyl (-CH=CH), allyl (-CHCH=CH), and the like. Preferably, alkenyl has 2 to 10 carbon atoms. More preferably, alkenyl has 2 to 4 carbon atoms.

[0020] "Alkenylene" refers to a straight- or branched-chain divalent hydrocarbon radical of 2 to 20 carbon atoms having at least one site of unsaturation, i.e., a carbon-carbon double bond, where alkenyl radicals include radicals having "cis" and "trans" configurations, or alternatively, "E" and "Z" configurations. Examples include, but are not limited to, ethylenylene. Alternatively, vinylene (-CH=CH-), allylene (-CH2CH=CH-), etc. are included. Preferably, alkenylene has 2 to 10 carbon atoms. More preferably, alkenylene has 2 to 4 carbon atoms.

[0021] "Alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon radical of 2 to 20 carbon atoms having at least one site of unsaturation, i.e., a carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably, alkynyl has 2 to 10 carbon atoms. More preferably, alkynyl has 2 to 4 carbon atoms.

[0022] "Alkynylene" refers to a straight- or branched-chain divalent hydrocarbon radical of 2 to 20 carbon atoms having at least one site of unsaturation, i.e., a carbon-carbon triple bond. Examples include, but are not limited to, ethynylene, propynylene, 1-butynylene, 2-butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, hexynylene, and the like. Preferably, the alkynylene has 2 to 10 carbon atoms. More preferably, the alkynylene has 2 to 4 carbon atoms.

[0023] The terms "carbocycle," "carbocyclyl," and "carbocyclic ring" refer to a monovalent non-aromatic, saturated, or partially unsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms may be arranged, for example, as bicyclo[4,5], [5,5], [5,6], or [6,6] systems, and bicyclic carbocycles having 9 or 10 ring atoms may be arranged as bicyclo[5,6] or [6,6] systems or as bridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like.

[0024] The terms "cyclic alkyl" and "cycloalkyl" may be used interchangeably. As used herein, this term refers to a saturated ring radical. In preferred embodiments, a cycloalkyl has from 3 to 10 carbon atoms in its ring structure, and more preferably 5 to 7 carbon atoms in the ring structure. In some embodiments, two cyclic rings may share more than one atom; for example, the rings are "fused rings." Suitable cycloalkyls include cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. In some embodiments, a cycloalkyl is a monocyclic group. In some embodiments, a cycloalkyl is a bicyclic group. In some embodiments, a cycloalkyl is a tricyclic group.

[0025] The term "cycloalkylene" refers to a divalent radical of a saturated carbocyclic ring. In preferred embodiments, cycloalkylenes have from 3 to 10 carbon atoms in their ring structure, and more preferably 5 to 7 carbon atoms in the ring structure. In some embodiments, two cyclic rings may share more than one atom; for example, the rings are "fused rings." Suitable cycloalkylenes include cycloheptylene, cyclohexylene, cyclopentylene, cyclobutylene, and cyclopropylene. In some embodiments, a cycloalkylene is a monocyclic group. In some embodiments, a cycloalkylene is a bicyclic group. In some embodiments, a cycloalkylene is a tricyclic group.

[0026] The term "cyclic alkenyl" refers to a carbocyclic ring radical having at least one double bond in the ring structure.

[0027] The term "cyclic alkynyl" refers to a carbocyclic ring radical having at least one triple bond in the ring structure.

[0028] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. Aryl groups include phenyl, phenol, aniline, and the like. The term "aryl" also includes "polycyclyl," "polycyclic," and "polycyclic" ring systems having two or more rings in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings," where at least one of the rings is aromatic; for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, or cycloalkynyl. In some preferred embodiments, the polycyclic ring has two or three rings. In certain preferred embodiments, the polycyclic ring system has two cyclic rings in which both rings are aromatic. Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring. For example, aryl groups include, but are not limited to, phenyl (benzene), tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. In some embodiments, an aryl is a monocyclic aromatic group. In some embodiments, an aryl is a bicyclic aromatic group. In some embodiments, an aryl is a tricyclic aromatic group.

[0029] As used herein, an "arylene" is a divalent radical of an aryl group as defined above.

[0030] As used herein, the terms "heterocycle," "heterocyclyl," and "heterocyclic ring" refer to a substituted or unsubstituted non-aromatic ring structure consisting of a 3- to 18-membered ring, preferably a 3- to 10-membered ring, and more preferably a 3- to 7-membered ring, wherein the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, and more preferably 1 to 2 heteroatoms. In certain embodiments, the ring structure can have two cyclic rings. In some embodiments, the two cyclic rings can share two or more atoms, e.g., the rings are "fused rings." Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Examples of heterocyclic rings include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiene, tetrahydropyran, dihydropyran, tetrahydrothiopyranyl, thiomorpholine, thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine, oxepane, thiepane, oxazepine, diazepine, thiazepine, 2-pyrroline, 3-pyrroline, indoline, 2H-pyran, 4H-pyran, dioxanyl, 1,3-dioxolane, pyrazoline, dithiane, dithiolane, dihydropyran, dihydrothiene, dihydrofuran, pyrazolidinylimidazoline, imidazolidine, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptane, and azabicyclo[2.2.2]hexane. Spiro moieties are also included within the scope of this definition. Examples of heterocyclic groups in which ring atoms are substituted with oxo (=O) moieties are pyrimidinyl, methyl ... non- and 1,1-dioxo-thiomorpholine.

[0031] The term "heterocyclylene" refers to a divalent radical of a heterocyclic group as defined above.

[0032] As used herein, the term "heteroaryl" refers to a substituted or unsubstituted aromatic monocyclic structure, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring, whose ring structure contains at least one heteroatom (e.g., O, N, or S), preferably 1 to 4 or 1 to 3 heteroatoms, more preferably 1 or 2 heteroatoms. When two or more heteroatoms are present in a heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "polycyclyl," "polycyclic," and "polycyclic" ring systems having two or more cyclic rings in which two or more atoms are common to two adjacent rings, e.g., rings are "fused rings," where at least one of the rings is heteroaromatic; e.g., the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl. In some preferred embodiments, preferred polycycles have two to three rings. In certain embodiments, preferred polycyclic ring systems have two cyclic rings in which both rings are aromatic. In certain embodiments, each ring of the polycycle contains 3 to 10 atoms, preferably 5 to 7 atoms. For example, heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole, benzofuran, benzothiophene, cinnoline, phthalazine, quinazoline, carbazole, phenoxazine, quinoline, purine, and the like. In some embodiments, heteroaryl is a monocyclic aromatic group. In some embodiments, heteroaryl is a bicyclic aromatic group. In some embodiments, heteroaryl is a tricyclic aromatic group.

[0033] The term "heteroarylene" refers to a divalent radical of a heteroaryl group as defined above.

[0034] A heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked where such is possible. By way of example and not limitation, a carbon-bonded heterocycle or heteroaryl is bonded at the 2-, 3-, 4-, 5-, or 6-position of a pyridine, at the 3-, 4-, 5-, or 6-position of a pyridazine, at the 2-, 4-, 5-, or 6-position of a pyrimidine, at the 2-, 3-, 5-, or 6-position of a pyrazine, at the 2-, 3-, 4-, or 5-position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, at the 2-, 4-, or 5-position of an oxazole, imidazole, or thiazole, at the 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole, at the 2- or 3-position of an aziridine, at the 2-, 3-, or 4-position of an azetidine, at the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of a quinoline, or at the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of an isoquinoline.

[0035] By way of example and not limitation, nitrogen-linked heterocycles or heteroaryls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, at the 2-position of isoindole or indoline, at the 4-position of morpholine, and at the 9-position of carbazole or O-carboline.

[0036] Heteroatoms present in heteroaryl or heterocyclyl include oxidized forms such as NO, SO, and SO2.

[0037] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0038] The alkyl, alkylene, alkenylene, alkyne, alkynylene, cyclic alkyl, cycloalkylene, carbocyclyl, aryl, arylene, heterocyclyl, heterocycloalkylene, heteroaryl, and heteroarylene may be optionally substituted with one more (e.g., 2, 3, 4, 5, 6, or more) substituent.

[0039] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon atoms in the backbone of the compound. It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permissible valencies of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatom. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Illustratively, monofluoroalkyl is alkyl substituted with a fluoro substituent, and difluoroalkyl is alkyl substituted with two fluoro substituents. When there is more than one substitution on a substituent, it should be understood that each non-hydrogen substituent may be the same or different (unless otherwise stated). "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the application includes instances in which the circumstance occurs and instances in which the circumstance does not occur.For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus its application includes structures in which the non-hydrogen substituent is present and structures in which the non-hydrogen substituent is not present. When a carbon of a substituent is described as being optionally substituted with one or more of a list of substituents, then one or more of the hydrogens on that carbon (if present) can be replaced separately and / or together with independently selected optional substituents. When a nitrogen of a substituent is described as being optionally substituted with one or more of a list of substituents, then one or more of the hydrogens on that nitrogen (if present) can each be replaced with independently selected optional substituents. One exemplary substituent can be depicted as -NR'R", where R' and R" together with the nitrogen atom to which they are attached can form a heterocyclic ring. The heterocyclic ring formed by R′ and R″ together with the nitrogen atom to which they are attached can be partially or fully saturated. In some embodiments, the heterocyclic ring consists of 3 to 7 atoms. In other embodiments, the heterocyclic ring is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl, and thiazolyl.

[0040] The terms "substituent" and "group" are used interchangeably herein.

[0041] When a group of substituents is described collectively as being optionally substituted with one or more of a list of substituents, the group includes (1) non-substitutable substituents, (2) It may include (3) substitutable substituents that are not substituted by optional substituents, and / or (4) substitutable substituents that are substituted by one or more of the optional substituents.

[0042] When a substituent is described as being optionally substituted with up to a specified number of non-hydrogen substituents, the substituent may be either (1) unsubstituted or (2) substituted with up to the specified number of non-hydrogen substituents or up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as being heteroaryl optionally substituted with up to three non-hydrogen substituents, then a heteroaryl having fewer than three substitutable positions may be optionally substituted with up to the same number of non-hydrogen substituents as the heteroaryl has substitutable positions. Such substituents include, by way of non-limiting example, straight-chain, branched-chain, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [—NH(C═NH)NH], —OR 100 , N.R. 101 R 102 , -NO2, -NR 101 COR 102 , -SR 100 , -SOR 101 sulfoxides, represented by -SO2R 101 Sulfones, sulfonates -SO3M, sulfates -OSO3M, -SO2NR 101 R 102 Sulfonamide, cyano, azido, -COR 101 , -OCOR 101 , -OCONR 101 R 102 and polyethylene glycol units (-CH2CH2O) n R 101 (wherein M is H or a cation (Na + or K + etc.);R 101 , R 102 and R are each independently selected from H, a straight-chain, branched-chain, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, a polyethylene glycol unit (-CH2CH2O) n -R 104(wherein n is an integer of 1 to 24), aryl having 6 to 10 carbon atoms, heterocyclic ring having 3 to 10 carbon atoms, and heteroaryl having 5 to 10 carbon atoms; R 104 is H or a straight or branched chain alkyl having 1 to 4 carbon atoms, 100 , R 101 , R 102 , R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl in the group represented by are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, —OH, —CN, —NO2, and unsubstituted straight- or branched-chain alkyl having 1 to 4 carbon atoms. Preferably, the substituents for the above optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl include halogen, —CN, —NR 102 R 103 , -CF3, -OR 101 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101 and -SO3M.

[0043] The terms "compound" or "cytotoxic compound" are used interchangeably. They are intended to include compounds whose structures or formulas, or any derivatives, are disclosed in the present invention, or whose structures or formulas, or any derivatives, are incorporated by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts), and prodrugs, as well as prodrug salts, of compounds of all formulas disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. Specific reference to "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salts," "prodrugs," "prodrug salts," "conjugates," "conjugate salts," "solvates," "hydrates," or "polymorphs" in particular embodiments of the invention described in this application should not be construed as intending to exclude these forms in other embodiments of the invention in which the term "compound" is used without mention of these other forms.

[0044] As used herein, the term "conjugate" refers to a compound or cell-bound molecule described herein. It refers to a derivative linked to a combination drug.

[0045] As used herein, the term "linkable to a cell binding agent" refers to the compounds described herein or derivatives thereof containing at least one linking group or precursor thereof suitable for linking these compounds or derivatives thereof to a cell binding agent.

[0046] The term "precursor" of a given group refers to any group that can, by any deprotection, chemical modification, or coupling reaction, become that group.

[0047] The term "linked to a cell binding agent" refers to a conjugate molecule comprising at least one compound described herein, or a derivative thereof, attached to a cell binding agent via a suitable linking group or precursor thereof.

[0048] The term "chiral" refers to a molecule that has the property of not being superimposable on its mirror image partner, and the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0049] The term "stereoisomers" refers to compounds that have identical chemical constitution and connectivity but differ with regard to the arrangement of their atoms in space and cannot be interconverted by rotation about a single bond.

[0050] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography.

[0051] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0052] The definitions and conventions of stereochemistry used herein generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and, therefore, can exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including, but not limited to, diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e., have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to describe the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to designate the sign of rotation of plane-polarized light by a compound; (-) or l means the compound is levorotatory. A compound with the prefix (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. Specific stereoisomers may be referred to as enantiomers, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, lacking optical activity.

[0053] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions via rearrangement of some of the bond electrons.

[0054] The term "prodrug" as used herein refers to a precursor or derivative form of a compound of the present invention that can be activated or converted, enzymatically or by hydrolysis, to a more active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical See, "Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, ester-containing prodrugs, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which can be converted to more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention include, but are not limited to, the compounds of the present invention and chemotherapeutic agents, as described above.

[0055] The term "prodrug" is also meant to include derivatives of compounds that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to yield compounds of the present invention. Prodrugs may become active only upon such reaction under biological conditions, or may have activity in their unreacted, reacted form. Examples of prodrugs contemplated by the present invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulas disclosed herein that contain a biohydrolyzable moiety, such as a biohydrolyzable amide, biohydrolyzable ester, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ureide, and biohydrolyzable phosphate analog. Other examples of prodrugs include derivatives of compounds of any one of the formulas disclosed herein that contain a -NO, -NO2, -ONO, or -ONO2 moiety. Prodrugs can typically be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed.); see also Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs."

[0056] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, and glucuronic acid. Pharmaceutically acceptable salts include saccharinate, formate, benzoate, glutamate, methanesulfonate ("mesylate"), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may include the inclusion of another molecule such as acetate, succinate, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. When multiple charged atoms are part of the pharmaceutically acceptable salt, multiple counterions are possible. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0057] When the compound of the present invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, or a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid.

[0058] If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base, such as a (primary, secondary, or tertiary) amine, alkali metal hydroxide, or alkaline earth metal hydroxide. Examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines such as piperidine, morpholine, and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0059] As used herein, the term "solvate" refers to a compound that further contains a stoichiometric or non-stoichiometric amount of a solvent, such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2-propanol, etc., bound by non-covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by adding at least one molar equivalent of a hydroxylic solvent, such as methanol, ethanol, 1-propanol, 2-propanol, or water, to the compound to cause solvation or hydration of the imine moiety.

[0060] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. As used herein, "abnormal cell growth," unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example: (1) tumor cells (tumors) that grow due to the expression of mutant tyrosine kinases or the overexpression of receptor tyrosine kinases; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumor that grows due to receptor tyrosine kinases; (4) any tumor that grows due to aberrant serine / threonine kinase activation; and (5) abnormal growth of benign and malignant cells of other proliferative diseases in which aberrant serine / threonine kinase activation occurs.

[0061] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" is one or more comprises a plurality of cancer cells and / or benign or precancerous cells.

[0062] "Therapeutic agent" includes both biological agents such as antibodies, peptides, proteins, enzymes or chemotherapeutic agents.

[0063] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.

[0064] A "metabolite" is a product produced by metabolism in the body of a specified compound, its derivative, or conjugate, or salt thereof. Metabolites of a compound, its derivative, or conjugate can be identified using routine techniques known in the art, and their activity can be determined using tests such as those described herein. Such products can result, for example, from oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of an administered compound. Thus, the present invention includes metabolic products of a compound, its derivative, or conjugate of the present invention, including compounds, its derivative, or conjugate, produced by a process comprising contacting a compound, its derivative, or conjugate of the present invention with a mammal for a period of time sufficient to yield a metabolic product thereof.

[0065] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammal being treated therewith.

[0066] The term "protecting group" or "protecting moiety" refers to a substituent commonly used to block or protect a particular functional group while reacting other functional groups on a compound, its derivatives, or its conjugates. For example, an "amine protecting group" or "amino protecting moiety" is a substituent attached to an amino group that blocks or protects the amino functionality in a compound. Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7, J. Wiley & Sons, NJ) and are exemplified by carbamates such as methyl and ethyl carbamates, FMOC, substituted ethyl carbamates, carbamates that cleave by 1,6-β-elimination (also referred to as "self-immolative"), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a review of protecting groups and their use, see P.G.M.Wuts & T.W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.

[0067] The term "leaving group" refers to a charged or uncharged moiety that leaves during substitution or replacement. Such leaving groups are well known in the art and include, but are not limited to, halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates, nitriles, azides, carbamates, disulfides, thioesters, thioethers, and diazonium compounds.

[0068] The term "bifunctional crosslinker," "bifunctional linker," or "crosslinker" refers to a modifying agent that has two reactive groups; one of which can react with a cell-binding agent and the other of which can react with a cytotoxic compound, linking the two moieties together. Such bifunctional crosslinkers are well known in the art (see, e.g., Isalm and Dent in Bioconjugation chapter 5, p. 218-363, Groves (See Dictionaries Inc., New York, 1999). For example, bifunctional crosslinkers that allow linkage via a thioether bond include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) for introducing a maleimide group, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) for introducing an iodoacetyl group. Other bifunctional crosslinkers that introduce maleimide or haloacetyl groups into cell-binding agents are well known in the art (see U.S. Patent Application Publication Nos. 2008 / 0050310 and 20050169933; Pierce Biotechnology Inc., PO Box 117, Rockland, IL). 61105, USA), which include, but are not limited to, bis-maleimide polyethylene glycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(β-maleimidopropyloxy) succinimide ester (BMPS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, the "long chain" analogue of SMCC (LC-SMCC) N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidyl-2,These include 3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS), N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

[0069] Heterobifunctional crosslinkers are bifunctional crosslinkers with two different reactive groups. Heterobifunctional crosslinkers containing both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link the cytotoxic compounds described herein to cell-binding agents (e.g., antibodies). Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0070] Bifunctional crosslinkers that allow for the linkage of cell-binding agents and cytotoxic compounds via disulfide bonds are known in the art, including N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-5-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-6-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-7-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-8-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-9-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-1 ... Examples of suitable crosslinkers include N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB). Other bifunctional crosslinkers that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Patent Nos. 6,913,748, 6,716,821, and U.S. Patent Application Publication Nos. 20090274713 and 20100129314, all of which are incorporated herein by reference. Alternatively, crosslinkers that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, can be used.

[0071] As defined herein, a "reactive moiety" or "reactive group" refers to a chemical moiety that forms a covalent bond with another chemical group. For example, a reactive moiety can react with a specific group on a cell-binding agent (CBA) to form a covalent bond. In some embodiments, the reactive moiety is an amine-reactive group that can form a covalent bond with the ε-amine of a lysine residue located on the CBA. In other embodiments, the reactive moiety is an aldehyde-reactive group that can form a covalent bond with an aldehyde group located on the CBA. In yet other embodiments, the reactive moiety is a thiol-reactive group that can form a covalent bond with the thiol group of a cysteine ​​residue located on the CBA.

[0072] As defined herein, a "linker," "linker moiety," or "linking group" refers to a moiety that connects two groups, such as a cell-binding agent and a cytotoxic compound, together. Typically, a linker is substantially inert under the conditions under which the two groups it connects are linked. A bifunctional crosslinker may contain two reactive groups, one at each end of the linker moiety, such that one reactive group can first react with a cytotoxic compound to provide a compound bearing a linker moiety, and then the second reactive group can react with a cell-binding agent. Alternatively, one end of the bifunctional crosslinker can first react with a cell-binding agent to provide a cell-binding agent bearing a linker moiety, and then the second reactive group can react with a cytotoxic compound. The crosslinking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, peptidase-labile bonds, and esterase-labile bonds (see, e.g., U.S. Patent Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026, and 7,414,073). Disulfide bonds, thioethers, and peptidase-labile bonds are preferred. Other linkers that can be used in the present invention include non-cleavable linkers such as those detailed in U.S. Patent Application Publication No. 20050169933, or charged or hydrophilic linkers described in U.S. Patent Application Publication Nos. 2009 / 0274713, 2010 / 01293140, and WO2009 / 134976, each of which is expressly incorporated herein by reference.

[0073] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, serinocysteine, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium compounds. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but contain the same amino acids as naturally occurring amino acids. The amino acids retain the same basic chemical structure as those of the naturally occurring amino acids. One particular amino acid that can be used is citrulline, a derivative of arginine that is involved in the production of urea in the liver. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of amino acids but function similarly to naturally occurring amino acids. The term "unnatural amino acid" is intended to refer to the "D" stereochemical form of the 20 naturally occurring amino acids listed above. Furthermore, it is understood that the term unnatural amino acid includes analogs of natural amino acids or their D isomers, as well as synthetically modified forms of natural amino acids. Synthetic modified forms include, but are not limited to, amino acids with side chains shortened or extended by up to two carbon atoms, amino acids containing optionally substituted aryl groups, and amino acids containing halogenated groups, preferably alkyl and aryl halogenated groups, as well as N-substituted amino acids, such as N-methyl-alanine. The amino acid or peptide can be attached to a linker / spacer or cell-binding agent via the terminal amine or terminal carboxylic acid of the amino acid or peptide. Amino acids can also be attached to linkers / spacers or cell binding agents through side chain reactive groups such as, but not limited to, the thiol group of cysteine, the epsilon amine lysine of lysine, or the side chain hydroxyl of serine or threonine.

[0074] In some embodiments, the amino acid is NH2-C(R aa’ R aa )—C(═O)OH (wherein R aa and R aa’ are each independently H, an optionally substituted straight, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, or heterocyclyl, or R aa and the N-terminal nitrogen atom may together form a heterocyclic ring (e.g., as in proline). The term "amino acid residue" refers to -NH-C(R aa’ R aa )-C(=O)O- refers to the corresponding residue where one hydrogen atom has been removed from the amine and / or a hydroxyl group has been removed from the carboxy terminus of an amino acid.

[0075] As used herein, amino acids may be L or D isomers. Unless otherwise specified, when an amino acid is referred to, it may be an L or D isomer or a mixture thereof. In certain embodiments, when a peptide is referred to by its amino acid sequence, each of the amino acids may be an L or D isomer unless otherwise specified. When one of the amino acids in a peptide is designated as a D isomer, the other amino acid(s) are L isomers unless otherwise specified. For example, the peptide D-Ala-Ala means D-Ala-L-Ala.

[0076] Amino acids and peptides may be protected by blocking groups. A blocking group is an atom or chemical moiety that protects the N-terminus of an amino acid or peptide from undesired reactions and can be used during the synthesis of a drug-ligand conjugate. It should remain attached to the N-terminus throughout the synthesis and can be removed by chemical or other conditions that selectively achieve its removal after the synthesis of the drug conjugate is complete. Blocking groups suitable for N-terminal protection are well known in the field of peptide chemistry. Exemplary blocking groups include, but are not limited to, methyl esters, tert-butyl esters, 9-fluorenylmethylcarbamate (Fmoc), and carbobenzoxy (Cbz).

[0077] The term "protease-cleavable peptide" refers to a peptide containing a cleavage recognition sequence for a protease. As used herein, a protease is an enzyme that can cleave a peptide bond. A cleavage recognition sequence for a protease is a specific amino acid sequence that is recognized by the protease during proteolytic cleavage. Many protease cleavage sites are known in the art, and these and other cleavage sites can be included in the linker moiety. See, for example, Matayoshi et al. Science 247:954 (1990); Dunn et al. Meth. Enzymol. 241:254 (1994); Seidah et al. Meth. Enzymol. 244:175 (1999). 4);Thornberry,Meth.Enzymol.244:615(1994);Weber et al.Meth.Enzymol.244:595(1994);Smith et al.Meth.Enzymol.244:412(1994);Bouvier et al.Meth.Enzymol.248:614(1995),Hardy et al,in AMYLOID PROTEIN PRECURSOR See IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, ed. Masters et al. pp. 190-198 (1994).

[0078] The peptide sequence is selected based on its ability to be cleaved by a protease, non-limiting examples of which include cathepsin B, C, D, H, L and S, and furin. Preferably, the peptide sequence can be cleaved in vitro by a suitable isolated protease, which can be tested using in vitro protease cleavage assays known in the art.

[0079] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by lysosomal proteases. Lysosomal proteases are proteases that are primarily located in lysosomes, but can also be located in endosomes. Examples of lysosomal proteases include, but are not limited to, cathepsins B, C, D, H, L, and S, and furin.

[0080] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by tumor-associated proteases, such as proteases found on the surface of cancer cells or extracellularly near tumor cells, non-limiting examples of which include thimet oligopeptidase (TOP), CD10 (neprilysin), matrix metalloproteases (such as MMP2 or MMP9), type II transmembrane serine proteases (such as hepsin, testisin, TMPRSS4, or matriptase / MT-SP1), legumain, and enzymes described in the following reference (Current Topics in Developmental Biology: Cell Surface Proteases, vol. 54 Zucker S. 2003, Boston, MA). The ability of a peptide to be cleaved by a tumor-associated protease can be tested using in vitro protease cleavage assays known in the art.

[0081] The term "cation" refers to an ion that has a positive charge. A cation is a monovalent ion (e.g., Na + , K. + etc.), divalent (e.g., Ca 2+ , Mg2+ ) or multivalent (e.g., Al 3+ In some embodiments, the cation is monovalent.

[0082] The term "therapeutically effective amount" refers to the amount of an active compound or conjugate that elicits a desired biological response in a subject. Such responses include alleviating symptoms of the disease or disorder being treated, preventing, inhibiting, or delaying the recurrence of disease symptoms or the disease itself, increasing the subject's lifespan compared to the absence of treatment, or preventing, inhibiting, or delaying the progression of disease symptoms or the disease itself. Determining an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. The toxicity and therapeutic efficacy of Compound I can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of a compound or conjugate or other therapeutic agent of the present invention administered to a subject depends on the stage, category, and condition of multiple myeloma, as well as the subject's characteristics, such as general health, age, sex, weight, and drug tolerance. The effective amount of a compound or conjugate or other therapeutic agent of the present invention administered also depends on the route of administration and dosage form. Dosage amount and interval can be adjusted individually to achieve plasma levels of the active compound sufficient to produce the desired therapeutic effect.

[0083] Cell-binding agent-cytotoxic drug conjugates In a first aspect, the present invention provides a cell-binding agent-cytotoxic drug conjugate comprising a cell-binding agent as described herein covalently attached to one or more molecules of a cytotoxic compound as described herein.

[0084] In a first embodiment, the conjugate of the invention has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, CB is a cell binding agent; L2 is absent or a spacer; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~6 alkyl (e.g., R 1 and R 2 are each independently H or C 1~3 alkyl); L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0085] In one embodiment, L1 is -L1'-C(=O)-; L1' is alkylene, alkenylene, alkynylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, wherein the -C(=O)- group in L1 is connected to D.

[0086] In another embodiment, R 1 and R 2 At least one of R is H. In more specific embodiments, 1 and R 2 One of them is H and the other is Me.

[0087] In a first specific embodiment, for the conjugate of formula (I), R 1 and R 2 are each independently H or Me. In more specific embodiments, R 1 and R 2 are both H.

[0088] In a second specific embodiment, for the conjugate of formula (I), L is -L'-C(=O)-; L is alkylene or cycloalkylene, where the -C(=O)- group in L is connected to D; and the remaining variables are as described above in the first embodiment or the first specific embodiment. In a more specific embodiment, L is C 1~10In another more specific embodiment, L is C 1~20 It is alkylene.

[0089] In a third specific embodiment, for the conjugate of formula (I), L is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me; the remaining variables are as described above in the first embodiment or first specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me.

[0090] In a fourth specific embodiment, for the conjugate of formula (I), L is -CR 3 R 4 -(CH2) 2~5 -C(=O)- or -CR 3 R 4 -(CH2) 3~5 -C(= O)-;R 3 and R 4 are each independently H or Me; the remaining variables are as described above in the first embodiment or first specific embodiment. In a more specific embodiment, R 3 and R 4 and R are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0091] In a fifth specific embodiment, for the conjugate of formula (I), L is -(CH) 4~6 is —C(═O)—; and the remaining variables are as defined above in the first embodiment or first specific embodiment.

[0092] In a sixth specific embodiment, for the conjugate of formula (I), L2 has the following structural formula: [ka] Represented by [In the formula, R A is alkylene, cycloalkylalkylene, arylene, heteroarylene, or heterocyclylene; R B and R C are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W does not exist or [ka] where s2' represents the site connected to V, and R A or J CB and s3' is R B , V', R C or J A Show the part that connects to; J CB is -C(=O)-, [ka] where s1 represents the site attached to the cell-binding agent CB and s2 represents the site attached to R A indicates the part connected to; R a , R b , R c , and R e is, independently for each occurrence, H or alkyl; J A is -C(=O)-; the remaining variables are as defined above in the first embodiment or the first, second, third, fourth or fifth specific embodiment.

[0093] In a more specific embodiment of the sixth specific embodiment, RA is alkylene, cycloalkylalkylene, or arylene; W is absent or [ka] and; J CB is -C(=O)-, [ka] is.

[0094] In more specific embodiments, p is 0 and R C is not present; the remaining variables are as described above in the sixth specific embodiment.

[0095] In another more specific embodiment, J CB is -C(=O)- or [ka] and The remaining variables are as described above in the sixth specific embodiment.

[0096] In a seventh embodiment, for the conjugate in formula (I), L2 has the following structural formula: [ka] Represented by [In the formula, R x , R y , R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + , or -OH, halogen, -SO3H, -NR 40 R 41 R 42 + C optionally replaced with1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; l and k each independently represent an integer of 1 to 10; s1 represents the site connected to CBA, and s3 represents the site connected to group A; The remaining variables are as described above in the first embodiment or the first, second, third, fourth or fifth specific embodiment.

[0097] In more specific embodiments, R x , R y , R x’ and R y’ are all H; the remaining variables are as described above in the seventh specific embodiment.

[0098] In another more specific embodiment, l and k are each independently an integer from 2 to 6; the remaining variables are as described above in the seventh specific embodiment.

[0099] In an even more specific embodiment, R x , R y , R x’ and R y’ are all H; l and k are each independently an integer from 2 to 6; and the remaining variables are as described above in the seventh specific embodiment.

[0100] In another more specific embodiment, L2 has the following structural formula: [ka] Represented by [In the formula, R x and R y are both H; l and l1 each independently represent an integer of 1 to 10; k1 is an integer from 1 to 12 (e.g., 2, 4, 6, 8, 10, or 12)].

[0101] In one embodiment, l and l1 are each an integer from 2 to 6; and k1 is an integer from 2 to 6 (eg, 2, 4, or 6).

[0102] In an eighth specific embodiment, for the conjugate in formula (I), A is a peptide cleavable by a protease; the remaining variables are as described above in the first embodiment or the first, second, third, fourth, fifth, sixth, or seventh specific embodiments. In a more specific embodiment, A is a peptide cleavable by a protease expressed in tumor tissue.

[0103] In a ninth specific embodiment, for the conjugates in formula (I), A is selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, each independently as an L or D isomer, -NH-CR 1 R 2 and the remaining variables are as described above in the first embodiment or the first, second, third, fourth, fifth, sixth or seventh specific embodiment. In a more specific embodiment, -NH-CR 1 R 2 The amino acid connected to -S-L1-D is an L amino acid.

[0104] In a tenth specific embodiment, for the conjugates in formula (I), A is selected from Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Al a-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-A la-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D- Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Al aD-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is connected to the L2 group and the last amino acid in each peptide is selected from the group consisting of -NH-CR 1 R 2 -S-L1-D; the remaining variables are as described above in the first embodiment or the first, second, third, fourth, fifth, sixth, or seventh specific embodiment. In a more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In another more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0105] In an eleventh specific embodiment, for the conjugates in formula (I), D is a maytansinoid; and the remaining variables are as described above in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth specific embodiments. In a more specific embodiment, D is represented by the following formula: [ka] is expressed by

[0106] In more specific embodiments, D is of the formula: [ka] is expressed by

[0107] In a twelfth specific embodiment, the conjugate of the invention has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, [ka] is a cell-binding agent connected to the L2 group via an amine group (e.g., a Lys amine group); [ka] is a cell-binding agent attached to the L2 group via a thiol group (e.g., a Cys thiol group); R 3 and R 4 are each independently H or Me; m1, m3, p1, n1, r1, and t1 each independently represent an integer of 1 to 10; m2, n2, p2, r2, and t2 each independently represent an integer of 1 to 19; t3 is an integer from 1 to 12; D1 is expressed by the following formula: [ka] is represented by A is as defined above in the eighth, ninth, or tenth specific embodiments.

[0108] In more specific embodiments, m1, m3, p1, n1, and r1 are each independently an integer from 1 to 6; m2, n2, p2, and r2 are each independently an integer from 1 to 7.

[0109] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0110] In more specific embodiments, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); the remaining variables are as described above in the twelfth specific embodiment.

[0111] In another more specific embodiment, m1, r1, n1, p1, and m3 are each independently and m2, p2, n2, and r2 are each independently an integer from 3 to 5. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer from 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer from 3 to 6.

[0112] In more specific embodiments, m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In more specific embodiments, m2, n2, p2, r2, and t2 are each independently an integer of 3 to 6. In more specific embodiments, m2, n2, p2, r2, and t2 are each independently 5.

[0113] In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 2 to 10, and m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 3 to 6, and m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 3 to 6, and m2, n2, p2, r2, and t2 are each independently an integer of 3 to 6.

[0114] In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 12. In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 6. In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 4. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 12. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 6. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 4.

[0115] In more specific embodiments, r2 and r1 are each independently an integer of 2 to 6. In more specific embodiments, r2 is an integer of 3 to 5, and r1 is an integer of 2 to 6. In more specific embodiments, r2 is an integer of 3 to 5, and r1 is an integer of 2 to 4. In more specific embodiments, r2 is 4, and r1 is 2. In more specific embodiments, r2 is 4, and r1 is 3. In more specific embodiments, r2 is 4, and r1 is 4. In more specific embodiments, r2 is 4, and r1 is 5. In more specific embodiments, r2 is 4, and r1 is 6.

[0116] In yet another more specific embodiment, R 3 and R 4 are both Me. Alternatively, R 3 and R 4 are both H.

[0117] In another specific embodiment, the conjugate has the following formula: [ka] Represented by [In the formula, r1 and t1 are each an integer of 2 to 10; r2 and t2 are each an integer of 2 to 19; t3 is an integer from 2 to 12 (e.g., t3 is 2, 4, 6, 8, 10, or 12)].

[0118] In more specific embodiments, r1 and t1 are each an integer from 2 to 6; r2 and t2 are each an integer from 2 to 5; and t3 is an integer from 2 to 6 (e.g., t3 is 2, 4, or 6).

[0119] In a thirteenth specific embodiment, the conjugate of the invention has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala- tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); D1 is expressed by the following formula: [ka] is expressed by

[0120] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0121] Also, in a thirteenth specific embodiment, the conjugate of the invention has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala- Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, DA la-Pro, or D-Ala-tBu-Gly), D1 is expressed by the following formula: [ka] [represented by]

[0122] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0123] In another more specific embodiment, the conjugate has the following formula: [ka] [ka] Represented by [In the formula, D1 is expressed by the following formula: [ka] [represented by]

[0124] In some embodiments, for the conjugates of the invention described above (e.g., the conjugates described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), the cell binding agent (CBA) may be any one of the cell binding agents (CBAs) described herein.

[0125] In some embodiments, for the conjugates of the invention described above (e.g., the conjugates described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), the cell binding agent (CBA) binds to a cell selected from the group consisting of tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; CA6, CAK1, CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, The antibody binds to a target cell selected from a cell expressing CD38, CD40, CD44, CD56, CD123, CD138, CanAg, CALLA, CEACAM5, FGFR3, LAMP1, p-cadherin, CA6, TROP-2, DLL-3, CDH6, AXL, SLITRK6, ENPP3, BCMA, tissue factor (TF), CD352, Her-2, or Her-3 antigen; or a cell expressing insulin growth factor receptor, epidermal growth factor receptor, nectin-4, mesothelin, GD3, prolactin receptor, and folate receptor.

[0126] In some embodiments, for the conjugates of the invention described above (e.g., the conjugates described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), the cell binding agent is an antibody or antigen-binding fragment thereof, a single-chain antibody, a single-chain antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a lymphokine, a hormone, a vitamin, a growth factor, a colony-stimulating factor, a nutrient transport molecule, a Bicycles® peptide, or pentarin.

[0127] In some embodiments, for the conjugates of the invention described above (e.g., the conjugates described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), the cell binding agent is an antibody or an antigen-binding fragment thereof. In other embodiments, the cell binding agent is a resurfaced antibody or a resurfaced antibody fragment thereof. In some embodiments, the cell binding agent is a monoclonal antibody or a monoclonal antibody fragment thereof. In some embodiments, the cell binding agent is a humanized antibody or a humanized antibody fragment thereof. In other embodiments, the cell binding agent is a chimeric antibody or a chimeric antibody fragment thereof. In some embodiments, the cell binding agent is an anti-folate receptor antibody or or an antibody fragment thereof, an anti-EGFR antibody or an antibody fragment thereof, an anti-CD33 antibody or an antibody fragment thereof, an anti-CD19 antibody or an antibody fragment thereof, an anti-Muc1 antibody or an antibody fragment thereof, or an anti-CD37 antibody or an antibody fragment thereof.

[0128] In one embodiment, the conjugate of the invention has the following formula: [ka] Represented by [wherein Ab is an anti-folate receptor antibody].

[0129] In another embodiment, the conjugate of the invention comprises: Below formula: [ka] Represented by wherein Ab is an anti-folate receptor antibody. In a specific embodiment, the anti-folate receptor antibody comprises (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO: 4); a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO: 5); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 6); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO: 7); a light chain CDR2 comprising RASNLEA (SEQ ID NO: 8); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO: 9); wherein Xaa1 is selected from K, Q, H, and R; Xaa2 is selected from Q, H, N, and R; and Xaa3 is selected from G, E, T, S, A, and V. Preferably, the heavy chain CDR2 sequence comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 10). In another specific embodiment, the anti-folate receptor antibody comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In another specific embodiment, the anti-folate receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 13 (huFOLRl). In another specific embodiment, the anti-folate receptor antibody is encoded by plasmid DNA deposited with the ATCC on April 7, 2010, and having ATCC accession numbers PTA-10772 and PTA-10773 or 10774. See WO2011 / 106528 (incorporated herein by reference).

[0130] In some embodiments, for the conjugates of the invention described above (e.g., the conjugates described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), q is 1 to 10, is an integer from 1 to 8, or from 2 to 5. In some embodiments in conjugates covalently attached to a cytotoxic drug via a Cys thiol group, q is 1 or 2. In one embodiment, q is 2.

[0131] In some embodiments, for compositions (e.g., pharmaceutical compositions) comprising a conjugate of the invention described above (e.g., a conjugate described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiments), the average number of cytotoxic drugs per cell-binding agent (CBA, e.g., antibody) in the composition (i.e., the average value of q), also known as the drug-antibody ratio (DAR), is within the range of 1.0 to 8.0. In some embodiments, the DAR is within the range of 1.0 to 5.0, 1.0 to 4.0, 1.0 to 3.4, 1.0 to 3.0, 1.5 to 2.5, 2.0 to 2.5, or 1.8 to 2.2.

[0132] Compounds of the Invention In a second aspect, the present invention provides maytansinoid derivatives as described herein.

[0133] In a second embodiment, the compound of the present invention has formula (II), (III) or (IV): L2'-A-NH-CR 1 R 2 -S-L1-D(II), A'-NH-CR 1 R 2 -S-L1-D(III), or L3-A-NH-CR 1 R 2 -S-L1-D(IV) or a pharmaceutically acceptable salt thereof [In the formula, L2' is absent or a spacer having a reactive moiety capable of forming a covalent bond with a cell-binding agent; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~6 alkyl (e.g., R 1 and R 2 are each independently H or C 1~3 alkyl); L1 is a spacer; D-L1-SH is a cytotoxic drug; q is an integer from 1 to 20; A' is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; L3 is given by the following formula: [ka] Represented by; R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + , or -OH, halogen, SO3H or NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; and k is an integer between 1 and 10.

[0134] In one embodiment, L1 is -L1'-C(=O)-; L1' is alkylene, alkenylene, alkynylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, wherein the -C(=O)- group in L1 is connected to D.

[0135] In another embodiment, R 1 and R 2 At least one of R is H. In more specific embodiments, 1 and R 2 One of the groups is H and the other is Me.

[0136] In a fourteenth specific embodiment, for compounds of formula (II), (III) and (IV), R 1 and R 2 are each independently H or Me; the remaining variables are as described above in the second embodiment. In more specific embodiments, R 1 and R 2 are both H.

[0137] In a fifteenth specific embodiment, for compounds of formula (II), (III), and (IV), L is -L'-C(=O)-; L is alkylene or cycloalkylene, where the -C(=O)- group in L is connected to D; and the remaining variables are as described above in the second or fourteenth specific embodiment. In a more specific embodiment, L is C 1~10 In another more specific embodiment, L is C 1~20 It is alkylene.

[0138] In a sixteenth specific embodiment, for compounds of formula (II), (III) and (IV), L is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4are each independently H or Me; the remaining variables are as described above in the second embodiment or the fourteenth specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me.

[0139] In a seventeenth specific embodiment, for compounds of formula (II), (III) and (IV), L is -CR 3 R 4 -(CH2) 2~5 -C(=O)- or -CR 3 R 4 -(CH2) 3~5 -C(=O)-; R 3 and R 4 are each independently H or Me; the remaining variables are as described above in the second embodiment or the fourteenth specific embodiment. In a more specific embodiment, R 3 and R 4 and R are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0140] In an eighteenth specific embodiment, for compounds of formula (II), (III) and (IV), L is -(CH) 4~6 the remaining variables are as defined above in the second embodiment or the fourteenth specific embodiment.

[0141] In a 19th specific embodiment, for compounds of formula (II), (III), and (IV), A or A' is a peptide cleavable by a protease; the remaining variables are as described above in the second embodiment or the 14th, 15th, 16th, 17th, or 18th specific embodiments. In a more specific embodiment, A or A' is a peptide cleavable by a protease expressed in tumor tissue.

[0142] In a twentieth specific embodiment, for compounds of formula (II), (III), and (IV), A or A′ is independently selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, each as an L or D isomer, -NH-CR 1 R 2 and the remaining variables are as described above in the second embodiment or the fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth specific embodiments. In a more specific embodiment, -NH-CR 1 R 2 The amino acid connected to -S-L1-D is an L amino acid.

[0143] In a twenty-first specific embodiment, for compounds of formula (II), (III) and (IV), A or A′ is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, D- Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Al a-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-A and D-Ala-tBu-Gly, D-Val-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is connected to the L2 group and the last amino acid in each peptide is connected to the L2 group. 1 R 2 -S-L1-D; the remaining variables are as described above in the second embodiment or the fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth specific embodiments. In a more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In another more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0144] When a specific amino acid or peptide sequence is referred to for A herein, this is one in which a hydrogen atom has been removed from the amino terminus of the amino acid connected to the L2 group and the hydroxyl group has been replaced with -NH-CR 1 R2 -S-L1-D means an amino acid residue or a peptide containing amino acid residues removed from the carboxy terminus of the amino acid connected to S-L1-D. For example, when A is represented by Ala-Ala-Ala, this refers to -NH-CH(CH3)-C(=O)-NH-CH(CH3)-C(=O)-NH-CH(CH3)-C(=O)-.

[0145] When a specific amino acid or peptide sequence is referred to for A' herein, this means that the hydroxyl group is -NH-CR 1 R 2 -S-L1-D means an amino acid residue or a peptide containing amino acid residues removed from the carboxy terminus of the amino acid connected to A. For example, when A' is represented by Ala-Ala-Ala, this refers to NH-CH(CH)-C(=O)-NH-CH(CH)-C(=O)-NH-CH(CH)-C(=O)-.

[0146] In a 22nd specific embodiment, for compounds of formula (II), (III), and (IV), D is a maytansinoid; the remaining variables are as described above in the second embodiment or the 14th, 15th, 16th, 17th, 18th, 19th, 20th, or 21st specific embodiments. In a more specific embodiment, D is a group represented by the following formula: [ka] is expressed by

[0147] In another more specific embodiment, D is of the formula: [ka] is expressed by

[0148] In a twenty-third specific embodiment, for compounds of formula (II), L2' has the following structural formula: [ka] Represented by [In the formula, R A is alkylene, cycloalkylalkylene, arylene, heteroarylene, or heterocyclylene; R B and R C are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W does not exist or [ka] where s2' is V, R A or J CB s3' indicates the site connected to R B , V', R C or J A Show the part that connects to; J CB ' is -C(=O)OH-, -COE, [ka] X 1 -CR b R c -C(=O)-, X 1 -CR b R c -C(=O)-NR e -, [ka] and R a , R b , R c , and R e is, independently for each occurrence, H or alkyl; X 1 is a halogen (e.g., —Cl, —Br, or —I); COE is a reactive ester; J A is —C(═O)—; and the remaining variables are as defined above in the second embodiment or the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, or twenty-second specific embodiment.

[0149] In a more specific embodiment, for the compound of the twenty-third specific embodiment, R A is alkylene, cycloalkylalkylene, or arylene; W is absent or [ka] and; J CB ' is -C(=O)OH, -COE, [ka] and; The remaining variables are as described above in the twenty-third specific embodiment.

[0150] In more specific embodiments, p is 0 and R c does not exist; The remaining variables are as described above in the twenty-third specific embodiment.

[0151] In other more specific embodiments, J CB ' is -C(=O)OH, -COE or [ka] and The remaining variables are as described above in the twenty-third specific embodiment.

[0152] In a twenty-fourth embodiment, for compounds of Formula (II), L2' has the following structural formula: [ka] [ka] is expressed by [In the formula, R x , R y , R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + , or -OH, halogen, -SO3H, NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; l and k each independently represent an integer of 1 to 10; J CB is -C(=O)OH or -COE; The remaining variables are as described above in the second embodiment or the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second or twenty-third specific embodiments.

[0153] In more specific embodiments, R x , R y , R x’ and R y’ are all H; the remaining variables are as described above in the twenty-fourth specific embodiment.

[0154] In another more specific embodiment, l and k are each independently an integer from 2 to 6; the remaining variables are as described above in the twenty-fourth specific embodiment.

[0155] In an even more specific embodiment, R x , R y , Rx’ and R y’ are all H; l and k are each independently an integer from 2 to 6; and the remaining variables are as described above in the twenty-fourth specific embodiment.

[0156] In another more specific embodiment, L2' has the following structural formula: [ka] Represented by [In the formula, R x and R y are both H; l and l1 are each an integer from 1 to 10; k1 is an integer between 1 and 12.

[0157] In even more specific embodiments, l and l1 are each integers from 2 to 6.

[0158] In a twenty-fifth specific embodiment, for compounds of formula (IV), R x’ and R y’ are both H; and the remaining variables are as described above in the second embodiment or the fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, or twenty-second specific embodiments.

[0159] In a more specific embodiment, k is an integer from 2 to 6; the remaining variables are as described above in the twenty-fifth specific embodiment.

[0160] In another more specific embodiment, k is 3; the remaining variables are as described above in the twenty-fifth specific embodiment.

[0161] In a 26th specific embodiment, the compound of formula (II) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, p1, and t1 each independently represent an integer of 1 to 10; m2, n2, r2, p2, and t2 each independently represent an integer of 1 to 19; t3 is an integer between 1 and 12; J CB ' is -C(=O)OH or -COE; D1 is expressed by the following formula: [ka] Represented by; The remaining variables are as described above in the second embodiment or the nineteenth, twentieth or twenty-first specific embodiments.

[0162] In more specific embodiments, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In another more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0163] In more specific embodiments, m1, m3, p1, n1, and r1 are each independently an integer from 1 to 6; m2, n2, p2, and r2 are each independently an integer from 1 to 7.

[0164] In another more specific embodiment, m1, p1, r1, n1, and m3 are each independently an integer from 2 to 4; m2, p2, n2, and r2 are each independently an integer from 3 to 5. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer from 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer from 3 to 6.

[0165] In more specific embodiments, m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In more specific embodiments, m2, n2, p2, r2, and t2 are each independently an integer of 3 to 6. In more specific embodiments, m2, n2, p2, r2, and t2 are each independently 5.

[0166] In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 2 to 10, and m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 3 to 6, and m2, n2, p2, r2, and t2 are each independently an integer of 2 to 10. In a more specific embodiment, m1, m3, p1, n1, r1, and t1 are each independently an integer of 3 to 6, and m2, n2, p2, r2, and t2 are each independently an integer of 3 to 6.

[0167] In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 12. In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 6. In a more specific embodiment, r2 and t2 are each independently an integer from 2 to 6, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 4. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 12. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 6. In a more specific embodiment, r2 and t2 are each independently an integer from 3 to 5, r1 and t1 are each independently an integer from 2 to 6, and t3 is an integer from 1 to 4.

[0168] In a more specific embodiment, r2 and r1 are each independently an integer of 2 to 6. In a more specific embodiment, r2 is an integer of 3 to 5, and r1 is an integer of 2 to 6. In a more specific embodiment, r2 is an integer of 3 to 5, and r1 is an integer of 2 to 4. In a more specific embodiment, r2 is 4, and r1 is 2. In a more specific embodiment, r2 is 4, and r1 is 3. In a more specific embodiment, , r2 is 4 and r1 is 4. In a more specific embodiment, r2 is 4 and r1 is 5. In a more specific embodiment, r2 is 4 and r1 is 6.

[0169] In yet another more specific embodiment, R 3 and R 4 are both Me. Alternatively, R 3 and R 4 are both H.

[0170] In another more specific embodiment, the compound has the following formula: [ka] Represented by [In the formula, r1 and t1 are each an integer of 2 to 10; r2 and t2 are each an integer of 2 to 19; t3 is an integer from 2 to 12 (e.g., t3 is 2, 4, 6, 8, 10, or 12)].

[0171] In more specific embodiments, r1 and t1 are each an integer from 2 to 6; r2 and t2 are each an integer from 2 to 5; and t3 is an integer from 2 to 6 (e.g., t3 is 2, 4, or 6).

[0172] In a 27th specific embodiment, the compound of formula (II) has the following formula: [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); J CB ' is -C(=O)OH or -COE; D1 is expressed by the following formula: [ka] [represented by]

[0173] In an even more specific embodiment, D1 is of the following formula: [ka] is expressed by

[0174] In a twenty-seventh specific embodiment, the compound of formula (II) also has the following formula: [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); J CB ' is -C(=O)OH or -COE; D1 is expressed by the following formula: [ka] is expressed by

[0175] In an even more specific embodiment, D1 is of the following formula: [ka] is expressed by

[0176] In another more specific embodiment, the compound has the following formula: [ka] Represented by [In the formula, D1 is expressed by the following formula: [ka] Represented by

[0177] In some embodiments, the -COE is a reactive ester selected from an N-hydroxysuccinimide ester, an N-hydroxysulfosuccinimide ester, a nitrophenyl (e.g., 2- or 4-nitrophenyl) ester, a dinitrophenyl (e.g., 2,4-dinitrophenyl) ester, a sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) ester, and a pentafluorophenyl ester. More specifically, the -COE is an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

[0178] In a twenty-eighth specific embodiment, the compound of formula (III) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 3 and R 4 are each independently H or Me; m2 each independently represents an integer of 1 to 19; D1 is expressed by the following formula: [ka] Represented by: The remaining variables are as described above in the second embodiment or the nineteenth, twentieth or twenty-first specific embodiments.]

[0179] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0180] In more specific embodiments, A is Ala-Ala-Ala, Ala-D-Ala-A In an even more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In an even more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0181] In a more specific embodiment, m2 is an integer from 2 to 10. In another more specific embodiment, m2 is an integer from 1 to 7. In another more specific embodiment, m2 is an integer from 2 to 6. In another more specific embodiment, m2 is an integer from 2 to 5. In another more specific embodiment, m2 is 4.

[0182] In yet another more specific embodiment, R 3 and R 4 are both Me. Alternatively, R 3 and R 4 are both H.

[0183] In a twenty-ninth specific embodiment, the compound of formula (III) has the following formula: [ka] or a pharmaceutically acceptable salt thereof, A' is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); D1 is expressed by the following formula: [ka] [represented by]

[0184] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0185] In a 29th specific embodiment, the compound of formula (III) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, A' is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); D1 is expressed by the following formula: [ka] [represented by]

[0186] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0187] In a thirtieth specific embodiment, the compound of formula (IV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, R 3 and R 4 are each independently H or Me; m3 is an integer from 1 to 10; m2 is an integer from 1 to 19; D1 is expressed by the following formula: [ka] Represented by; the remaining variables are as described above in the second embodiment or the nineteenth, twentieth, or twenty-first specific embodiments. In a more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly. In another more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0188] In a more specific embodiment, m3 is an integer from 1 to 6, and m2 is an integer from 1 to 7. In another more specific embodiment, m3 is an integer from 2 to 4; and m2 is an integer from 3 to 5. In a more specific embodiment, m3 is an integer from 2 to 10, and m2 is an integer from 2 to 10. In a more specific embodiment, m3 is an integer from 3 to 6, and m2 is an integer from 2 to 10. In a more specific embodiment, m3 is an integer from 3 to 6, and m2 is an integer from 3 to 6.

[0189] In yet another more specific embodiment, R 3 and R 4 are both Me. Alternatively, R 3 and R 4 are both H.

[0190] In a thirty-first specific embodiment, the compound of formula (IV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); D1 is expressed by the following formula: [ka] [represented by]

[0191] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0192] In a thirty-first specific embodiment, the compound of formula (IV) has the following formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, D-Ala-Ala-Ala, Ala-Ala-D-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly (more specifically, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly); D1 is expressed by the following formula: [ka] [represented by]

[0193] In more specific embodiments, D1 is of the following formula: [ka] is expressed by

[0194] Metabolites In certain embodiments, the conjugate of the invention comprises a peptide moiety represented by variable A, which undergoes bond cleavage followed by -NH-CR 1 R 2 The -S- moiety can undergo self-immolation to release a free cytotoxic drug (e.g., a maytansinoid) through which the free cytotoxic drug bears a thiol group that may be further methylated. [ka]

[0195] Thus, in a third embodiment, the present invention provides a compound of formula (V): D-L1-SZ 0 (V) Targeted at [In the formula, L1 is a spacer; Z 0 is H or Me, except that Z 0 is H, L1 is -C(=O)-(CH2) q - or is not -C(=O)-CH2-CH2-C(CH3)2-, where q is an integer from 1 to 3; Z 0 is Me, then L1 is not -C(=O)-(CH2)2- or -C(=O)-CH2-CH2-C(CH3)2-; D-L1-SH is a cytotoxic drug].

[0196] In a thirty-second specific embodiment, L is -L-C(=O)-; L is alkylene or cycloalkylene. More specifically, L is C 1~10 Al In another more specific embodiment, L is C 1~20 It is alkylene.

[0197] In a thirty-third specific embodiment, L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me.

[0198] In a thirty-fourth specific embodiment, L1 is -CR 3 R 4 -(CH2) 2~5 -C(=O)- or -CR 3 R 4 -(CH2) 3~5 In more specific embodiments, R 3 and R 4 and R are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0199] In a thirty-fifth specific embodiment, L1 is -(CH2) 2~10 In more specific embodiments, L is -C(=O)-. 4~6 In another more specific embodiment, L1 is -(CH2)5-C(=O)-. In another more specific embodiment, L1 is -(CH2)6-C(=O)-. In another more specific embodiment, L1 is -(CH2)7-C(=O)-. In another more specific embodiment, L1 is -(CH2)8-C(=O)-. In another more specific embodiment, L1 is -(CH2) 10 In another more specific embodiment, L is -C(=O)-. 15 -C(=O)-.

[0200] In a thirty-sixth specific embodiment, for compounds of formula (V), D is of the following formula: [ka] wherein the remaining variables are as defined in the second embodiment or the thirty-second, thirty-third, or thirty-fourth, or thirty-fifth specific embodiments.

[0201] More specifically, D is expressed by the following formula: [ka] is expressed by

[0202] In a thirty-seventh specific embodiment, the compound of formula (V) has the following formula: [ka] [ka] is expressed by

[0203] Cell-binding agents The effectiveness of the conjugates of the invention as therapeutic agents depends on the careful selection of an appropriate cell-binding agent. Cell-binding agents can be of any type now known or to be discovered, including peptides and non-peptides. Generally, they can be antibodies (such as polyclonal and monoclonal antibodies, particularly monoclonal antibodies), lymphokines, hormones, growth factors, vitamins (such as folic acid, which can bind to its cell surface receptor, e.g., the folate receptor), nutrient transport molecules (such as transferrin), or any other cell-binding molecule or substance.

[0204] The selection of an appropriate cell-binding agent is a matter of choice that depends in part on the particular cell population being targeted, but in many (but not all) cases, a human monoclonal antibody is a good choice if one is available. For example, the monoclonal antibody MY9 is a murine IgG1 antibody that specifically binds to the CD33 antigen (J.D. Griffin et al., Leukemia Res., 8:521 (1984)), and can be used when the target cells may express CD33, such as in acute myeloid leukemia (AML) disease.

[0205] In certain embodiments, the cell-binding agent is not a protein. For example, in certain embodiments, the cell-binding agent may be a vitamin that binds to a vitamin receptor, such as a cell surface receptor. In this regard, vitamin A binds to retinol-binding protein (RBP) to form a complex, which in turn binds with high affinity to the STRA6 receptor, increasing vitamin A uptake. In another example, folic acid / folate compounds / vitamin B9 bind with high affinity to cell surface folate receptors (FR), such as FRα. Folic acid or antibodies that bind to FRα can be used to target folate receptors expressed on ovarian and other tumors. In addition, vitamin D and its analogs bind to vitamin D receptors.

[0206] In other embodiments, the cell-binding agent is a protein or polypeptide, or a compound containing a protein or polypeptide, including an antibody, a non-antibody protein, or a polypeptide. Preferably, the protein or polypeptide contains one or more Lys residues with a side chain -NH2 group. The Lys side chain -NH2 group can be covalently bound to a bifunctional cross-linker, which in turn binds to the dimeric compound of the present invention, thereby conjugating the cell-binding agent to the dimeric compound of the present invention. Each protein-based cell-binding agent may contain multiple Lys side chain -NH2 groups available for linking the compound of the present invention via a bifunctional cross-linker.

[0207] In some embodiments, GM-CSF, a ligand / growth factor that binds to myeloid cells, can be used as a cell-binding agent for diseased cells from acute myeloid leukemia. IL-2, which binds to activated T cells, can be used to prevent graft rejection, treat and prevent graft-versus-host disease, and treat acute T-cell leukemia. MSH, which binds to melanocytes, can be a melanoma-directed antibody and can be used to treat melanoma. Epidermal growth factor can be used to target squamous cell carcinomas, such as lung and head and neck and gingival squamous cell carcinoma. Somatostatin can be used to target neuroblastoma and other tumor types. Estrogen (or estrogen analogs) can be used to target breast cancer. Androgen (or androgen analogs) can be used to target the testes.

[0208] In certain embodiments, the cell binding agent may be a lymphokine, hormone, growth factor, colony-stimulating factor, or nutrient transport molecule.

[0209] In certain embodiments, the cell binding agent is an ankyrin repeat protein, centilin, or an antibody mimetic such as an adnectin / monobody.

[0210] In other embodiments, the cell binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment (or "antigen binding portion") that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment (or "antigen binding portion") that specifically binds to a target cell, a domain antibody (e.g., an sdAb), or a domain antibody fragment that specifically binds to a target cell.

[0211] In certain embodiments, the cell binding agent is a humanized antibody, a humanized single chain antibody, or a humanized anti- The humanized antibody is a humanized antibody fragment (or "antigen-binding portion") of a humanized antibody. In a specific embodiment, the humanized antibody is huMy9-6 or another related antibody, which is described in U.S. Patent Nos. 7,342,110 and 7,557,189. In another specific embodiment, the humanized antibody is an anti-folate receptor antibody, which is described in WO 2011 / 106528 and U.S. Patent Nos. 8,557,966, 9,133,275, 9,598,490, 9,657,100, 9,670,278, 9,670,279, and 9,670,280. The teachings of all of these applications are incorporated herein by reference in their entirety.

[0212] In certain embodiments, the cell binding agent is a resurfaced antibody, a resurfaced single chain antibody, a resurfaced antibody fragment (or "antigen-binding portion"), or a bispecific antibody.

[0213] In certain embodiments, the cell binding agent is a minibody, avibody, diabody, tribody, tetrabody, nanobody, probody, domain antibody, or unibody.

[0214] In other words, exemplary cell binding agents include antibodies, single chain antibodies, antibody fragments that specifically bind to target cells, monoclonal antibodies, single chain monoclonal antibodies, monoclonal antibody fragments that specifically bind to target cells, chimeric antibodies, chimeric antibody fragments that specifically bind to target cells, bispecific antibodies, domain antibodies, domain antibody fragments that specifically bind to target cells, interferons (e.g., α, β, γ), lymphokines (e.g., IL-2, IL-3, IL-4, and IL-6), hormones (e.g., insulin, thyrotropin-releasing hormone (TRH), melanocyte-stimulating hormone (MSH), and steroid hormones (e.g., androgens and estrogens)), vitamins (e.g., folic acid), growth factors (e.g., EGF, TGF-alpha, FGF, VEGF), colony-stimulating factors, nutrient transport molecules (e.g., transferrin; O'Keefe et al. al. (1985) J. Biol. Chem. 260:932-937, incorporated herein by reference), centirin (a scaffolding protein based on the consensus sequence of fibronectin type III (FN3) repeats; see U.S. Patent Application Publication Nos. 2010 / 0255056, 2010 / 0216708, and 2011 / 0274623, incorporated herein by reference), ankyrin repeat proteins (e.g., designed ankyrin repeat proteins known as DARPins; see U.S. Patent Application Publication Nos. 2004 / 0132028, 2009 / 0082274, 2011 / 0118146, and 2011 / 0224100, incorporated herein by reference; C. Zahnd et al., Cancer Res.(2010)70:1595-1605;Zahnd et al., J.Biol.Chem.(2006)281(46):35167-35175, and Binz, HK, Amstutz, P. & Pluckthun, A., Nature Biotechnology (2005) 23:1257-1268, which are incorporated herein by reference), ankyrin-like repeat proteins or synthetic peptides (see, e.g., U.S. Patent Application Publication No. 2007 / 0238667, U.S. Patent No. 7,101,675, WO2007 / 147213, and WO2007 / 062466, which are incorporated herein by reference), adnectins (Fcn, These include fibronectin domain scaffold proteins; see U.S. Patent Application Publication Nos. 2007 / 0082365, 2008 / 0139791 (incorporated herein by reference), avibodies (including diabodies, tribodies, and tetrabodies; see U.S. Patent Application Publication Nos. 2008 / 0152586 and 2012 / 0171115), dual receptor retargeting (DART) molecules (PA Moore et al., Blood, 2011;117(17):4542-4551; Veri MC, et al., Arthritis Rheum, 2010 Mar 30;62(7):1933-43; Johnson S, et al. J Mol Biol, 2010 Apr 9;399(3):436-49), cell-permeable supercharged proteins (Metho. ds in Enzymol. 502, 293-319 (2012), and other cell-binding molecules or substances.

[0215] In certain embodiments, the cell-binding agent may be a ligand that binds to a moiety on a target cell, such as a cell surface receptor. For example, the ligand may be a growth factor or a fragment thereof that binds to a growth factor receptor; or a cytokine or a fragment thereof that binds to a cytokine receptor. In certain embodiments, the growth factor receptor or cytokine receptor is a cell surface receptor.

[0216] In certain embodiments, when the cell binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives), or certain antibody mimetics, the CBA can bind to a ligand on the target cell, such as a cell surface ligand including a cell surface receptor.

[0217] Specific exemplary antigens or ligands include renin; growth hormones (e.g., human growth hormone and bovine growth hormone); growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors (e.g., vmc factor, factor IX, tissue factor, and von Willebrand factor); anticoagulants (e.g., protein C); atrial natriuretic factor; pulmonary surface Surfactants; plasminogen activators (e.g., urokinase, human urinary or tissue-type plasminogen activator); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and beta; enkephalinase; RANTES (i.e., regulator of normal T cell expression and secretory activation); human macrophage inflammatory protein-1-alpha; serum albumin (human serum albumin); Müllerian inhibitory factor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial protein proteins (beta-lactamases); DNases; IgE; cytotoxic T lymphocyte-associated antigens (e.g., CTLA-4); inhibins; activins; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors (e.g., bone-derived neurotrophic factor, neurotrophin-3, -4, -5, or -6) or nerve growth factors (e.g., NGF-β); platelet-derived growth factor; fibroblast growth factors (e.g., aFGF and bFGF); fibroblast growth factor receptor 2; epidermal growth factor; Transforming growth factors (e.g., TGF-alpha, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5), insulin-like growth factors I and II, des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein, melanotransferrin; CA6, CAK1, CAEEA, CAECAM5, GD3; FET3; PSMA; PSCA; MUC1; STEAP; CEA; TENB2; EphA receptors; EphB receptors; folate receptors; FOLR1; mesothelin; Cryptogen; alpha vbeta6; integrins; VEGF; VEGFR; EGFR; FGFR3; LAMP1, p-cadherin, transferrin receptor; IRTA1; IRTA2; IRTA3; IRTA4; IRTA5; CD proteins (e.g., CD2, CD3, CD4, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD123, CD 134, CD137, CD138, and CD152), one or more tumor-associated antigens or cell surface receptors (see U.S. Patent Application Publication No. 2008 / 0171040 or U.S. Patent Application Publication No. 2008 / 0305044, which are incorporated by reference in their entireties); erythropoietin; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins; interferons (e.g., interferon-alpha, -beta, and -gamma); colony stimulating factors (e.g., M-CSF, GM-CSF, and G-CSF); interleukins (e.g., IL-1 through IL-10); superoxide dismutase (SDS); These polypeptides may include cytoside dismutase; T-cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens (e.g., parts of the HIV envelope); transport proteins; homing receptors; addressins; regulatory proteins; integrins (e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM); tumor-associated antigens (e.g., HER2, HER3, and HER4 receptors); endoglin; c-Met; c-kit; 1GF1R; PSGR; NGEP; PSMA; PSCA; TMEFF2; LGR5; B7H4; TROP-2, DLL-3, CDH6, AXL, SLITRK6, ENPP3, BCMA, tissue factor, CD352, and fragments of any of the foregoing polypeptides.

[0218] As used herein, the term "antibody" includes immunoglobulin (Ig) molecules. In certain embodiments, antibodies are full-length antibodies comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region, which in turn consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs). Interspersed with these regions are more highly conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0219] In certain embodiments, the antibody is IgG, IgA, IgE, IgD, or IgM. In certain embodiments, the antibody is IgG1, IgG2, IgG3, or IgG4, or is IgA1 or IgA2.

[0220] In certain embodiments, the cell binding agent is an "antigen-binding portion" of a monoclonal antibody, which portion shares sequences essential for antigen binding with the antibody (such as huMy9-6 or related antibodies thereof described in U.S. Patent Nos. 7,342,110 and 7,557,189; huMovl9 or related antibodies thereof described in U.S. Patent Nos. 8,557,966, 9,133,275, 9,598,490, 9,657,100, 9,670,278, 9,670,279, and 9,670,280, and WO2011106528, all of which are incorporated herein by reference).

[0221] As used herein, the term "antigen-binding portion" of an antibody (sometimes synonymously and interchangeably referred to as "antibody fragment") includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been found that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains (e.g., digestion of an antibody with papain produces three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind antigen); (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., digestion of an antibody with pepsin produces two fragments: a bivalent antigen-binding F(ab')2 fragment and a pFc' fragment that does not bind antigen), and its associated F(ab') monovalent unit; (iii) a Fd fragment (i.e., the portion of the heavy chain contained in a Fab) consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody and associated disulfide-linked Fvs; and (v) a dAb (domain antibody) or sdAb (single domain antibody) fragment (Ward et al., J. Immunol. 2002, 10:111-114). al., Nature 341:544-546, 1989) (which fragment consists of the VH domain), and (vi) isolated complementarity-determining regions (CDRs). In certain embodiments, the antigen-binding portion is an sdAb (single domain antibody).

[0222] In certain embodiments, antigen-binding portions also include certain engineered or recombinant derivatives (or "derivative antibodies"), which also contain one or more fragments of an antibody that retain the ability to specifically bind to an antigen, in addition to elements or sequences that may not be found in a naturally occurring antibody.

[0223] For example, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be combined using standard recombinant methods with a synthetic linker that enables them to form a single protein chain in which the VL and VH regions can pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).

[0224] In all embodiments described herein, the N-terminus of the scFv may be a VH domain (i.e., N-VH-VL-C) or a VL domain (i.e., N-VL-VH-C).

[0225] Bivalent (or divalent) single-chain variable fragments (di-scFv, bi-scFv) can be engineered by linking two scFvs. This generates a single peptide chain with two VH and two VL regions, resulting in tandem scFvs (tascFv). Higher tandem repeats, e.g., tri-scFv, can be similarly generated by linking three or more scFvs in a head-to-tail fashion.

[0226] In certain embodiments, scFvs may be dimerized to form diabodies by linking the two variable regions via a linker peptide that is too short (approximately 5 amino acids) for the two variable regions to fold together (see, e.g., Holliger et al., 2002). (See, e.g., Poljak et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994). Diabodies can be bispecific or monospecific. Diabodies have been shown to have dissociation constants up to 40-fold lower than the corresponding scFvs, i.e., they have much higher affinity for their targets.

[0227] Shorter linkers (one or two amino acids) form trimers, so-called triabodies or tribodies. Tetrabodies have also been produced similarly. These exhibit even higher affinity for targets than diabodies. Diabodies, triabodies, and tetrabodies are sometimes collectively referred to as "AVIBODY™" cell binding agents (or simply "AVIBODYs"). That is, AVIBODYs with two, three, or four target-binding regions (TBRs) are commonly known as diabodies, triabodies, and tetrabodies. See, for example, U.S. Patent Application Publication Nos. 2008 / 0152586 and 2012 / 0171115 for details, the entire teachings of which are incorporated herein by reference.

[0228] All of these formats may be composed of variable fragments with specificities for two or more different antigens, in which case they are of the bi- or multispecific antibody variety, for example, certain bispecific tandem di-scFvs are known as bispecific T cell engagers (BiTEs).

[0229] In certain embodiments, tandem scFv or diabody / triabody / tetrabody Each scFv of the di may have the same or different binding specificities and may each independently have an N-terminal VH or an N-terminal VL.

[0230] Single-chain Fvs (scFvs) can also be fused to an Fc portion, such as a human IgG Fc portion, to have IgG-like properties while still being encoded by a single gene. Transient production of such scFv-Fc proteins in mammals can readily yield milligram quantities, making this derivative antibody format particularly suitable for many research applications.

[0231] Fcabs are antibody fragments engineered from the Fc constant region of an antibody. Fcabs can be expressed as soluble proteins, or they can be engineered back into full-length antibodies such as IgG to create mAb2s. mAb2s are full-length antibodies that have an Fcab instead of the normal Fc region. Using these additional binding sites, mAb2 bispecific monoclonal antibodies can simultaneously bind to two different targets.

[0232] In certain embodiments, engineered antibody derivatives are antigen-binding Ig-derived recombinant proteins reduced in size ("miniaturization" of full-length mAbs) produced by removing domains believed to be non-essential for function, one of the best examples of which is the SMIP.

[0233] Small modular immunopharmaceuticals (SMIPs) are artificial proteins constructed primarily from antibody moieties (immunoglobulins) and intended for pharmaceutical use. SMIPs have similar biological half-lives to antibodies but are smaller and therefore may have better tissue penetration properties. SMIPs are single-chain proteins containing a binding region, a hinge region as a connector, and an effector domain. The binding region comprises a modified single-chain variable fragment (scFv), and the remainder of the protein can be constructed from the Fc region (e.g., CH2 and CH3 as the effector domain) and hinge region of an antibody, e.g., IgG1. Genetically modified cells produce SMIPs as antibody-like dimers that are approximately 30% smaller than actual antibodies.

[0234] Another example of such an engineered miniaturized antibody is a "unibody," in which the hinge region has been removed from an IgG4 molecule. IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with each other. Deletion of the hinge region completely prevents heavy-chain pairing, leaving a highly specific monovalent light / heavy chain heterodimer while retaining the Fc region to ensure stability and half-life in vivo.

[0235] Single domain antibodies (sdAbs, including but not limited to those referred to as nanobodies by Ablynx) are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, single domain antibodies are capable of selectively binding to a specific antigen, but are much smaller, with a molecular weight of only 12-15 kDa. In certain embodiments, single domain antibodies are engineered from heavy chain antibodies (hcIgG). The first such sdAb was engineered based on hcIgG found in camels, and is a V H In certain embodiments, single domain antibodies are referred to as V fragments. NAR They are engineered from IgNARs ("Immunoglobulin Novel Antigen Receptors", see below) using fragments. Cartilaginous fish (such as sharks) have such heavy chain IgNAR antibodies. In certain embodiments, sdAbs are engineered by splitting the dimeric variable domain of a common immunoglobulin G (IgG), e.g., of human or mouse origin, into monomers. In certain embodiments, nanobodies are derived from heavy chain variable domains. In certain embodiments, nanobodies are derived from light chain variable domains. In certain embodiments, sdAbs are obtained by screening libraries of single domain heavy chain sequences (e.g., human single domain HCs) for binders to the target antigen.

[0236] Single variable novel antigen receptor domain antibody fragment (V NAR , or V NAR The single domain proteins are derived from cartilaginous fish (e.g., shark) immunoglobulin novel antigen receptor antibodies (IgNARs). Being one of the smallest known immunoglobulin scaffold proteins, such single domain proteins exhibit desirable size and potential epitope recognition capabilities. Mature IgNAR antibodies contain one variable novel antigen receptor (V NAR ) domain and five constant novel antigen receptors (C NAR) domains. This molecule has high stability and efficient binding properties. Its inherent stability is likely due to both (i) the underlying Ig scaffold, which provides a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in murine antibodies, and (ii) structural stabilizing features in the complementarity-determining region (CDR) loops, including a pattern of inter-loop disulfide bridges and intra-loop hydrogen bonds.

[0237] Minibodies are engineered antibody fragments containing an scFv linked to a CH domain, such as CH3γ1 (the CH3 domain of IgG1) or CH4ε (the CH4 domain of IgE). For example, a carcinoembryonic antigen (CEA)-specific scFv linked to CH3γ1 has been generated, and this minibody has already demonstrated rapid in vivo clearance as well as excellent tumor targeting (Hu et al., Cancer Res. 56:3055-3061, 1996). The scFv may have an N-terminal VH or VL. The linker may be a short peptide (e.g., a two-amino acid linker such as ValGlu) resulting in a noncovalent hingeless minibody. Alternatively, the linker may be an IgG1 hinge and GlySer linker peptide to generate a covalent hinge minibody.

[0238] Natural antibodies are monospecific but bivalent in that they express two identical antigen-binding domains. In contrast, in certain embodiments, certain engineered antibody derivatives are bispecific or multispecific molecules with two or more distinct antigen-binding domains, each with a different target specificity. Bispecific antibodies can be generated by fusing two antibody-producing cells, each with a different specificity. These "quadromas" have produced multiple molecular species because the two distinct light chains and two distinct heavy chains freely recombine in various configurations within the quadroma. Since then, bispecific Fabs, scFvs, and full-size mAbs have been produced using a variety of techniques (see above).

[0239] Dual variable domain immunoglobulin (DVD-Ig) proteins are a type of bispecific IgG that simultaneously targets two antigens / epitopes (DiGiammarino et al., Methods Mol Biol. 899:145-56, 2012). This molecule contains an Fc region and a constant region in an arrangement similar to that of conventional IgG. However, DVD-Ig proteins are unique in that each arm of the molecule contains two variable domains (VDs). The VDs within an arm are linked in tandem, allowing them to have different binding specificities.

[0240] Trispecific antibody derivative molecules can also be produced, for example, by expressing a bispecific antibody with two different Fabs and one Fc. One example is a mouse IgG2a anti-Ep-CAM, rat IgG2b anti-CD3 quadroma called BiUII, which allows the coexistence of Ep-CAM-expressing tumor cells, CD3-expressing T cells, and FcγRI-expressing macrophages, and is therefore thought to enhance immune cell co-stimulation and anti-tumor function.

[0241] Probodies remain inactive in healthy tissues but become activated especially in disease microenvironments. The masked monoclonal antibody is a fully recombinant, masked monoclonal antibody that is cleaved (e.g., by protease cleavage by proteases that are abundant or specific to the disease microenvironment). See, e.g., Sci Transl. Med., 5:207, 144, 2013. Similar masking techniques can be used with any of the antibodies or antigen-binding portions thereof described herein.

[0242] Intrabodies are antibodies that have been modified to localize intracellularly so that they can function intracellularly and bind to intracellular antigens. Intrabodies may remain in the cytoplasm, or may contain a nuclear localization signal or a KDEL sequence for ER targeting. Intrabodies may be single-chain fragments (scFvs), modified immunoglobulin VL domains with ultrastability, selected antibodies that tolerate a more reducing intracellular environment, or expressed as fusion proteins with maltose-binding protein or other stable intracellular proteins. Such optimization improves the stability and structure of intrabodies and is generally applicable to any of the antibodies or antigen-binding portions thereof described herein.

[0243] The antigen-binding portions or derivative antibodies of the present invention may have substantially the same or identical (1) light chain and / or heavy chain CDR3 regions; (2) light chain and / or heavy chain CDR1, CDR2, and CDR3 regions; or (3) light chain and / or heavy chain regions compared to the antibody from which they are derived / engineered. The sequences within these regions may contain conservative amino acid substitutions, including substitutions within the CDR regions. In certain embodiments, there are no more than one, two, three, four, or five conservative substitutions. Alternatively, the antigen-binding portions or derivative antibodies have light chain and / or heavy chain regions that are at least about 90%, 95%, 99%, or 100% identical to the antibody from which they are derived / engineered. These antigen-binding portions or derivative antibodies may have substantially the same binding specificity and / or affinity for the target antigen compared to the antibody. In certain embodiments, the K of the antigen-binding portion or derivative antibody may be 0.01 or 0.02. d and / or k off Values ​​are within 10-fold (higher or lower), 5-fold (higher or lower), 3-fold (higher or lower), or 2-fold (higher or lower) of the antibodies described herein.

[0244] In certain embodiments, the antigen-binding portion or derivative antibody may be derived from / engineered from a fully human antibody, a humanized antibody, or a chimeric antibody, and may be produced according to any art-recognized method.

[0245] Monoclonal antibody technology allows for the production of highly specific cell-binding agents in the form of specific monoclonal antibodies. Particularly well-known in the art is the technique of producing monoclonal antibodies by immunizing mice, rats, hamsters, or any other mammals with an antigen of interest, such as intact target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses, and viral proteins such as viral coat proteins. Sensitized human cells can also be used. Another method for producing monoclonal antibodies is the use of phage libraries of scFvs (single-chain variable regions), particularly human scFvs (see, e.g., Griffiths et al., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO 92 / 01047; Liming et al., WO 99 / 06587). In addition, the resurfaced antibodies disclosed in US Pat. No. 5,639,641 can also be used as chimeric and humanized antibodies.

[0246] Cell-binding agents can be synthesized using phage display (see, e.g., Wang et al., Proc. Natl. Acad. Sci. USA (2011) 108(17), 6909-6914) or peptide library technology (see, e.g., Dane et al., M The peptide may be derived from the human ovarian cancer cell line (see, for example, Vol. Cancer. Ther. (2009) 8(5):1312-1318).

[0247] In certain embodiments, CBAs of the invention also include antibody mimetics, e.g., DARPins, affibodies, affilins, affitins, anticalins, avimers, finomers, Kunitz domain peptides, monobodies, nanofitins, Bicycles® peptides such as those described in U.S. Patent Application Publication No. 2014 / 0163201 (incorporated herein by reference), and pentalines such as those described in Abstract 3674, AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA (incorporated herein by reference).

[0248] As used herein, the terms "DARPin" and "(designed) ankyrin repeat protein" are used interchangeably and refer to specific engineered antibody-mimetic proteins that typically exhibit preferential (and sometimes specific) target binding. The target may be a protein, carbohydrate, or other chemical moiety, and the binding affinity can be quite high. DARPins may be derived from natural ankyrin repeat-containing proteins and preferably consist of at least three, usually four or five, ankyrin repeat motifs (typically 33 residues in each ankyrin repeat motif) of these proteins. In certain embodiments, DARPins may contain approximately four or five repeats and have molecular weights of approximately 14 or 18 kDa, respectively. For use in selecting DARPins that bind to desired targets with picomolar affinity and specificity (e.g., acting as receptor agonists or antagonists, inverse agonists, enzyme inhibitors, or simple target protein binders), DARPins can be isolated using various techniques, such as ribosome display or signal recognition particle (SRP) phage display. 12Libraries of DARPins with randomized interaction residues with potential targets can be generated at the DNA level, with a diversity of over 1000 mutants. For example, the preparation of DARPins is described in U.S. Patent Application Publication Nos. 2004 / 0132028, 2009 / 0082274, 2011 / 0118146, and 2011 / 0224100, WO02 / 20565, and WO06 / 083275 (the entire teachings of which are incorporated herein by reference), as well as C. Zahnd et al. (2010) Cancer Res., 70:1595-1605; Zahnd et al. (2006) J. Biol. Chem., 281(46):35167-35175, and Binz, HK, Amstutz, P. & Pluckthun, A. (2005) Nature 10:1002-10055; Biotechnology, 23:1257-1268 (all of which are incorporated herein by reference). Also see U.S. Patent Application Publication No. 2007 / 0238667, U.S. Patent No. 7,101,675, WO2007 / 147213, and WO2007 / 062466 (the entire teachings of which are incorporated herein by reference) for related ankyrin-like repeat proteins or synthetic peptides.

[0249] Affibody molecules are small proteins engineered to bind with high affinity to numerous target proteins or peptides, thus mimicking monoclonal antibodies. Affibodies consist of three alpha helices with 58 amino acids and a molecular weight of approximately 6 kDa. Affibodies have been shown to tolerate high temperatures (90°C) or acidic and alkaline conditions (pH 2.5 or 11), and binders with affinities down to the subnanomolar range have been obtained from naive library selections, and binders with picomolar affinities have been obtained after affinity maturation. In certain embodiments, affibodies are conjugated to weak electrophiles for covalent binding to targets.

[0250] Monobodies (also known as Adnectins) are genetically engineered antibody-mimetic proteins capable of binding to antigens. In certain embodiments, monobodies The monobody consists of 94 amino acids and has a molecular weight of approximately 10 kDa. The monobody is based on the structure of human fibronectin, more specifically, the tenth extracellular type III domain of human fibronectin. This domain has a structure similar to that of an antibody variable domain, with seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three complementarity-determining regions. By modifying the BC loop (between the second and third beta sheets) and the FG loop (between the sixth and seventh beta sheets), monobodies with specificity for various proteins can be tailored.

[0251] Tribodies are self-assembling antibody mimics designed based on the C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP), which self-assemble into parallel trimeric complexes. Tribodies are highly stable trimeric targeting ligands created by fusing specific target-binding moieties with the trimerization domain from CMP. The resulting fusion proteins can efficiently self-assemble into well-defined, highly stable parallel homotrimers. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly improved target binding strength compared to the corresponding monomers. Cell-binding studies confirmed that such tribodies possess superior binding strength to their respective receptors.

[0252] Centilin is another antibody mimetic that can be obtained using a library constructed on the framework of the consensus FN3 domain sequence (Diem et al., Protein Eng. Des. Sel., 2014). This library utilizes various positions within the C strand, CD loop, F strand, and FG loop of the FN3 domain, allowing high-affinity centrinin mutants to be selected against specific targets.

[0253] In some embodiments, the cell-binding agent is an anti-folate receptor antibody. More specifically, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 (also known as folate receptor alpha (FR-α)). As used herein, the terms "human folate receptor 1," "FOLR1," or "folate receptor alpha (FR-α)" refer to any naturally occurring human FOLR1 unless otherwise specified. Thus, all of these terms can refer to any of the proteins or nucleic acid sequences set forth herein. The term "FOLR1" encompasses "full-length," unprocessed FOLR1, as well as any form of FOLR1 obtained by intracellular processing. The FOLR1 antibody comprises (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO: 4), a heavy chain CDR2 comprising RIHPYDGDTFYNQXaa1FXaa2Xaa3 (SEQ ID NO: 5), and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO: 6), and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO: 7), a light chain CDR2 comprising RASNLEA (SEQ ID NO: 8), and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO: 9), where Xaa1 is selected from K, Q, H, and R, Xaa2 is selected from Q, H, N, and R, and Xaa3 is selected from G, E, T, S, A, and V. Preferably, the heavy chain CDR2 sequence comprises RIHPYDGDTFYNQKFQG (SEQ ID NO: 10).

[0254] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 and has the amino acid sequence: [ka] In some embodiments, the heavy chain amino acid sequence has a C-terminal lysine after the last glycine of SEQ ID NO:11.

[0255] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof encoded by the plasmid DNA deposited with the ATCC on April 7, 2010, and having ATCC deposit number PTA-10772 and PTA-10773 or 10774. In one embodiment, the anti-folate receptor antibody comprises a heavy chain HC encoded by the plasmid DNA having ATCC deposit number PTA-10772 and a light chain LC encoded by the plasmid DNA having ATCC deposit number PTA-10773 or 10774. In another embodiment, the anti-folate receptor antibody comprises a heavy chain HC encoded by the plasmid DNA having ATCC deposit number PTA-10772 and a light chain LC encoded by the plasmid DNA having ATCC deposit number PTA-10773. In another embodiment, the anti-folate receptor antibody comprises a heavy chain HC encoded by plasmid DNA having ATCC deposit number PTA-10772 and a light chain LC encoded by plasmid DNA having ATCC deposit number PTA-10774.

[0256] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1 and has the amino acid sequence: [ka] The light chain comprises a

[0257] In another embodiment, the anti-folate receptor antibody is a humanized antibody that specifically binds to human folate receptor 1. An antibody or antigen-binding fragment thereof, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 13 (hu FOLR1). In some embodiments, the heavy chain sequence of hu FOLR1 (huMovl9) comprises a C-terminal lysine after the last glycine of SEQ ID NO: 11.

[0258] In another embodiment, the anti-folate receptor antibody is a humanized antibody or antigen-binding fragment thereof that specifically binds to human folate receptor 1, [ka] a heavy chain variable domain at least about 90%, 95%, 99% or 100% identical to [ka] and a light chain variable domain at least about 90%, 95%, 99% or 100% identical to

[0259] In another embodiment, the anti-folate receptor antibody is huMov19 or M9346A or M antibody (see, e.g., U.S. Patent Nos. 8,709,432, 8,557,966, 9133275, 9598490, 9657100, 9670278, 9670279, and 9670280 and WO2011106528, all of which are incorporated by reference herein).

[0260] In another embodiment, the cell binding agent is an anti-EGFR antibody or an antibody fragment thereof. In some embodiments, the anti-EGFR antibody is a non-antagonist antibody, including, for example, the antibody described in WO2012058592 (incorporated herein by reference). In another embodiment, the anti-EGFR antibody is a non-functional antibody, such as humanized ML66 or EGFR-8. More particularly, the anti-EGFR antibody is huML66.

[0261] In yet another embodiment, the anti-EGFR antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 17 and a light chain having the amino acid sequence of SEQ ID NO: 18. As used herein, double-underlined sequences represent the variable regions of the heavy or light chain sequence (i.e., heavy chain variable region or HCVR, and light chain variable region or LCVR), and bolded sequences represent the CDR regions (i.e., CDR1, CDR2, and CDR3, respectively, from the N-terminus to the C-terminus of the heavy or light chain sequence). [Table 1a]

[0262] In yet another embodiment, the anti-EGFR antibody comprises heavy chain CDR1-CDR3 of SEQ ID NO: 17 and / or light chain CDR1-CDR3 of SEQ ID NO: 18, and preferably specifically binds to EGFR.

[0263] In yet another embodiment, the anti-EGFR antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 17 and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 18, and preferably specifically binds to EGFR.

[0264] In another embodiment, the anti-EGFR antibody is an antibody described in 8,790,649 and WO2012 / 058588, which are incorporated by reference. In some embodiments, the anti-EGFR antibody is a huEGFR-7R antibody.

[0265] In some embodiments, the anti-EGFR antibody [ka] an immunoglobulin heavy chain region having the amino acid sequence [ka] an immunoglobulin light chain region having the amino acid sequence of [ka] The immunoglobulin light chain region has the amino acid sequence:

[0266] In another embodiment, the anti-EGFR antibody comprises an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:19 and an immunoglobulin light chain region having the amino acid sequence set forth in SEQ ID NO:20.

[0267] In another embodiment, the anti-EGFR antibody comprises an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:19 and an immunoglobulin light chain region having the amino acid sequence set forth in SEQ ID NO:21.

[0268] In yet another embodiment, the anti-EGFR antibody comprises heavy chain CDR1-CDR3 of SEQ ID NO: 19 and / or light chain CDR1-CDR3 of SEQ ID NO: 20 or 21, and preferably specifically binds to EGFR.

[0269] In yet another embodiment, the anti-EGFR antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 19 and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 20 or 21, and preferably specifically binds to EGFR.

[0270] In another embodiment, the cell binding agent is an anti-CD19 antibody, such as those described in U.S. Patent No. 8,435,528 and WO 2004 / 103272 (incorporated by reference). In some embodiments, the anti-CD19 antibody is [ka] an immunoglobulin heavy chain region having the amino acid sequence [ka] The immunoglobulin light chain region has the amino acid sequence:

[0271] In another embodiment, the anti-CD19 antibody is a huB4 antibody.

[0272] In yet another embodiment, the anti-CD19 antibody comprises heavy chain CDR1-CDR3 of SEQ ID NO: 22 and / or light chain CDR1-CDR3 of SEQ ID NO: 23, and preferably specifically binds to CD19.

[0273] In yet another embodiment, the anti-CD19 antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 22 and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 23, and preferably specifically binds to CD19.

[0274] In yet another embodiment, the cell binding agent is an anti-Muc1 antibody, such as those described in U.S. Patent No. 7,834,155, WO2005 / 009369, and WO2007 / 024222, which are incorporated herein by reference. In some embodiments, the anti-Muc1 antibody is [ka] an immunoglobulin heavy chain region having the amino acid sequence [ka] The immunoglobulin light chain region has the amino acid sequence:

[0275] In another embodiment, the anti-Muc1 antibody is a huDS6 antibody.

[0276] In yet another embodiment, the anti-Muc1 antibody comprises the heavy chain CDR1-CDR3 of SEQ ID NO: 24 and / or the light chain CDR1-CDR3 of SEQ ID NO: 25, and preferably specifically binds to Muc1.

[0277] In another embodiment, the anti-Muc1 antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 24 and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 25, and preferably specifically binds to Muc1.

[0278] In another embodiment, the cell binding agent is an anti-CD33 antibody or fragment thereof, such as an antibody or fragment thereof described in U.S. Patent Nos. 7,557,189, 7,342,110, 8,119,787, and 8,337,855, and WO2004 / 043344 (incorporated herein by reference). In another embodiment, the anti-CD33 antibody is the huMy9-6 antibody.

[0279] In some embodiments, the anti-CD33 antibody [ka] an immunoglobulin heavy chain region having the amino acid sequence [ka] The immunoglobulin light chain region has the amino acid sequence:

[0280] In yet another embodiment, the anti-CD33 antibody comprises the heavy chain CDR1-CDR3 of SEQ ID NO: 26 and / or the light chain CDR1-CDR3 of SEQ ID NO: 27, and preferably specifically binds to CD33.

[0281] In yet another embodiment, the anti-CD33 antibody has a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO:26 and / or a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO:27. It contains a light chain variable region (LCVR) sequence and preferably binds specifically to CD33.

[0282] In another embodiment, the cell binding agent is an anti-CD37 antibody or fragment thereof, such as an antibody or antibody fragment thereof described in U.S. Patent No. 8,765,917 and WO2011 / 112978 (incorporated herein by reference). In some embodiments, the anti-CD37 antibody is a huCD37-3 antibody.

[0283] In some embodiments, the anti-CD37 antibody [ka] an immunoglobulin light chain region having the amino acid sequence [ka] an immunoglobulin heavy chain region having the amino acid sequence of [ka] The immunoglobulin heavy chain region has the amino acid sequence:

[0284] In another embodiment, the anti-CD37 antibody comprises an immunoglobulin light chain region having the amino acid sequence set forth in SEQ ID NO:28 and an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:29.

[0285] In yet another embodiment, the anti-CD37 antibody comprises an immunoglobulin light chain region having the amino acid sequence set forth in SEQ ID NO:28 and an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:30.

[0286] In yet another embodiment, the anti-CD37 antibody comprises the heavy chain CDR1-CDR3 of SEQ ID NO: 29 or 30 and / or the light chain CDR1-CDR3 of SEQ ID NO: 28, and preferably specifically binds to CD37.

[0287] In yet another embodiment, the anti-CD37 antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 29 or 30, and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 28, and preferably specifically binds to CD37.

[0288] In yet another embodiment, the anti-CD37 antibody is [ka] an immunoglobulin light chain region having the amino acid sequence [ka] The immunoglobulin heavy chain region has the amino acid sequence:

[0289] In yet another embodiment, the anti-CD37 antibody comprises the heavy chain CDR1-CDR3 of SEQ ID NO: 32 and / or the light chain CDR1-CDR3 of SEQ ID NO: 31, and preferably specifically binds to CD37.

[0290] In yet another embodiment, the anti-CD37 antibody comprises a heavy chain variable region (HCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 32 and / or a light chain variable region (LCVR) sequence at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 31, and preferably specifically binds to CD37.

[0291] In yet another embodiment, the anti-CD37 antibody is a huCD37-50 antibody.

[0292] In one embodiment, the cell binding agent is an anti-CD123 antibody or antibody fragment thereof, such as those described in WO2017 / 004026 (incorporated herein by reference).

[0293] In one embodiment, the anti-CD123 antibody or antibody fragment thereof comprises: a) a heavy chain variable region CDR1 having the amino acid sequence of SSIMH (SEQ ID NO: 33), a heavy chain variable region CDR2 having the amino acid sequence of YIKPYNDGTKYNEKFKG (SEQ ID NO: 34), and a heavy chain variable region CDR3 having the amino acid sequence of EGGNDYYDTMDY (SEQ ID NO: 35); and b) a light chain variable region CDR1 having the amino acid sequence of RASQDINSYLS (SEQ ID NO: 36), a light chain variable region CDR2 having the amino acid sequence of RVNRLVD (SEQ ID NO: 37), and a light chain variable region CDR3 having the amino acid sequence of LQYDAFPYT (SEQ ID NO: 38).

[0294] In another embodiment, the anti-CD123 antibody or antibody fragment thereof [ka] a heavy chain variable region having the amino acid sequence [ka] In certain embodiments, the second residue from the N-terminus of SEQ ID NO: 39, X (or Xaa), is Phe (F). In certain embodiments, X (or Xaa) in SEQ ID NO: 39 is Val (V).

[0295] In another embodiment, the anti-CD123 antibody or antibody fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and [ka] The light chain variable region has the amino acid sequence:

[0296] In another embodiment, the anti-CD123 antibody is [ka] and a heavy chain having the amino acid sequence [ka] In some embodiments, the second residue from the N-terminus of SEQ ID NO: 42, X (or Xaa), is Val.

[0297] In another embodiment, the anti-CD123 antibody is [ka] and a light chain having the amino acid sequence of SEQ ID NO: 43. In some embodiments, the second residue from the N-terminus, X (or Xaa), of SEQ ID NO: 44 is Val.

[0298] In certain embodiments, cell-binding agents (eg, antibodies) of the invention have an N-terminal serine, which may be oxidized with an oxidizing agent to form an oxidized cell-binding agent having an N-terminal aldehyde group.

[0299] Any suitable oxidizing agent can be used in step (a) of the above method. In certain embodiments, the oxidizing agent is a periodate compound. More specifically, the oxidizing agent is a periodate compound. It is sodium hypochlorite.

[0300] An excess of molar equivalents of oxidizing agent relative to the cell-binding agent can be used. In certain embodiments, about 2-100, 5-80, 10-50, 1-10, or 5-10 molar equivalents of oxidizing agent can be used. In certain embodiments, about 10 or about 50 equivalents of oxidizing agent can be used. When using a large amount of oxidizing agent, a short reaction time is used to avoid overoxidation. For example, when 50 equivalents of oxidizing agent are used, the oxidation reaction is carried out for about 5 to about 60 minutes. Alternatively, when 10 equivalents of oxidizing agent are used, the reaction is carried out for about 30 minutes to about 24 hours. In some embodiments, 5 to 10 molar equivalents of oxidizing agent are used, and the oxidation reaction is carried out for about 5 to about 60 minutes (e.g., about 10 to about 30 minutes, about 20 to about 30 minutes).

[0301] In certain embodiments, the oxidation reaction does not result in significant non-target oxidation, e.g., an insignificant amount (e.g., less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1%) of methionine and / or glycans are oxidized during the N-terminal serine oxidation process, resulting in an oxidized cell-binding agent having an N-terminal aldehyde group.

[0302] In certain embodiments, cell-binding agents (e.g., antibodies) of the invention have an engineered Cys residue, e.g., a Cys residue at position 442 of the EU / OU numbering system of the antibody. Thus, the term "cysteine ​​engineered antibody" includes antibodies with at least one Cys not normally present at a given residue in the antibody light or heavy chain. Such a Cys, sometimes referred to as an "engineered Cys," may be engineered using any conventional molecular biology or recombinant DNA technique (e.g., by replacing the coding sequence for a non-Cys residue at the target residue with the coding sequence for a Cys). For example, if the original residue is Ser with a coding sequence of 5'-UCU-3', the coding sequence can be mutated (e.g., by site-directed mutagenesis) to 5'-UGU-3', which encodes a Cys. In certain embodiments, Cys-engineered antibodies of the invention have an engineered Cys in the heavy chain. In certain embodiments, the engineered Cys is in or near the CH3 domain of the heavy chain. The engineered antibody heavy (or light) chain sequence can be inserted into a suitable recombinant expression vector to generate an engineered antibody with an engineered Cys residue in place of the original Ser residue.

[0303] Cytotoxicity of compounds and conjugates The cytotoxic compounds and cell-binding agent-drug conjugates of the present invention can be evaluated in vitro for their ability to inhibit the growth of various cancer cell lines. For example, cell lines such as human choriocarcinoma JEG-3 cells can be used to evaluate the cytotoxicity of these compounds and conjugates. The cells to be evaluated can be exposed to the compound or conjugate for 1-5 days, and cell viability can be measured in a direct assay using known methods. The results of the assay can then be used to determine the IC 50Alternatively, or in addition, in vitro cell-based susceptibility screening methods, such as those described by the National Cancer Institute (see Voskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9:42227-4239, incorporated herein by reference), can be used as a guide to determining the types of cancer that may be susceptible to treatment with the compounds or conjugates of the invention.

[0304] An example of the in vitro efficacy and target specificity of antibody-cytotoxic drug conjugates of the invention is described in Example 6. Antigen-negative cell lines remained viable when exposed to the same conjugates.

[0305] Compositions and Methods of Use The present invention includes compositions (e.g., pharmaceutical compositions) comprising the novel maytansinoid compounds described herein, their derivatives, or conjugates thereof (and / or solvates, hydrates, and / or salts thereof) and a carrier (a pharmaceutically acceptable carrier). The present invention also includes compositions (e.g., pharmaceutical compositions) comprising the novel maytansinoid compounds described herein, their derivatives, or conjugates thereof (and / or solvates, hydrates, and / or salts thereof) and a carrier (a pharmaceutically acceptable carrier). The compositions are useful for inhibiting abnormal cell growth or treating proliferative disorders in mammals (e.g., humans).

[0306] The present invention includes a method for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a novel maytansinoid compound, derivative thereof, or conjugate thereof, (and / or solvates and salts thereof), or composition thereof, as described herein.

[0307] The present invention also provides a method of treatment comprising administering to a subject in need thereof an effective amount of any of the conjugates described above.

[0308] Similarly, the present invention provides a method for inducing cell death in a selected cell population, comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent comprising any of the cytotoxic compound-cell binding agents of the present invention, their salts or solvates. The target cells are cells to which the cell binding agent can bind.

[0309] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those skilled in the art, where the clinical situation warrants.

[0310] Examples of suitable carriers, diluents, and / or additives include (1) Dulbecco's phosphate buffered saline (pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin), (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose; they may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

[0311] The methods for inducing cell death in selected cell populations can be performed in vitro, in vivo, or ex vivo.

[0312] Examples of in vitro applications include the treatment of autologous bone marrow prior to transplant into the same patient to kill diseased or malignant cells; the treatment of bone marrow prior to transplant to kill competent T cells and prevent graft-versus-host disease (GVHD); the treatment of cell cultures to kill all cells except for desired variants that do not express the target antigen; or the killing of variants that express undesirable antigens.

[0313] Conditions for non-clinical in vitro use are readily determined by one skilled in the art.

[0314] Examples of clinical ex vivo applications are for the removal of tumor cells or lymphocytes from bone marrow prior to autologous transplantation in cancer treatment or in the treatment of autoimmune diseases, or for the removal of T cells and other lymphocytes from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent GVHD. Treatment can be performed as follows: Bone marrow is harvested from a patient or other individual and then incubated at about 37°C for about 30 minutes to about 48 hours in a medium containing serum to which a cytotoxic drug of the present invention has been added at a concentration ranging from about 10 μM to 1 pM. The exact conditions for the concentration and time of incubation, i.e., the dose, can be easily determined by one skilled in the art. After incubation, the bone marrow is then cultured using known methods. The bone marrow cells are then washed with serum-containing medium and returned intravenously to the patient. If the patient undergoes other treatments, such as a course of ablative chemotherapy or total body irradiation, between the time of bone marrow harvest and the time of reinfusion of the treated cells, the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0315] For clinical in vivo use, the cytotoxic drugs of the present invention will be supplied as solutions or lyophilized powders that have been tested for sterility and endotoxin levels.

[0316] In some embodiments, the compounds and conjugates of the invention are used in the treatment of cancer (e.g., kidney cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, brain cancer, prostate cancer, endometrial cancer, cervical cancer, renal cancer, pancreatic cancer, ovarian cancer, head and neck cancer, melanoma, colorectal cancer, gastric cancer, squamous cell carcinoma, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), testicular cancer, choriocarcinoma, Merkel cell carcinoma, sarcoma (e.g., osteosarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma), It can be used to treat glioblastoma, neuroblastoma, lymphoma (e.g., non-Hodgkin's lymphoma), myelodysplastic syndrome (MDS), peritoneal cancer, fallopian tube cancer, uterine cancer, or leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (e.g., B-ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML)).

[0317] Analogs and Derivatives Those skilled in the art of cytotoxic drugs will readily understand that each of the cytotoxic drugs described herein can be modified so that the resulting compound still retains the specificity and / or activity of the starting compound. Those skilled in the art will also understand that many of these compounds can be used in place of the cytotoxic drugs described herein. That is, the cytotoxic drugs of the present invention include analogs and derivatives of the compounds described herein.

[0318] All references cited herein and in the examples below are incorporated by reference in their entirety. [Example]

[0319] Example 1. Preparation of DM-H(7) stock solution [ka] Maytansinol (5.0 g, 8.85 mmol) was dissolved in anhydrous DMF (125 mL) and cooled in an ice bath. N-carboxyanhydride of N-methylalanine (5.7 g, 44.25 mmol), anhydrous DIPEA (7.70 mL, 44.25 mmol), and zinc trifluoromethanesulfonate (22.5 g, 62 mmol) were then added under an argon atmosphere with magnetic stirring. The ice bath was removed, and the reaction was allowed to warm with stirring. After 16 hours, deionized water (10 mL) was added. After 30 minutes, a 1:1 solution of saturated aqueous sodium bicarbonate:saturated aqueous sodium chloride (190 mL) and ethyl acetate (250 mL) was added with vigorous stirring. The mixture was transferred to a separatory funnel, and the organic layer was retained. The aqueous layer was extracted with ethyl acetate (100 mL), and the organic layers were then combined and washed with saturated sodium chloride. The solution was washed with aqueous maytansinol (50 mL). The organic layer was concentrated to one-quarter of its volume by rotary evaporation under vacuum without heating the evaporating bath and was not purified. The concentration of the solution was estimated by dividing the millimoles (1.77 mmol) of maytansinol used in the reaction by the volume (150 mL) to obtain a DM-H stock solution (0.06 mmol / mL). Aliquots of the stock solution were immediately dispensed and then used in reactions or stored in a -80°C freezer and thawed when needed.

[0320] Example 2. Synthesis of thio-peptide-maytansinoids Compounds of the type FMoc-peptide-NH—CH 2 —OAc were prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH—CH 2 —OAc.

[0321] FMoc-peptide-OAc compounds FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-OAc (9a): FMoc-L-Ala-L-Ala-L-Ala-Gly-OH (500 mg, 0.979 mmol) was dissolved in DMF (2 mL) and copper(II) acetate (17.8 mg, 0.098 mmol) and acetic acid (84 μL, 1.47 mmol) were added under argon with magnetic stirring. Once the solid dissolved, lead tetraacetate (434 mg, 0.979 mmol) was added. The reaction proceeded at 60 °C for 20 min and then purified on a C18, 30 micron, 450 g column cartridge eluted with deionized water containing 0.1% formic acid and a linear acetonitrile gradient from 5% to 55% over 26 min at a flow rate of 125 mL / min. Fractions containing the pure desired product were frozen and lyophilized to give 178 mg (34% yield) of a white solid. HRMS (M+Na) + Calculated value 547.2163; measured value 547.2160. 1H NMR (400 MHz, DMSO-d6) δ 1.20 (qd, J = 7.5, 6.9, 4.2 Hz, 9H), 1.91 - 2.05 (m, 3H), 3.26 - 3.38 (m, 1H), 4.3 Hz, 4.05 (q (td, J = 11.9, 10.7, 6.4 Hz, 5H), 5.07( ddd, J = 11.2, 6.9, 4.3 Hz, 2H), 7.32 (q, J = 7.5 Hz, 2H), 7.41 (q, J = 7.4 Hz, 2H), 7.52 ( t, J = 6.8 Hz, 1H), 7.71 (q, J = 7.5, 7.0 Hz, 2H), 7.82 – 8.08 (m, 4H), 8.84 (q, J = 7.1 Hz , 1H).

[0322] FMoc-D-Ala-L-Ala-L-Ala-NH-CH2-OAc(9b):HRMS(M+Na) + Phone: 547.2163, Exchange address 1 H NMR (400 MHz, DMSO-d6) δ 1.23 (dd, J = 12.5, 7.4 Hz, 9H), 1.95 (s, 2H), 4.00 - 4.13 (m, 1H), 4.17 - 4.38 (m, Hz), 06H 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.62 (d, J = 6.8 Hz, 1H), 7.71 (t, J = 8.6 Hz, 2H), 7.85 - 8.01 (m, 3H), 8.21 (d, J = 7.0 Hz, 1H), 8.69, 61 (d). J = Hz.

[0323] FMoc-L-Ala-D-Ala-L-Ala-NH-CH2-OAc(9c):HRMS(M+Na) +The calculated value is 547.2163, and the measured value is 547.2168. 1 H NMR (400 MHz, DMSO-d6) δ 1.16 - 1.24 (m, 9H), 1.9 7 (s, 3H), 4.07 (q, J = 7.0 Hz, 1H), 4.16 - 4.34 (m, 5H), 5.00 - 5.16 (m, 2H), 7.33 (td, J = 7.4, 1.1 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.58 (d, J = 7.0 Hz, 1H), 7.72 (t, J = 8.1 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.03 (d, J = 7 .5 Hz, 1H), 8.14 (d, J = 7.2 Hz, 1H), 8.85 (t , J = 6.9 Hz, 1H).

[0324] FMoc-L-Ala-L-Ala-D-Ala-NH-CH2-OAc(9d):HRMS(M+Na) + The calculated value is 547.2163, and the measured value is 547.2167. 1 H NMR (400 MHz, DMSO-d6) δ 1.18 - 1.25 (m, 9H), 1. 97 (s, 3H), 3.96 - 4.15 (m, 1H), 4.17 - 4.36 (m, 5H), 5.09 (d, J = 6.9 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.57 (d, J = 7.2 Hz, 1H), 7.71 (d, J = 7.3 Hz, 2H), 7.90 (d, J = 7.5 Hz, 2H), 8.07 (s, 2H), 8.86 (s, 1H).

[0325] FMoc-L-Ala-D-Ala-NH-CH2-OAc(9f):HRMS(M+Na) +476.1792,movement 1 H NMR (400 MH z, DMSO-d6) δ 1.13 (dd, J = 7.1, 1.4 Hz, 6H), 1.89 (s, 3H), 3.99 (q, J = 7.1 Hz, 1H), 4.10 - 4.29 (m, 4H), 4.95 - 5.08 (m, 2H), 7.26 (t, J = 7.4, 1.3 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H ), 7.49 (d, J = 7.2 Hz, 1H), 7.66 (t, J = 7.6 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H), 8.11 (d, J = 7.7 Hz, 1H), 8.76 (t, J = 7.0 Hz, 1H).

[0326] FMoc-D-Ala-L-Ala-NH-CH2-OAc(9g):HRMS(M+Na) + 476.1792, 476.1788. 1 H NMR (400 MH z, DMSO-d6) δ 1.21 (dd, J = 7.1, 1.4 Hz, 6H), 1.96 (s, 3H), 4.08 (t, J = 7.1 Hz, 1H), 4.17 - 4.36 (m, 4H), 5.05 - 5.14 (m, 2H), 7.26 - 7 .38 (m, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.56 ( d, J = 7.3 Hz, 1H), 7.73 (t, J = 7.6 Hz, 2H), 7.90 (d, J = 7.6 Hz, 2H), 8.18 (d, J = 7.8 Hz, 1H), 8.83 (t, J = 6.9 Hz, 1H).

[0327] FMoc-D-Ala-D-Ala-NH-CH2-OAc(9h):HRMS(M+H) + Calculated value: 455.4877, measured value: 455.2051. 1 H NMR (400 MH z, DMSO-d6) δ 1.14 (dd, J = 7.1, 3.3 Hz, 6H), 1.21 (d, J = 7.2 Hz, 1H), 1.81 (s, 1H), 1.91 (s, 2H), 4.01 (q, J = 7.7 Hz, 1H), 4.09 - 4.2 7 (m, 5H), 4.95 - 5.10 (m, 1H), 7.26 (td, J = 7.4, 1.2 Hz, 3H), 7.35 (t, J = 7.4 Hz, 3H), 7 .45 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.1 Hz, 3H), 7.82 (d, J = 6.4 Hz, 2H), 7.96 (d, J = 7.4 Hz, 1H), 8.78 (t, J = 7.0 Hz, 1H).

[0328] FMoc-peptide-COOH compounds FMoc-peptide-NH-CH2-S-(CH2) n Compounds of the -CO2H type were prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO2H.

[0329] FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-C OH (10a): 6-Mercaptohexanoic acid (287 μL, 2.07 mmol) was dissolved in a 1:4 TFA:dichloromethane (5 mL) solution and then added to a vial containing FMoc-L-Ala-L-Ala-L-Ala-NH-CH-OAc (178 mg, 0.339 mmol). The reaction proceeded for 20 minutes at room temperature under an argon atmosphere with magnetic stirring. The crude material was concentrated under vacuum, redissolved in a minimum volume of DMF, and purified on a C18, 30 micron, 30 g cartridge eluted with deionized water containing 0.1% formic acid and a linear gradient of 5% to 95% acetonitrile over 13 minutes at 35 mL / min. Fractions containing the pure desired product were frozen and lyophilized to give 200 mg (96% yield) of a white solid. HRMS (M+H) + Calculated value 613.2690; measured value 613.2686. 1 H NMR (400 MHz, DMSO-d6) δ 1.20 (dt, J = 7.1, 4.9 Hz, 10H), 1.31 (tt, J = 10.1, 6.0 Hz, 2H), 1.49 (dq, J = 12.5, 7.4 Hz, 4 H), 2.18 (t, J = 7.3 Hz, 2H),4.05 (t, J = 7.3 Hz, 1H), 4.16 - 4.30 (m, 7H), 7.33 (td, J = 7 .4, 1.2 Hz, 2H), 7.42 (td, J = 7.3, 1.1 Hz, 2 H), 7.54 (d, J = 7.4 Hz, 1H), 7.72 (t, J = 7.0 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 7.94 - 8.07 (m, 2H), 8.44 (t, J = 6.1 Hz, 1H).

[0330] FMoc-D-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO2H(10b):HRMS(M+Na) + Calculated value 635.2510, measured value 635.2515. 1H NMR (400 MHz, DMSO-d6) δ 1.15 (d, J = 6.8 Hz, 3H), 1.18 − 1.25 (m, 10H), 2.18 (q, J = 7.5 Hz, 4H), 2.40 - 2.48 (m, 1H), 2.70 (t, J = 7.2 Hz, 1H), 4.15 - 4.30 (m, 6H), 6.29 (s , 2H), 7.34 (q, J = 7.3 Hz, 3H), 7.42 (t, J = 7.4 Hz, 3H), 7.63 - 7.78 (m, 1H), 7.85 (d, J = 7.3 Hz, 2H), 7.89 (d, J = 7.5 Hz, 3H), 8.37 - 8.46 (m, 1H).

[0331] FMoc-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO2H(10c):HRMS(M+Na) + Phone: 635.2510, address 1 H NMR (400 MHz, DMSO-d6) δ 1.18 − 1.23 (m, 10H), 1.34 (q, J = 3.4 Hz, 5H), 2.24 (s, 2H), 2.44 (s, 2H), 4.05 (t, J = 7.1 Hz, 1H), 4.16 – 4.35 (m, 8H), 7.33 (t, J = 7.4 Hz, 2H) , 7.42 (t, J = 7.5 Hz, 2H), 7.58 (d, J = 7.0 H z, 1H), 7.71 (t, J = 8.4 Hz, 2H), 7.90 (s, 1H ), 7.98 (d, J = 7.5 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H), 8.39 (t, J = 6.2 Hz, 1H), 11.98 (s, 1H).

[0332] FMoc-L-Ala-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO2H(10d):HRMS(M+Na) + The calculated value is 635.2510, and the measured value is 635.2510. 1 H NMR (400 MHz, DMSO-d6) δ 1.15 (d, J = 6.9 Hz, 3H), 1.21 (d, J = 7.1 Hz, 9H), 1.28 - 1.38 (m, 3H), 1.44 - 1.60 (m, 5H), 2.13 - 2.22 (m, 3H), 3.33 (q, J = 6.9 Hz, 1H), 4.20 (s, 2H), 6.29 (s, 2H), 7.29 - 7.40 (m, 3H), 7.38 - 7.47 (m, 3H), 7.85 (d, J = 7.5 Hz, 2H), 7.8 9 (d, J = 7.5 Hz, 2H), 8.26 (d, J = 7.6 Hz, 1H ), 8.48 (d, J = 6.2 Hz, 1H).

[0333] FMoc-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO2H(10g):HRMS(M+H) + The calculated value is 542.2319, and the measured value is 542.2316. 1 H NMR (400 MHz, DMSO-d6) δ 1.13 (dd, J = 7.1, 1.7 Hz, 6H), 1.16 - 1.25 (m, 2H), 1.32 - 1.47 (m, 4H), 2.08 (t, J = 7.3 Hz, 2H), 3.25 (s, 2 H), 3.99 (p, J = 7.0 Hz, 1H), 4.07 - 4.27 (m, 6H), 7.26 (t, J = 7.4, 1.2 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.52 (d, J = 7.0 Hz, 1H), 7.65 (t, J = 7.3 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H ), 8.08 (d, J = 7.7 Hz, 1H), 8.27 (t, J = 6.2 Hz, 1H), 11.82 (s, 1H).

[0334] FMoc-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO2H(10f):HRMS(M+H) + The calculated value is 542.2319, and the measured value is 542.2321. 1 H NMR (400 MHz, DMSO-d6) δ 1.13 (dd, J = 7.1, 1.8 Hz, 7H), 1.17 - 1.26 (m, 2H), 1.32 - 1.48 (m, 5H), 2.08 (t, J = 7.3 Hz, 2H), 3.99 (p, J = 7.1 Hz, 1H), 4.07 - 4.26 (m, 7H), 7.26 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.53 (d, J = 7.1 Hz, 1H), 7.65 (t, J = 7.3 Hz, 2H ), 7.82 (d, J = 7.4 Hz, 2H), 8.10 (d, J = 7.7 Hz, 1H), 8.28 (t, J = 6.3 Hz, 1H).

[0335] FMoc-D-Ala-D-Ala-NH-CH2-S-(CH2)5-CO2H(10h):(16.7 mg, 0.031 mmol, yield 70%). HRMS (M+H) + The calculated value is 542.2319, and the measured value is 542.2318.

[0336] FMoc-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO2H(10j):HRMS(M+H)+ Phone: 585.2377, Phone: 1 H NMR (400 MHz, DMSO-d6) δ 1.14 − 1.26 (m, 9H), 1.75 (p, J = 7.3 Hz, 2H), 2.27 (t, J = 7.3 Hz, 2H), 2.54 (d, J = 7.7 Hz, 2H), 3.97 - 4.10 (m, 1H), 4.13 - 4.34 (m, 7H), 7.33 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.57 (d, J = 6.9 Hz, 1H), 7.71 (t, J = 8.4 Hz, 2H) , 7.89 (d, J = 7.6 Hz, 2H), 7.97 (d, J = 7.5 H z, 1H), 8.14 (d, J = 7.0 Hz, 1H), 8.41 (s, 1H ), 12.06 (s, 1H).

[0337] FMoc-D-Ala-L-Ala-NH-CH2-S-(CH)2-CO2H(10i):HRMS(M+H) + Exchange rate 500.1850, exchange rate 1 HN MR (400 MHz, DMSO-d6) δ 1.20 (dd, J = 7.2, 1.9 Hz, 6H), 2.53 (d, J = 7.1 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 4.4, 1.7 (H0), J = 4.26 (m, 4H), 4.29 (d, J = 6.8 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2 H), 7.56 (d, J = 7.1 Hz, 1H), 7.72 (t, J = 7.7 Hz, 2H), 7.89 (d, J = 7.5 Hz, 2H), 8.14 (d, J = 7.6 Hz, 1H), 8.42 (t, J = 6.3 Hz, 1H), 12.22 (s, 1H).

[0338] Synthesis of FMOc-L-Ala-D-Ala-L-Ala-NH-CH2-OAc (9c): [ka] Step 1: FMoc-L-Ala-D-Ala-OtBu (9c1): FMoc-L-alanine (10 g, 32.1 mmol) and d-Ala-OtBu, HCl (7.00 g, 38.5 mmol) were dissolved in CHCl (100 mL) and treated with COMU (20.63 g, 48.2 mmol) and DIPEA (11.22 mL, 64.2 mmol). The reaction was allowed to proceed under argon at room temperature. After 2 h, the reaction was shown to be complete by UPLC and was diluted with 2-MeTHF (50 mL) and washed with 10% aqueous citric acid (2 × 100 mL), water (100 mL), and then brine (100 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated to give crude FMoc-L-Ala-d-Ala-OtBu. Estimated yield: 100%.

[0339] Step 2: FMoc-L-Ala-D-Ala (9c2) FMoc-EAla-DAla-OtBu (11.25 g, 25.7 mmol) was treated with TFA:water (95:5) (50 ml). The reaction proceeded at room temperature under an argon atmosphere. After 4 hours, the reaction was shown to be complete by UPLC and was diluted with toluene (25 mL) and co-evaporated (3 times) to give FMoc-L-Ala-D-Ala. Estimated yield: 100%.

[0340] Step 3: FMoc-L-Ala-Gly-OtBu (9c3) ZL-Ala-ONHS (10 g, 31.2 mmol) and tert-butyl glycinate (6.28 g, 37.5 mmol) were dissolved in CHCl (100 mL) and treated with DIPEA (10.91 mL, 62.4 mmol). The reaction was allowed to proceed under argon at room temperature. After 2 h, UPLC indicated completion, and the reaction was diluted with 2-MeTHF (50 mL) and washed with 10% aqueous citric acid (100 mL), saturated sodium bicarbonate (2 × 100 mL), water (100 mL), and brine (100 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated to give ZL-Ala-Gly-OtBu. Estimated yield: 100%.

[0341] Step 4. L-Ala-Gly-OtBu (9c4) Z-Ala-Gly-OtBu (10.05 g, 29.9 mmol) was dissolved in 95:5 MeOH:water (50 ml), transferred to a reduction flask, and treated with Pd / C (1.272 g, 11.95 mmol). The reduction flask was placed on a shaker and the air was removed by vacuum while shaking the flask. Hydrogen was charged to 30 psi, the flask was shaken for 2 minutes, and the hydrogen was removed by vacuum. This was repeated two more times. Hydrogen was charged to 30 psi and the flask was shaken. After 4 hours, UPLC showed completion, and the reaction was filtered in vacuo through a Celite plug, dissolved in 2-MeTHF, and concentrated to give LAla-Gly-OtBu. Estimated yield: 100%.

[0342] Step 5: FMoc-L-Ala-D-Ala-L-Ala-Gly-OtBu (9c5) FMoc-LAla-D-ALa-OH (0.959 g, 2.508 mmol) and L-Ala-Gly-OtBu (0.718 g, 3.01 mmol) were dissolved in CHCl (10 mL) and treated with COMU (1.181 g, 2.76 mmol) and DIPEA (0.876 mL, 5.02 mmol). The reaction was allowed to proceed under argon at room temperature. After 2 h, the reaction was shown to be complete. The reaction was concentrated to remove CHCl, redissolved in 2 mL of DMF, and purified by CombiFlash using a linear gradient. The combined product gave FMoc-L-Ala-D-Ala-L-Ala-Gly-OtBu (660 mg, 46% yield).

[0343] Step 6. FMoc-L-Ala-D-Ala-L-Ala-Gly-OH (9c6) FMoc-LAla-DAla-LAla-GlyOtBu (200 mg, 0.353 mmol) was treated with TFA:water (95:5) (2 mL). The reaction was allowed to proceed under argon at room temperature. After 1 hour, the reaction was shown to be complete by UPLC. Dilution with toluene (1 mL) and coevaporation with toluene twice gave FMoc-L-Ala-D-Ala-L-Ala-Gly-OH. Estimated yield: 100%.

[0344] Step 7. FMoc-L-Ala-D-Ala-L-Ala-CH2-OAc (9c7) FMoc-L-Ala-D-Ala-L-Ala-Gly-OH (2.65 g, 5.19 mmol) was dissolved in DMF (20 mL) and treated with copper(II) acetate (0.094 g, 0.519 mmol) and acetic acid (0.446 mL, 7.79 mmol). Once all reagents were dissolved, the reaction was treated with lead tetraacetate (3.45 g, 7.785 mmol). The reaction proceeded under argon at 60°C for 30 minutes. The crude reaction mixture was purified by Combiflash Rf 200i using a C18 450 g column at a flow rate of 125 mL / min with deionized water containing 0.1% formic acid and acetonitrile as the solvent, using the following gradient (time (min), percent acetonitrile): (0, 5) (8, 50) (26, 55). The desired product had a retention time of 11 min, and the product fraction was immediately frozen and lyophilized to give FMoc-L-Ala-D-Ala-L-Ala-CH2-OAc (843 mg, 1.607 mmol, 31.0% yield). HRMS (M+Na) + Calculated value 547.2163, measured value 547.2167. 1 H NMR (400 MHz, DMSO-d6) δ 1.23 (dd, J = 12.5, 7.4 Hz, 9H), 1.95 (s, 2H), 4.00 - 4.13 (m, 1H), 4.17 - 4.38 (m, 6H), 5.06 (q, J = 8.8 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.62 (d, J = 6.8 Hz, 1H), 7.71 (t, J = 8.6 Hz , 2H), 7.85 - 8.01 (m, 3H), 8.21 (d, J = 7.0 Hz, 1H), 8.69 (d, J = 6.9 Hz, 1H).

[0345] FMoc-peptide-May-NMA compound FMoc-peptide-NH-CH2-S-(CH2) nCompounds of the -CO2-DM type were prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0346] FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (11a): To the DM-H stock solution (8.2 mL, 0.49 mmol) was added FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-COOH (300 mg, 0.49 mmol), EDC (94 mg, 0.490 mmol), and DIPEA (90 μL, 0.49 mmol). The reaction was allowed to proceed with magnetic stirring at room temperature under an argon atmosphere for 2 hours. The crude material was concentrated under vacuum by rotary evaporation, and the residue was taken up in a minimal volume of DMF and then purified on a C18, 30 micron, 30 g cartridge, eluting with deionized water containing 0.1% formic acid and a linear gradient of 5% to 50% acetonitrile over 25 minutes. Fractions containing the pure desired product were frozen and lyophilized to give 151 mg (37.2% yield) of a white solid. HRMS (M+Na) + Calculated value 1266.5170; Measured value 1266.5141. 1 H NMR (400 MHz, DMSO-d6) δ 0.77 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 1.14 - 1.22 (m, 12H), 1.22 - 1.30 (m, 3H), 1.35 - 1.49 (m, 4H), 1.50 - 1.55 ( m, 1H), 1.59 (s, 3H), 2.00 - 2.07 (m, 1H), 2.14 (ddd, J = 15.6, 8.7, 5.9 Hz, 1H), 2.40 (dtd, J = 17.0, 7.9, 7.0, 4.9 Hz, 3H), 2.69 (s, 3H), 2.79 (d, J = 9.6 Hz, 1H), 3.08 (s, 3H), 3.20 (d, J = 12.6 Hz, 1H), 3.24 (s, 3H), 3.43 (d, J = 12.4 Hz, 2H), 3.48 (d, J = 8.9 Hz, 1H ), 3.92 (s, 3H), 4.08 (ddd, J = 20.8, 10.8, 5.0 Hz, 3H), 4.14 - 4.24 (m, 4H), 4.26 (d, J = 6.0 Hz, 3H), 4.52 (dd, J = 12.0, 2.8 Hz, 1H), 5.34 (q, J = 6.7 Hz, 1H), 5.56 (dd, J = 14.7, 9.0 Hz, 1H), 5.91 (s, 1H), 6.50 - 6.66 (m, 3 H), 6.88 (s, 1H), 7.17 (d, J = 1.8 Hz, 1H), 7 .33 (td, J = 7.5, 1.2 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.53 (d, J = 7.4 Hz, 1H), 7.72 (t, J = 7.0 Hz, 2H), 7.89 (d, J = 7.5 Hz, 3H), 7.99 (d, J = 7.3 Hz, 1H), 8.36 (t, J = 6.3 Hz, 1H ).

[0347] FMoc-D-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)s-CO-DM(11b): HRMS(M+Na) + Calculated value: 1266.5170, Measured value: 1266.5164. 1 H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1.14 (dd, J = 14.6, 6.5 Hz, 6H), 1.22 (t, J = 6.8 Hz, 10H), 1.33 - 1.57 (m, 4H), 1.59 (s, H), 2.04 (d, J = 13.5 Hz, 1H), 2.27 - 2. 44 (m, 1H), 2.69 (s, 3H), 2.80 (d, J = 9.7 Hz It should be noted that there is an "H" in the line " 1.59 (s, H), 2.04 (d, J = 13.5 Hz, 1H)" which seems to be a typo in the original text. It might be intended to be "1.59 (s, 3H)" as in the previous similar description., 1H), 3.08 (s, 3H), 3.14 - 3.28 (m, 5H), 3.37 - 3.55 (m, 3H), 3.92 (s, 3H), 3.98 - 4.16 ( m, 3H), 4.20 (dd, J = 15.6, 7.6 Hz, 7H), 4.52 (d, J = 12.7 Hz, 1H), 5.34 (d, J = 6.9 Hz, 1H ), 5.57 (dd, J = 14.7, 9.0 Hz, 1H), 5.92 (s, 1H), 6.46 - 6.72 (m, 4H), 6.88 (s, 1H), 7.17 (s, 1H), 7.33 (t, J = 7.5 Hz, 3H), 7.41 (t, J = 7.4 Hz, 3H), 7.60 - 7.75 (m, 4H), 7.80 - 7. 93 (m, 4H), 8.12 (t, 1H), 8.29 (d, J = 6.9 Hz , 1H).

[0348] FMoc-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(11c): HRMS(M+Na) + Calculated value 1266.5170, measured value 1266.5170. 1 1H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.96 - 1.16 (m, 10H), 1.16 - 1.51 (m, 10H), 1.52 (s, 4H), 1.82 - 2.16 (m, 1H), 2.17 - 2.56 (m, 11H), 2.62 (d, J = 5.8 Hz, 4H), 2.6 8 - 2.87 (m, 3H), 2.92 - 3.04 (m, 4H), 3.09 - 3.22 (m, 7H), 3.24 (d, J = 7.4 Hz, 1H), 3.33 - 3.50 (m, 2H), 3.73 - 3. .07 (m, 2H), 4.07 - 4.25 (m, 2H), 4.45 (dd, J = 12.0, 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.40 - 5.55 (m, 1H), 5.85 (s, 1H), 6.33 - 6 .66 (m, 4H), 6.81 (s, 2H), 7.03 - 7.19 (m, 1H), 7.19 - 7.43 (m, 2H), 7.62 (d, J = 11.6 Hz, 1H), 7.73 - 7.85 (m, 1H).

[0349] FMoc-L-Ala-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(11d):HRMS(M+Na) + The calculated value is 1266.5170, and the measured value is 1266.5158. 1 H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1.06 - 1.33 (m, 16H), 1.44 (d, J = 10.3 Hz, 11H), 1.59 (s, 3H), 1.99 - 2.22 (m, 3H), 2.35 - 2.45 (m, 2H), 2.55 (d, J = 1.8 Hz, 1H) , 2.69 (s, 3H), 2.80 (d, J = 9.6 Hz, 1H), 3.0 8 (s, 2H), 3.25 (s, 3H), 3.39 - 3.52 (m, 3H), 3.92 (s, 3H), 3.99 - 4.40 (m, 4H), 4.52 (d, J = 11.1 Hz, 1H), 5.34 (d, J = 6.8 Hz, 1H), 5.57 (dd, J = 14.5, 9.2 Hz, 1H), 5.92 (s, 1H), 6.53 - 6.64 (m, 2H), 6.88 (s, 2H), 7.17 (d, J = 1.9 Hz, 1H), 7.33 (t, J = 7.3 Hz, 3H), 7.42 (t, J = 7.4 Hz, 3H), 7.57 (d, J = 7.4 Hz, 1H) , 7.72 (s, 3H), 7.89 (d, J = 7.6 Hz, 3H), 7.9 9 (d, J = 7.6 Hz, 1H), 8.07 (s, 1H), 8.35 (s, 1H).

[0350] FMoc-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(11g): HRMS(M+H) + Calculated value: 1173.4980, Measured value: 1173.4964. 1 H NMR (400 MHz, DMSO-d6) δ 0.79 (s, 3H), 1. 06 - 1.34 (m, 13H), 1.36 - 1.54 (m, 4H), 1.60 (s, 2H), 1.88 - 2.10 (m, 1H), 2.10 - 2.23 (m , 1H), 2.31 - 2.51 (m, 13H), 2.71 (s, 3H), 2.80 (d, J = 9.6 Hz, 1H), 3.10 (s, 3H), 3.26 (s , 4H), 3.33 - 3.66 (m, 3H), 3.98 - 4.32 (m, 4 H), 4.53 (dd, J = 12.0, 2.8 Hz, 1H), 5.35 (q, J = 6.7 Hz, 1H), 5.49 - 5.65 (m, 1H), 6.51 - 6.67 (m, 3H), 6.89 (s, 1H), 7.19 (d, J = 1.8 Hz, 1H), 8.25 (s, 2H), 8.34 (d, J = 7.1 Hz, 1 H), 8.58 (t, J = 6.3 Hz, 1H).

[0351] FMoc-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(11f):HRMS(M+H) + The calculated value is 1173.4980, and the measured value is 1173.4969.

[0352] FMoc-D-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(11h):HRMS(M+Na) + The calculated value is 1195.4907, and the measured value is 1195.4799. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1 .00 - 1.22 (m, 13H), 1.28 - 1.45 (m, 2H), 1.52 (s, 3H), 1.91 - 2.14 (m, 1H), 2.26 (t, J= 1 .9 Hz, 5H), 2.48 (t, J = 1.8 Hz, 2H), 2.62 (s , 3H), 2.66 - 2.77 (m, 2H), 3.01 (s, 2H), 3.10 - 3.21 (m, 5H), 3.28 - 3.47 (m, 2H), 3.86 ( d, J = 6.7 Hz, 4H), 3.93 - 4.25 (m, 10H), 4.3 7 - 4.54 (m, 1H), 5.27 (d, J = 6.7 Hz, 1H), 5.40 - 5.56 (m, 1H), 5.85 (s, 1H), 6.31 - 6.66 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.8 Hz, 1 H), 7.26 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.1 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H), 7.89 (d, J = 7.3 Hz, 1H).

[0353] FMoc-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(11j): HRMS(M+H) + Calculated value 1216.5038, measured value 1216.4999. 1 1H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 0.95 - 1.29 (m, 16H), 1.37 (d, J = 3.4 Hz , 1H), 1.46 (t, J = 12.5 Hz, 2H), 1.59 (s, 3H ), 1.62 - 1.90 (m, 1H), 1.99 - 2.07 (m, 1H), 2.08 (s, 2H), 2.18 - 2.43 (m, 1H), 2.50 - 2.59 (m, 1H), 2.69 (s, 3H), 2.73 - 2.83 (m, 1H), 3.10 (s, 2H), 3.25 (s, 3H), 3.38 - 3.55 (m, 2H), 3.91 (s, 3H), 3.99 - 4.13 (m, 4H), 4.12 - 4.35 (m, 7H), 4.52 (dd, J = 12.0, 2.9 Hz, 1 H), 5.34 (q, J = 6.7 Hz, 1H), 5.48 - 5.65 (m, 1H), 5.92 (s, 1H), 6.48 - 6.70 (m, 3H), 6.88 (s, 1H), 7.17 (d, J = 1.7 Hz, 1H), 7.33 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.58 (d, J = 7.0 Hz, 1H), 7.71 (t, J = 8.3 Hz, 2H), 7.89 (d, J = 7.5 Hz, 3H), 7.95 (d, J = 7.6 Hz , 1H), 8.15 (d, J = 7.2 Hz, 1H), 8.29 - 8.38 (m, 1H), 8.41 (s, 1H).

[0354] FMoc-D-Ala-L-Ala-NH-CH2-S-(CH2)2-CO-DM(11i): HRMS(M+H) + Calculated value: 1131.4510, measured value: 1131.4507. 1 H NMR (400 MHz, DMSO-d6) δ 0.76 (s, 3H), 1. 08 - 1.21 (m, 12H), 1.24 (d, J = 13.9 Hz, 1H) , 1.38 - 1.52 (m, 2H), 1.58 (s, 3H), 1.99 - 2 .09 (m, 1H), 2.33 - 2.44 (m, 1H), 2.68 (s, 3H), 2.80 (dd, J = 14.4, 8.6 Hz, 2H), 3.08 (s, 3H), 3.17 (d, J = 12.5 Hz, 1H), 3.23 (s, 3H), 3.46 (t, J = 10.3 Hz, 2H), 3.91 (s, 3H), 4.0 0 - 4.13 (m, 3H), 4.13 - 4.34 (m, 5H), 4.52 ( dd, J = 12.0, 2.9 Hz, 1H), 5.30 (q, J = 6.8 Hz, 1H), 5.55 (dd, J = 13.4, 9.1 Hz, 1H), 5.91 (s, 1H), 6.55 (dd, J = 7.4, 5.7 Hz, 3H), 6.87 (s, 1H), 7.16 (d, J = 1.8 Hz, 1H), 7.32 (tt, J = 7.4, 1.5 Hz, 2H), 7.41 (tt, J = 7.5, 1.5 Hz, 2H), 7.57 (d, J = 7.0 Hz, 1H), 7.71 (dd, J = 10.5, 7.5 Hz, 2H), 7.88 (d, J = 7.5 Hz, 2H) , 8.14 (d, J = 7.6 Hz, 1H), 8.37 (t, J = 6.3 H z, 1H).

[0355] Amino-peptide-maytansinoids H2N-peptide-NH-CH2-S-(CH2) n Compounds of the -CO2-DM type were prepared as exemplified by H2N-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0356] H2N-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (12a): FMoc-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (151 mg, 0.121 mmol) was treated with 20% morpholine in DMF (2 mL). The reaction was allowed to proceed with magnetic stirring under argon at room temperature for 1 hour. The crude material was purified on a C18, 30 micron, 150 g column cartridge eluted with deionized water containing 0.1% formic acid and a linear gradient of 5% to 50% acetonitrile over 26 minutes. Fractions containing the desired product were immediately frozen and lyophilized to give 46 mg (37.1% yield) of a colorless oil. HRMS (M+H) + Calculated value 1022.4670; Measured value 1022.4669. 1 H NMR (400 MHz , DMSO-d6) δ 0.78 (s, 3H), 1.12 (d, J = 6.3H z, 3H), 1.13 - 1.21 (m, 10H), 1.21 - 1.31 (m, 3H), 1.37 - 1.50 (m, 4H), 1.51 - 1.57 (m, 1H) ), 1.59 (s, 3H), 2.04 (dd, J = 14.4, 2.8 Hz, 1H), 2.15 (ddd, J = 15.9, 8.7, 6.0 Hz, 1H), 2.38 (td, J = 7.0, 3.6 Hz, 2H), 2.70 (s, 3H), 2.79 (d, J = 9.6 Hz, 1H), 3.09 (s, 3H), 3.21 (d, J = 12.5 Hz, 1H), 3.25 (s, 3H), 3.33 - 3.5 5 (m, 8H), 3.93 (s, 3H), 4.01 - 4.33 (m, 5H), 4.52 (dd, J = 12.0, 2.8 Hz, 1H), 5.34 (q, J = 6.7 Hz, 1H), 5.57 (dd, J = 14.6, 9.0 Hz, 1H) , 5.95 (s, 1H), 6.48 - 6.65 (m, 3H), 6.89 (s, 1H), 7.18 (d, J = 1.8 Hz, 1H), 8.07 (d, J = 7 .5 Hz, 1H), 8.13 (s, 1H), 8.31 (s, 1H), 8.40 (t, J = 6.3 Hz, 1H).

[0357] H2N - D - Ala - L - Ala - L - Ala - NH - CH2 - S - (CH2)5 - CO - DM(12b): HRMS(M + H) + Calculated value 1022.4670, measured value 1022.4675. 1 H NMR (400 MHz, DMSO - d6) δ 0.71 (s, 3H), 1.05 (dd, J = 6.7, 3.1 Hz, 7H), 1.08 - 1.1 6 (m, 10H), 1.19 (t, J = 8.1 Hz, 3H), 1.30 - 1.50 (m, 6H), 1.52 (s, 3H), 1.97 (d, J = 13.3 Hz, 1H), 2.01 - 2.21 (m, 2H), 2.34 (s, 3H), 2 .63 (s, 3H), 2.73 (d, J = 9.8 Hz, 1H), 3.02 ( s, 3H), 3.14 (d, J = 12.5 Hz, 1H), 3.33 - 3.4 8 (m, 2H), 3.86 (s, 3H), 3.95 - 4.23 (m, 7H), 4.45 (dd, J = 13.1 Hz, 1H), 5.27 (q, J = 6.8 Hz, 1H), 5.41 - 5.58 (m, 1H), 5.85 (s, 1H), 6.39 - 6.63 (m, 4H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.02 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.26 (s, 1H), 8.36 (t, J = 6.2 Hz, 1 H).

[0358] H2N-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(12c): HRMS(M+H) + Calculated value 1022.4670, measured value 1022.4680. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.01 - 1.26 (m, 19H), 1.25 - 1.50 (m, 6H), 1.52 (s, 3H), 1.97 (d, J = 13.7 Hz, 1H), 2.0 2 - 2.22 (m, 1H), 2.35 (dd, J = 17.2, 9.5 Hz, 2H), 2.47 (d, J = 11.5 Hz, 1H), 2.63 (s, 4H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.10 - 3.24 (m, 6H), 3.32 - 3.50 (m, 2H), 3.86 (s, 3H), 3.95 - 4.18 (m, 4H), 4.45 (dd, J = 12.1 , 2.6 Hz, 1H), 5.27 (q, J = 6.9 Hz, 1H), 5.44 - 5.55 (m, 1H), 5.85 (s, 1H), 6.42 - 6.59 (m, 4H), 6.81 (s, 1H), 7.12 (d, J = 1.7 Hz, 1H), 8.02 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 8.36 (t, J = 6.3 Hz, 1H).

[0359] H2N-L-Ala-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(12d):HRMS(M+H) + Calculated value 1022.4670, measured value 1022.4675. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.98 - 1.14 (m, 13H), 1.14 - 1.26 (m, 2H), 1.30 - 1.49 (m, 4H), 1.52 (s, 3H), 2.24 - 2. 41 (m, 2H), 2.44 (d, J = 1.8 Hz, 16H), 2.63 ( s, 2H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 2H ), 3.08 - 3.21 (m, 4H), 3.32 - 3.49 (m, 2H), 3.86 (s, 3H), 3.92 - 4.23 (m, 3H), 4.45 (d, J = 11.8 Hz, 1H), 5.26 (t, J = 6.7 Hz, 1H), 5.40 - 5.57 (m, 1H), 5.86 (s, 1H), 6.41 - 6.66 ( m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.7 Hz, 1H ), 8.02 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 8. 35 (t, J = 6.3 Hz, 1H).

[0360] H2N-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(12g):HRMS(M+H) +The calculated value is 951.4299, and the measured value is 951.4289. 1 H NMR (400 MHz, DMSO-d6) δ 0.79 (s, 3H), 1.06 - 1.34 (m, 13H), 1.36 - 1.54 (m, 4H), 1.60 (s, 2H), 1.88 - 2.10 (m, 1H), 2.10 - 2.23 (m, 1 H), 2.31 - 2.51 (m, 13H), 2.71 (s, 3H), 2.80 (d, J = 9.6 Hz, 1H), 3.10 (s, 3H), 3.26 (s, 4 H), 3.33 - 3.66 (m, 3H), 3.98 - 4.32 (m, 4H), 4.53 (dd, J = 12.0, 2.8 Hz, 1H), 5.35 (q, J = 6.7 Hz, 1H), 5.49 - 5.65 (m, 1H), 6.51 - 6.6 7 (m, 3H), 6.89 (s, 1H), 7.19 (d, J = 1.8 Hz, 1H), 8.25 (s, 2H), 8.34 (d, J = 7.1 Hz, 1H), 8.58 (t, J = 6.3 Hz, 1H).

[0361] H2N-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(12f):HRMS(M+H) + The calculated value is 951.4226, and the measured value is 951.1299. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.00 - 1.13 (m, 11H), 1.19 (t, J = 8.9 Hz, 3H), 1. 29 - 1.45 (m, 4H), 1.52 (s, 3H), 1.92 - 2.03 (m, 1H), 2.07 (dd, J = 15.7, 8.7 Hz, 1H), 2.23 - 2.39 (m, 1H), 2.63 (s, 3H), 2.73 (d, J = 9.7 Hz, 1H), 3.02 (s, 3H), 3.07 - 3.32 (m, 14H), 3.34 - 3.47 (m, 2H), 3.86 (s, 3H), 3.95 - 4.21 (m, 4H), 4.45 (dd, J = 11.9, 2.8 Hz, 1H), 5.27 (q, J = 6.8 Hz, 1H), 5.50 (dd, J = 14.7, 9.0 Hz, 1H), 5.85 (s, 1H), 6.40 - 6.61 (m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.41 (t, J = 6.1 Hz, 1H).

[0362] H2N-D-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(12h):HRMS(M+H) + Calculated value 950.4226, measured value 951.4299. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.96 - 1.14 (m, 14H), 1.19 (t, J = 8.9 Hz, 3H), 1. 38 (q, J = 10.5, 7.0 Hz, 5H), 1.52 (s, 3H), 1 .88 - 2.02 (m, 1H), 2.02 - 2.18 (m, 1H), 2.22 - 2.41 (m, 2H), 2.48 (s, 1H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.08 - 3.22 (m, 4H), 3.34 - 3.48 (m, 2H), 3.86 (s, [[ID=?]] 4H), 3.95 - 4.23 (m, 5H), 4.45 (dd, J = 11.9 , 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.41 - 5.60 (m, 1H), 5.85 (s, 1H), 6.40 - 6.65 (m, 4H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.44 (t, J = 6.1 Hz, 1H).

[0363] H2N-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(12j):HRMS(M+H) + The calculated value is 994.4357, and the measured value is 994.4330.

[0364] H2N-D-Ala-L-Ala-NH-CH2-S-(CH2)2-CO-DM(12i):HRMS(M+H) + The calculated value is 909.3830, and the measured value is 909.3826. 1 H NMR (400 MHz, DMSO-d6) δ 0.77 (s, 3H), 1.12 (d, J = 6.7 Hz, 6H), 1.17 (dd, J = 7.0, 5.2 Hz, 6H), 1.25 (d, J = 13.3 Hz, 1H), 1.40 - 1.51 (m, 2H), 1.59 (s, 3H), 2.04 (dd, J = 14.4, 2.9 Hz, 1H), 2.41 (ddt, J = 18.6, 10.1, 5.4 Hz, 1H), 2.61 - 2.70 (m, 1H), 2.72 (s, 3H), 2.76 - 2.90 (m, 3H), 3.09 (s, 3H), 3.20 (d, J = 12.4 Hz, 1H), 3.25 (s, 3H), 3.33 (q, J = 6.9 Hz , 1H), 3.39 - 3.64 (m, 3H), 3.93 (s, 3H), 4.0 3 - 4.16 (m, 2H), 4.24 (dt, J = 15.1, 7.6 Hz, 2H), 4.53 (dd, J = 12.0, 2.9 Hz, 1H), 5.32 (q, J = 6.8 Hz, 1H), 5.51 - 5.64 (m, 1H), 5.93 (s, 1H), 6.49 - 6.62 (m, 2H), 6.88 (s, 1H), 7.19 (d, J = 1.8 Hz, 1H), 8.10 (s, 1H), 8.55 (t , J = 6.3 Hz, 1H).

[0365] SPDB-peptide-maytansinoid SPDB-Peptide-NH-CH2-S-(CH2) n Compounds of the -CO2-DM type were prepared as exemplified by SPDB-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0366] SPDB-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (13a): H2N-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (46 mg, 0.045 mmol) was dissolved in DMF (2 mL), to which SPDB (14.7 mg, 0.045 mmol) was added and the reaction was allowed to proceed at room temperature with magnetic stirring under an argon atmosphere for 1 h. The crude material was purified on a C18, 430 micro, 30 g cartridge, eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 35 min. Fractions containing the pure desired product were frozen and lyophilized to give 38 mg (68.5% yield) of a white solid. HRMS (M+H) + Calculated value 1233.4796; Measured value 1233.4783. 1 H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1 .12 (d, J = 6.4 Hz, 3H), 1.14 - 1.21 (m, 10H) , 1.22 - 1.30 (m, 3H), 1.44 (qd, J = 10.2, 4. 5 Hz, 5H), 1.50 - 1.56 (m, 1H), 1.59 (s, 3H), 1.84 (p, J = 7.3 Hz, 2H), 2.04 (dd, J = 14.4, 2.7 Hz, 1H), 2.15 (ddd, J = 15.8, 8.6, 5.9 H z, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.39 (dtdd, J = 18.1, 13.2, 8.1, 4.7 Hz, 3H), 2.70 (s, 3 H), 2.76 - 2.86 (m, 3H), 3.09 (s, 3H), 3.21 (d, J = 12.5 Hz, 1H), 3.25 (s, 3H), 3.43 (d, J = 12.4 Hz, 1H), 3.48 (d, J = 9.0 Hz, 1H), 3.9 2 (s, 3H), 4.13 (s, 2H), 4.19 (h, J = 6.6 Hz, 4H), 4.52 (dd, J = 12.1, 2.8 Hz, 1H), 5.34 (q, J = 6.8 Hz, 1H), 5.56 (dd, J = 14.7, 9.0 Hz, 1H), 5.92 (s, 1H), 6.49 - 6.66 (m, 3H), 6.85 - 6.97 (m, 2H), 7.18 (d, J = 1.8 Hz, 1H), 7.23 (ddd, J = 7.3, 4.8, 1.2 Hz, 1H), 7.76 (dt, J = 8.1, 1.2 Hz, 1H), 7.78 - 7.91 (m, 2H), 8. 00 (d, J = 7.1 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1 H), 8.33 (t, J = 6.3 Hz, 1H), 8.44 (dt, J = 4.7, 1.3 Hz, 1H), 8.50 (s, 1H).

[0367] SPDB-D-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(13b):HRMS(M+H) + Location: 1233.4796, address 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.01 - 1.22 (m, 13H), 1.27 - 1.45 (m, 2H), 1.52 (s, 3H), 1.91 - 2, 2.16 (m J = 7.4 Hz, 7H), 2.26 (t, J = 1.9 Hz, 4H), 2. 48 (t, J = 1.8 Hz, 2H), 2.57 – 2.65 (m, 3H), 2 .65 − 2.77 (m, 2H), 3.01 (s, 2H), 3.13 (d, J = 12.2 Hz, 1H), 3.18 (s, 3H), 3.32 – 3.47 (m, 2H), 3.86 (d, J = 6.7 Hz, 4H), 3.93 - 4.11 (m, 3H), 4.18 (t, J = 11.2 Hz, 7H), 4.39 (H) - 4.50 (m, 1H), 5.27 (Hd, J = Hz, 4H). J = 14.7, 8.8 Hz, 1H), 5.85 (s, 1H), 6.37 − 6 .61 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.8 H z, 1H), 7.26 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.1 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H), 7.89 (d, J = 7.3 Hz, 1H).

[0368] SPDB-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(13c):HRMS(M+H) + The calculated value is 1233.4796, and the measured value is 1233.4795. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.02 - 1.25 (m, 18H), 1.29 - 1.50 (m, 6H), 1.52 (s, 3H), 1.70 - 1.87 (m, 2H), 1.87 - 2 .14 ​​(m, 2H), 2.13 - 2.22 (m, 2H), 2.27 - 2.40 (m, 3H), 2.63 (s, 3H), 2.69 - 2.84 (m, 4H), 3.02 (s, 3H), 3.14 (d, J = 12.3 Hz, 1H), 3.18 (s, 3H), 3.32 - 3.45 (m, 2H), 3.85 (s, 3H), 3.95 - 4.07 (m, 2H), 4.07 - 4.19 (m, 4H), 4.45 (dd, J = 11.9, 2.7 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.55 (m, 1H), 5.85 (s, 1H), 6.42 - 6.59 (m, 3H), 6.81 (s, 1H), 7.11 (s, 1H), 7.13 - 7.19 (m, 1H), 7.68 (d, J = 8.2, 2. 7 Hz, 1H), 7.72 - 7.80 (m, 1H), 7.88 (t, J = 6.6 Hz, 1H), 8.04 (d, J = 6.4 Hz, 1H), 8.09 (d , J = 7.4 Hz, 1H), 8.25 (t, J = 6.3 Hz, 1H), 8 .37 (dd, J = 5.0, 1.9 Hz, 1H).

[0369] SPDB-L-Ala-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(13d): HRMS(M+H) + Calculated value: 1233.4796, measured value: 1233.4797. 1 H NMR (400 MHz, DMSO-d6) δ 0.72 (d, J = 3.3 Hz, 3H), 0.98 - 1.28 (m, 22H), 1.30 - 1.46 (m, 3H), 1.53 (s, 3H), 1.78 (q, J = 7.1 H z, 2H), 1.86 - 2.16 (m, 2H), 2.19 (q, J = 7.4 , 5.6 Hz, 2H), 2.26 - 2.41 (m, 2H), 2.41 - 2. 55 (m, 4H), 2.64 (d, J = 3.2 Hz, 2H), 2.81 - 2.92 (m, 1H), 3.02 (s, 2H), 3.14 (d, J = 12.0 Hz, 1H), 3.26 (s, 1H), 3.31 - 3.48 (m, 2H), 3.86 (s, 3H), 3.97 - 4.30 (m, 7H), 4.46 (dd, J = 11.8, 3.2 Hz, 1H), 5.24 - 5.36 (m, 1H), 5. 45 - 5.62 (m, 1H), 5.86 (s, 1H), 6.40 - 6.65 (m, 3H), 6.82 (d, J = 3.4 Hz, 1H), 7.11 (d, J = 3.2 Hz, 1H), 7.18 (d, J = 12.1, 6.1, 4.9 Hz , 2H), 7.69 (d, J = 8.1 Hz, 1H), 7.75 (t, J = 7.6 Hz, 2H), 7.89 (d, J = 7.8, 3.2 Hz, 1H), 7 .95 - 8.04 (m, 2H), 8.26 (d, J = 6.1 Hz, 1H), 8.33 - 8.47 (m, 1H).

[0370] SPDB-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(13g):HRMS(M+H) + The calculated value is 1162.4425, and the measured value is 1162.4405. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1. 08 (dt, J = 13.9, 6.9 Hz, 15H), 1.15 - 1.25 ( m, 3H), 1.28 - 1.44 (m, 5H), 1.52 (s, 3H), 1.77 (p, J = 7.2 Hz, 2H), 1.91 - 2.02 (m, 1H), 2.02 - 2.13 (m, 1H), 2.17 (t, J = 7.2 Hz, 2H), 2.22 - 2.40 (m, 2H), 2.63 (s, 3H), 2.68 - 2.80 (m, 3H), 3.02 (s, 3H), 3.13 (d, J = 12.3 Hz , 1H), 3.18 (s, 3H), 3.33 - 3.45 (m, 2H), 3.85 (s, 3H), 3.95 - 4.16 (m, 5H), 4.45 (dd, J = 12.1, 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.43 - 6.60 (m, 3H), 6.82 (s, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.12 - 7.18 (m, 1H), 7.65 - 7.79 (m, 2H), 8.06 - 8.16 (m, 2H), 8.30 (t, J = 6.3 Hz, 1H), 8.35 - 8.40 (m, 1H).

[0371] SPDB-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(13f):HRMS(M+H) + The calculated value is 1162.4399, and the measured value is 1162.455. 1H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.0 2 - 1.13 (m, 12H), 1.14 - 1.25 (m, 3H), 1.31 - 1.44 (m, 5H), 1.52 (s, 3H), 1.77 (p, J = 7. 3 Hz, 2H), 1.97 (d, J = 14.3, 2.7 Hz, 1H), 2. 02 - 2.13 (m, 1H), 2.17 (t, J = 7.2 Hz, 2H), 2.28 - 2.40 (m, 3H), 2.43 (m, J = 3.2 Hz, 3H), 2.63 (s, 3H), 2.69 - 2.80 (m, 3H), 3.02 (s, 3H), 3.13 (d, J = 12.4 Hz, 1H), 3.18 (s, 3H), 3.39 (dd, J = 21.0, 10.7 Hz, 2H), 3.85 (s, 3H), 3.96 - 4.18 (m, 5H), 4.45 (dd, J = 12.1, 2 .8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.45 - 5.55 (m, 1H), 5.85 (s, 1H), 6.43 - 6.60 (m, 3H), 6.81 (s, 1H), 7.10 (d, J = 1.8 Hz, 1H), 7. 16 (t, J = 7.2, 4.9 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.71 - 7.79 (m, 1H), 8.02 - 8.15 (m, 2H), 8.28 (t, J = 6.3 Hz, 1H), 8.37 (d, J = 4 .8, 1.7 Hz, 1H).

[0372] SPDB-D-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(13h):HRMS(M+H) + The calculated value is 1162.4399, and the measured value is 1162.455.1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.0 2 - 1.16 (m, 13H), 1.14 - 1.25 (m, 3H), 1.28 - 1.49 (m, 5H), 1.52 (s, 3H), 1.77 (p, J = 7. 2 Hz, 2H), 1.92 - 2.14 (m, 2H), 2.17 (t, J = 7.2 Hz, 2H), 2.23 - 2.40 (m, 2H), 2.46 - 2.54 (m, 1H), 2.63 (s, 3H), 2.65 - 2.85 (m, 4H), 3.02 (s, 3H), 3.03 - 3.16 (m, 2H), 3.18 (s, 3H), 3.28 - 3.45 (m, 2H), 3.85 (s, 3H), 3.95 - 4.20 (m, 5H), 4.45 (dd, J = 12.1, 2.8 Hz, 1H) , 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.55 (m, 1H), 5.82 - 5.88 (m, 1H), 6.42 - 6.59 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.9 Hz, 1H), 7.14 - 7.20 (m, 1H), 7.67 - 7.72 (m, 1H), 7.72 - 7 .80 (m, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.99 ( d, J = 7.1 Hz, 1H), 8.28 (t, J = 6.3 Hz, 1H), 8.35 - 8.40 (m, 1H).

[0373] SPDB-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(13j):HRMS(M+H) + The calculated value is 1203.4337, and the measured value is 1203.4315. 1H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.94 - 1.24 (m, 20H), 1.38 (s, 3H), 1.52 (s, 3H), 1.57 - 1.87 (m, 1H), 1.89 - 2.08 (m, 1H), 2.26 (t, J = 15.1 Hz, 1H), 2.50 (d, J = 5.2 Hz, 2H), 2.54 - 2.79 (m, 7H), 3.05 (d, J = 3.8 Hz, 3H), 3.18 (s, 5H), 3.29 - 3.46 (m, 3H), 3.86 (d, J = 6.1 Hz, 4H), 4.00 (s, 3H), 4.05 - 4.24 (m, 4H), 4.33 - 4.54 (m, 1H), 5.17 - 5.38 (m, 1H), 5.39 - 5.58 (m, 1H), 5.85 ( s, 1H), 6.29 - 6.58 (m, 4H), 6.63 (s, 1H), 6.81 (s, 1H), 7.04 - 7.19 (m, 1H), 7.90 (s, 1H), 8.14 - 8.39 (m, 1H), 8.45 (s, 1H).

[0374] SPDB-D-Ala-L-Ala-NH-CH2-S-(CH2)2-CO-DM(13i):HRMS(M+H) + The calculated value is 1120.3955, and the measured value is 1120.3951. 1 H NMR (400 MHz, DMSO-d6) δ 0.74 - 0.82 (m, 3H), 1.10 - 1.22 (m, 13H), 1.25 (d, J = 14.1 H z, 1H), 1.46 (t, J = 10.9 Hz, 2H), 1.56 - 1.6 3 (m, 3H), 1.85 (ddd, J = 14.4, 9.0, 5.1 Hz, 2H), 2.00 (ddd, J = 14.7, 9.3, 5.4 Hz, 9H), 2.24 (dt, J = 10.8, 5.0 Hz, 2H), 2.72 (d, J = 3.6 Hz, 2H), 2.94 (dq, J = 10.7, 7.2, 5.7 Hz, 9H), 3.10 (d, J = 3.7 Hz, 3H), 3.20 (d, J = 3.4 Hz, 1H), 3.25 (d, J = 3.6 Hz, 3H), 3.32 (d, J = 3.7 Hz, 1H), 3.47 (td, J = 10.7, 10.0, 3. 8 Hz, 2H), 3.93 (t, J = 4.6 Hz, 3H), 4.02 - 4.25 (m, 6H), 4.49 - 4.57 (m, 1H), 5.28 - 5.37 (m, 1H), 5.53 - 5.62 (m, 1H), 5.92 (d, J = 3. 6 Hz, 1H), 6.57 (q, J = 5.4, 4.5 Hz, 3H), 6.8 5 - 6.93 (m, 1H), 7.17 (d, J = 3.3 Hz, 1H), 7.25 (dq, J = 8.0, 4.9 Hz, 6H), 7.72 - 7.87 (m, 11H), 8.16 (dt, J = 15.4, 4.9 Hz, 2H), 8.45 (tt, J = 9.9, 5.9 Hz, 6H).

[0375] Thio-peptide-maytansinoids HS-(CH2)3CO-peptide-NH-CH2-S-(CH2) n Compounds of the -CO2-DM type were prepared as exemplified by HS-(CH2)3CO-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0376] HS-(CH2)3CO-L-Ala-L-Ala-L-Ala-NH-CH2-S-( (CH2)5-CO-DM (14a): SPDB-L-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM (38 mg, 0.031 mmol) was dissolved in DMSO (1 mL) and a solution of DTT (19 mg, 0.12 mmol) in 1 mL of 100 mM potassium phosphate, 2 mM EDTA (pH 7.5) buffer was added. The reaction was allowed to proceed at room temperature with magnetic stirring under argon for 1 h. The crude reaction was purified on a C18, 30 micron, 30 g cartridge eluted with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 35 min. Fractions containing the desired product were immediately frozen and lyophilized to give 18.2 mg of a white solid (52.5% yield). HRMS (M+H)+ calculated 1124.4809; found 1124.4798. 1 H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 1.14 - 1.21 (m, 10H), 1.22 - 1.30 (m, 3H), 1.37 - 1.50 (m, 5H), 1.51 - 1.57 (m, 1H), 1.59 (s, 3H), 1.74 (p, J = 7.2 Hz, 2H), 2.04 (dd, J = 14.4, 2.8 Hz, 1H), 2.09 - 2.18 (m, 1H), 2.18 - 2. 24 (m, 2H), 2.27 (t, J = 7.6 Hz, 1H), 2.38 (t d, J = 7.1, 4.7 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 2.70 (s, 3H), 2.79 (d, J = 9.6 Hz, 1H), 3.09 (s, 3H), 3.21 (d, J = 12.6 Hz, 1H), 3.25 (s, 3H), 3.43 (d, J = 12.4 Hz, 1H), 3.49 (d, J = 9.0 Hz, 1H), 3.93 (s, 3H), 4.08 (ddd, J = 21.6, 11.4, 4.1 Hz, 2H), 4.13 - 4.28 (m, 4H) , 4.52 (dd, J = 12.1, 2.8 Hz, 1H), 5.34 (q, J = 6.7 Hz, 1H), 5.56 (dd, J = 14.7, 9.0 Hz, 1H ), 5.91 (d, J = 1.4 Hz, 1H), 6.48 - 6.66 (m, 3H), 6.88 (s, 1H), 7.18 (d, J = 1.8 Hz, 1H), 7 .86 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 7.3 Hz, 1H), 8.05 (d, J = 7.1 Hz, 1H), 8.33 (t, J = 6.3 Hz, 1H).

[0377] HS-(CH2)3CO-D-Ala-L-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(14b): HRMS(M+Na) + Calculated value 1146.4629, measured value 1146.4591. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.03 - 1.25 (m, 19H), 1.30 - 1.45 (m, 6H), 1.52 (s, 4H), 1.65 (p, J = 7.3 Hz, 2H), 1.91 - 2.02 (m, 1H), 2.02 - 2.13 (m, 1H), 2.12 - 2.19 (m, 4H), 2.29 - 2.39 (m, 4H), 2 .63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 ( s, 3H), 3.14 (d, J = 12.5 Hz, 1H), 3.33 - 3.4 7 (m, 2H), 3.86 (s, 3H), 4.01 (td, J = 10.4, 9.7, 4.3 Hz, 2H), 4.04 - 4.16 (m, 5H), 4.45 (dd, J = 12.0, 2.9 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.43 - 5.56 (m, 1H), 5.85 (s, 1H), 6.38 - 6.61 (m, 4H), 6.81 (s, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.97 (t , J = 6.3 Hz, 1H), 8.10 (d, J = 6.0 Hz, 1H), 8 .25 (d, J = 6.9 Hz, 1H).

[0378] HS-(CH2)3CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(14c):HRMS(M+Na) + Calculated value 1146.4629 , measured value 1146.4553. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.99 - 1.26 (m, 21H), 1.31 - 1.45 (m, 5H), 1.52 (s, 3H), 1.67 (p, J = 7.2 Hz, 2H), 1.89 - 2.02 (m, 1H), 2.02 - 2.24 (m, 4H), 2.25 - 2.46 (m, 3H), 2.63 (s, 3H), 2.73 (d, J = 9.7 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 3H), 3.32 - 3.51 (m, 2H), 3.86 (s, 3H), 3.96 - 4.18 (m, 7H), 4.45 (dd, J = 12.0, 2.9 Hz, 1H), 5 .27 (q, J = 6.8 Hz, 1H), 5.44 - 5.63 (m, 1H), 5.85 (s, 1H), 6.37 - 6.59 (m, 4H), 6.81 (s, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.89 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 6.5 Hz, 1H), 8.08 (d, J = 7.3 Hz, 1H), 8.27 (t, J = 6.3 Hz, 1H).

[0379] HS-(CH2)3CO-L-Ala-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(14d):HRMS(M+Na) + Calculated value 1146.4629, measured value 1146.4519. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.95 - 1.24 (m, 20H), 1.27 - 1.45 (m, 5H), 1.52 (s, 3H), 1.67 (p, J = 7.3 Hz, 2H), 1.93 - 2.01 (m, 1H), 2.02 - 2.22 (m, 4H), 2.22 - 2.41 (m, 5H), 2.63 (s, 3H), 2.73 (d, [[ID=...]]J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 4H), 3.39 (dd, J = 21.4, 10.7 Hz, 2H), 3.86 (s, 3H), 3.94 - 4.24 (m, 6H), 4.45 (dd, J = 12.0, 2 .8 Hz, (此处原文似乎有误,推测为2.8 Hz)1H), 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.57 (m, 1H), 5.85 (s, 1H), 6.37 - 6.65 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7. 89 (d, J = 7.6 Hz, 1H), 7.93 - 8.05 (m, 2H), 8.26 (t, J = 6.4 Hz, 1H). 需注意,原文中“4.45 (dd, J = 12.0, 2 ”这部分似乎不完整,推测可能是“4.45 (dd, J = 12.0, 2.8 Hz, 1H)”,已在翻译中按此推测进行了补充。若实际并非如此,请根据正确内容进行调整。

[0380] HS-(CH2)3CO-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(14g):HRMS(M+H) + The calculated value is 1053.4438, and the measured value is 1053.4426. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.01 - 1.15 (m, 13H), 1.15 - 1.27 (m, 3H), 1.31 - 1.44 (m, 5H), 1.53 (s, 3H), 1.67 (p, J = 7.1 Hz, 2H), 1.93 - 2.03 (m, 1H), 2.03 - 2.23 (m, 4H), 2.22 - 2.41 (m, 5H), 2.63 (s, 3H), 2.73 (d, J = 9.7 Hz, 1H), 3.02 (s, 3H), 3.14 (d, J = 12.5 Hz, 1H), 3.18 (s, 3H), 3.32 - 3.46 (m, 2H), 3.86 (s, 3H), 3.92 - 4.20 (m , 6H), 4.45 (dd, J = 11.9, 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.42 - 5.58 (m, 1H), 5.8 5 (s, 1H), 6.42 - 6.60 (m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.05 (d, J = 6.5 Hz , 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.30 (t, J = 6.3 Hz, 1H).

[0381] HS-(CH2)3CO-L-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(14f):HRMS(M+H) + The calculated value is 1053.4366, and the measured value is 1053.4438.1 1H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.02 - 1.14 (m, 13H), 1.19 (t, J = 9.7 H z, 3H), 1.31 - 1.43 (m, 6H), 1.53 (s, 3H), 1.67 (p, J = 7.3 Hz, 2H), 1.91 - 2.02 (m, 1H), 2.02 - 2.22 (m, 4H), 2.34 - 2.39 (m, 4H), 2.63 (s, 3H), 2.73 (d, J = 9.5 Hz, 1H), 3.02 (s, 3H), 3.19 (d, J = 4.2 Hz, 4H), 3.30 - 3.47 (m, 2H), 3.86 (s, 3H), 3.94 - 4.20 (m, 6H), 4.45 (d, J = 11.8, 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.40 - 6.61 (m, 3H), 6.81 (s, 1H), 7.12 (s, 1H), 8.03 (d, J = 6.5 Hz, 1H), 8.08 (d, J = 7.8 Hz , 1H), 8.29 (t, J = 6.2 Hz, 1H).

[0382] HS-(CH2)3CO-D-Ala-D-Ala-NH-CH2-S-(CH2)5-CO-DM(14h):HRMS(M+H) + Calculated value 1053.4366, measured value 1053.4438. 1 1H NMR (400 MHz, DMSFO-d6) δ 0.71 (s , 3H), 1.02 - 1.15 (m, 13H), 1.14 - 1.24 (m, 3H), 1.30 - 1.45 (m, 5H), 1.53 (s, 3H), 1.67 (p, J = 7.1 Hz, 2H), 1.90 - 2.01 (m, 1H), 2.0 1 - 2.24 (m, 4H), 2.27 - 2.33 (m, 1H), 2.33 - 2.42 (m, 4H), 2.63 (s, 3H), 2.73 (d, J = 9.7 Hz, 1H), 3.02 (s, 3H), 3.10 - 3.21 (m, 4H), 3.33 - 3.46 (m, 2H), 3.86 (s, 3H), 3.95 - 4.18 (m, 6H), 4.45 (dd, J = 11.9, 2.8 Hz, 1H), 5. 27 (q, J = 6.7 Hz, 1H), 5.44 - 5.55 (m, 1H), 5.85 (s, 1H), 6.42 - 6.59 (m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.05 (d, J = 6.5 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 8.30 (t, J = 6.3 Hz, 1H).

[0383] HS-(CH2)3CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(14j):HRMS(M+H) + Calculated value 1096.4496, measured value 1096.4464. 1H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1.02 - 1.31 (m, 19H), 1.35 - 1.55 (m, 2H), 1.60 (s, 3H), 1.74 (p, J = 7.4 Hz, 3H), 1.78 - 1.93 (m, 1H), 2.14 - 2.33 (m, 4H), 2.41 - 2.49 (m, 2H), 2.71 (s, 3H), 2.80 (d, J = 9.6 Hz, 1H), 3.12 (s, 3H), 3.22 (d, J = 12.7 Hz, 1H), 3.26 (s, 3H), 3.47 (dd, J = 21.3, 10.6 Hz, 2H), 3.93 (s, 4H), 4.03 - 4.13 (m, 3H), 4.13 - 4.25 (m, 3H), 4.52 (dd, J = 12.0, 2.8 Hz, 1H), 5.35 (q, J = 6.8 Hz, 1H), 5.50 - 5.64 (m, 1H), 5.92 (s, 1H), 6.47 - 6.69 (m, 4H), 6.88 (s, 1H), 7.18 (d, J = 1.7 Hz, 1H), 7.94 (d, J = 7.3 Hz, 1H), 8.09 (d, J = 6.4 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H), 8.32 (t, J = 6 .3 Hz, 1H).

[0384] HS-(CH2)3CO-(CH2)3-CO-D-Ala-L-Ala-NH-CH2-S-(CH2)2-CO-DM(14i):HRMS(M+H) + Calculated value 1011.3 969, measured value 1011.3961. 1 1H NMR (400 MHz, DMSO-d6) δ 0.77 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 1.17 (dd, J = 7.0, 5.1 Hz, 9H), 1.25 (d, J = 13.0 Hz, 1H), 1.40 - 1.51 (m, 2H), 1.59 (s, 3H), 1.74 (q, J = 7.2 Hz, 2H), 2.00 - 2.08 (m, 1H) , 2.23 (dt, J = 16.8, 7.6 Hz, 3H), 2.43 (q, J = 7.4 Hz, 2H), 2.62 - 2.69 (m, 1H), 2.72 (s, 3H), 2.76 - 2.88 (m, 2H), 3.10 (s, 3H), 3.20 (d, J = 12.6 Hz, 1H), 3.25 (s, 3H), 3.31 (s, 3H), 3.39 - 3.54 (m, 2H), 3.93 (s, 3H), 4.01 - 4.26 (m, 5H), 4.53 (dd, J = 12.0, 2.8 Hz, 1 H), 5.32 (q, J = 6.8 Hz, 1H), 5.49 - 5.63 (m, 1H), 5.92 (d, J = 1.4 Hz, 1H), 6.48 - 6.62 (m , 3H), 6.88 (s, 1H), 7.18 (d, J = 1.8 Hz, 1H) , 8.10 (d, J = 6.5 Hz, 1H), 8.16 (d, J = 7.7 H z, 1H), 8.41 (t, J = 6.3 Hz, 1H).

[0385] NHS-L-Ala-D-Ala-L-Ala-Imm-C6-May HOOC-(CH2)3-CO-peptide-NH-CH2-S-(CH2) n Compounds of the type -CO2-DM were prepared as exemplified by HOOC-(CH2)3-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0386] HOOC-(CH)-CO-L-Ala-D-Ala-L-Ala-NH-CH-S-(CH)-CO-DM (19a): L-Ala-D-Ala-L-Ala-NH-CH-S-(CH)-CO-DM (17.25 mg, 0.017 mmol) was treated with glutaric anhydride (38.5 mg, 0.337 mmol) and allowed to react overnight at room temperature under argon with magnetic stirring. The crude reaction was purified by HPLC using an XDB-C18, 21.2 x 5 mm, 5 micron column eluted with deionized water containing 0.1% formic acid and a linear gradient of 5% to 95% acetonitrile over 30 minutes at 20 mL / min. Fractions containing the pure desired product were immediately combined, frozen, and lyophilized to give 3 mg (15% yield) of a white solid. HRMS (M+H) + Calculated value 1136.4987, measured value 1136.4954. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.92 - 1.27 (m, 20H), 1.26 - 1.48 (m, 5H), 1.52 (s, 3H), 1.63 (q, J = 7.1 Hz, 2H), 1.83 - 2.20 (m, 7H), 2.23 - 2.41 (m, 5H), 2.63 (s, 4H), 2.73 (d, J = 9.5 Hz, 1H), 3.02 (s, 3H), 3.36 - 3.50 (m, 2H), 3.86 (s, 3H), 3.91 - 4.24 (m, 7H), 4.45 (d, J = 11.8Hz , 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.41 - 5.57 (m, 1H), 5.86 (s, 1H), 6.32 - 6.66 (m, 3H), 6.81 (s, 1H), 7.12 (s, 1H), 8.06 (t, J = 9.1Hz , 2H), 8.35 (d, J = 11.6 Hz, 1H), 8.62 (s, 1H ).

[0387] HOOC-(CH2)3-CO-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(19g):HRMS(M+H) + The calculated value is 1136.4987, and the measured value is 1136.4962. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 0.97 - 1.14 (m, 13H), 1.14 - 1.26 (m, 3H), 1.28 - 1.45 (m, 5H), 1.52 (s, 3H), 1.6 2 (p, J = 7.5 Hz, 2H), 1.93 - 2.00 (m, 1H), 2.08 (dt, J = 13.1, 7.4 Hz, 6H), 2.25 - 2.41 (m , 3H), 2.63 (s, 3H), 2.73 (d, J = 9.5 Hz, 1H) , 3.02 (s, 3H), 3.18 (s, 3H), 3.31 - 3.48 (m, 2H), 3.86 (s, 3H), 3.93 - 4.19 (m, 6H), 4.45 (dd, J = 12.0, 2.8 Hz, 1H), 5.27 (q, J = 6.8 Hz, 1H), 5.43 - 5.58 (m, 1H), 5.85 (s, 1H), 6.40 - 6.61 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 6.5 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.34 (t, J = 6.3 Hz, 1H), 11.94 (s, 1H).

[0388] HOOC-(CH2)3-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(19i):HRMS(M+H) + The calculated value is 1108.4674, and the measured value is 1108.4634. 1H NMR (400 MHz, DMSO-d6) δ 0.78 (s, 3H), 1.04 - 1.32 (m, 16H), 1.45 (d, J = 12.6 Hz, 2H), 1.60 (s, 3H), 1.69 (p, J = 7.2 Hz, 3H), 1.77 - 1.95 (m, 1H), 1.99 - 2.0 7 (m, 1H), 2.11 - 2.20 (m, 4H), 2.20 - 2.39 ( m, 1H), 2.55 (s, 1H), 2.71 (s, 3H), 2.80 (d, J = 9.5 Hz, 1H), 3.12 (s, 3H), 3.40 (d, J = 21.0 Hz, 8H), 3.49 (d, J = 9.1 Hz, 1H), 3.93 (s , 3H), 4.02 - 4.27 (m, 6H), 4.48 - 4.61 (m, 1 H), 5.34 (q, J = 6.6 Hz, 1H), 5.48 - 5.65 (m, 1H), 5.92 (s, 1H), 6.50 - 6.71 (m, 3H), 6.88 (s, 1H), 7.18 (s, 1H), 7.99 (d, J = 7.6 Hz, 1 H), 8.08 (d, J = 6.5 Hz, 1H), 8.22 (d, J = 7.4 Hz, 1H), 8.30 (s, 1H), 8.42 (s, 1H).

[0389] NHS-OOC-(CH2)3-CO-peptide-NH-CH2-S-(CH2) n Compounds of the -CO2-DM type were prepared as exemplified by NHS-OOC-(CH2)3-CO-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0390] NHS-OOC-(CH)-CO-D-Ala-L-Ala-NH-CH-S-(CH)-CO-DM (20 g): HOOC-(CH)-CO-D-Ala-L-Ala-NH-CH-S-(CH)-CO-DM (8 mg, 7.5 μmol) was dissolved in DMSO (1 mL) and treated with NHS (0.9 mg, 7.51 μmol) and EDC (1.4 mg, 7.51 μmol). The reaction proceeded at room temperature with magnetic stirring under an argon atmosphere for 2 h. The crude material was purified by HPLC using an XDB-C18, 21.2 × 5 mm, 5 μm column eluted with deionized water containing 0.1% formic acid and a linear gradient of 5% to 95% acetonitrile over 30 min at 20 mL / min. Fractions containing the desired product were combined, immediately frozen, and then lyophilized to give 6.5 mg (74% yield) of a white solid. 1 H NMR (400MHz, DMSO-d6) δ 0.71 (s, 3H), 1.00 - 1.14 (m, 13H), 1.14 - 1.25 (m, 3H), 1.29 - 1.46 (m, 5H), 1.52 (s, 3H), 1.75 (p, J = 7.5 Hz, 2H), 1.92 - 2.12 (m, 2H), 2.16 (t, J = 7.3 Hz, 2H), 2.2 2 - 2.39 (m, 3H), 2.62 (d, J = 10.8 Hz, 5H), 2.73 (d, J = 10.5 Hz, 5H), 3.02 (s, 3H), 3.18 (s, 3H), 3.32 - 3.47 (m, 2H), 3.86 (s, 3H), 3.95 - 4.19 (m, 6H), 4.45 (dd, J = 12.0, 2.8 H z, 1H), 5.27 (q, J = 6.8 Hz, 1H), 5.42 - 5.57 (m, 1H), 5.82 - 5.87 (m, 1H), 6.41 - 6.60 (m, 4H), 6.81 (s, 1H), 7.11 (d, J = 1.7 Hz, 1H), 8.05 (d, J = 6.5 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 8.20 (d, J = 4.8 Hz, 1H), 8.29 (t, J = 6 .3 Hz, 1H).

[0391] NHS-OOC-(CH2)3-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(20g):HRMS(M+H) + Calculated value 1233.5151, measured value 1233.5135.

[0392] NHS-OOC-(CH2)3-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)3-CO-DM(20i):HRMS(M+H) + Calculated value 1205.4838, measured value 1205.4808.

[0393] H2N-O-CH2-CO-peptide-NH-CH2-S-(CH2) n Compounds of the -CO2-DM type were prepared as exemplified by H2N-O-CH2-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM.

[0394] H2N-O-CH2-CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-DM(22c): HN-L-Ala-D-Ala-L-Ala-CH-S-(CH)-CO-DM (23 mg, 0.022 mmol) was dissolved in DMF (1 mL) and treated with FMoc-aminooxyacetic acid (14.09 mg, 0.045 mmol) and EDC (8.62 mg, 0.045 mmol). The reaction proceeded for 3 hours at room temperature with magnetic stirring under an argon atmosphere. The crude material was treated with 20% morpholine in DMF (1 mL) and allowed to proceed for 2 hours. The crude material was purified by semi-preparative HPLC using an XDB-C18, 21.2 × 5 mm, 5 μm column, eluting with deionized water containing 0.1% formic acid and a linear gradient of 5% to 95% acetonitrile over 30 minutes at 20 mL / min. Fractions containing the desired product were pooled, immediately frozen, and then lyophilized to give 5.5 mg (22% yield) of a white solid. HRMS (M+H) + Calculated value 1095.4834, measured value 1095.4795. 1 H NMR (400MHz, DMSO-d6) δ 0.71 (s, 3H), 1.00 - 1.14 (m, 13H), 1.14 - 1.25 (m, 6H), 1.31 - 1.43 (m, 4H), 1.52 (s, 3H), 1.92 - 2.02 (m, 1H), 2.02 - 2.14 (m, 1H), 2 .23 - 2.39 (m, 3H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.14 (d, J = 12. 5 Hz, 1H), 3.18 (s, 3H), 3.29 - 3.46 (m, 3H), 3.86 (s, 3H), 3.90 (d, J = 2.0 Hz, 2H), 3.95 - 4.20 (m, 6H), 4.25 (p, J = 7.7, 7.2 Hz, 1H) , 4.45 (dd, J = 12.0, 2.8 Hz, 1H), 5.27 (q, J = 6.8 Hz, 1H), 5.44 - 5.58 (m, 1H), 5.85 (s, 1H), 6.30 (s, 2H), 6.43 - 6.60 (m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.7 Hz, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.29 (t, J = 6.3 Hz, 1H).

[0395] H2N-O-CH2CO-L-Ala-D-Ala-L-Ala-NH-CH2-S-( CH2)3-CO-DM(22i):HRMS(M+H) + The calculated value is 1067.4521, and the measured value is 1067.4484. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.01 - 1.26 (m, 18H), 1.30 - 1.46 (m, 2H), 1.52 (s, 3H), 1.55 - 1.69 (m, 1H), 1.69 - 1.84 (m, 1H), 1.97 (d, J = 14.4, 2.8 H z, 1H), 2.15 - 2.31 (m, 1H), 2.56 - 2.61 (m, 1H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.04 (s, 3H), 3.14 (d, J = 12.6 Hz, 1H), 3.18 (s, 3H), 3.36 (d, J = 12.3 Hz, 1H), 3.42 (dd , J = 9.1, 3.3 Hz, 1H), 3.85 (s, 3H), 3.90 (d , J = 2.3 Hz, 2H), 3.95 - 4.05 (m, 3H), 4.06 - 4.17 (m, 2H), 4.15 - 4.35 (m, 2H), 4.45 (dd, J = 12.0, 2.8 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1 H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.30 (s, 2H), 6.42 - 6.61 (m, 3H), 6.81 (s, 1H), 7.11 (d, J = 1.7 Hz, 1H), 7.82 (d, J= 7.3 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H), 8.22 - 8.40 (m, 1H).

[0396] Mal-(CH)-CO-L-Ala-D-Ala-L-Ala-NH-CH-S-(CH)-CO-DM (23c): HN-L-Ala-D-Ala-L-Ala-CH-S-(CH)-CO-DM (8 mg, 7.82 μmol) was dissolved in DMF (2 mL) and treated with 3-maleimidopropanoic acid (1.32 mg, 7.82 μmol), EDC (2.25 mg, 0.012 mmol), and HOBt (1.198 mg, 7.82 μmol). The reaction proceeded at room temperature with magnetic stirring under an argon atmosphere for 2 h. The crude material was purified by semi-preparative HPLC using an XDB-C18, 21.2 x 5 mm, 5 μm column, eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 30 min at 20 ml / min. Fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 1.8 mg (19.60% yield) of a white solid. HRMS (M+H) + Calculated value 1173.4940, measured value 1173.4931. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.02 - 1.14 (m, 15H), 1.16 - 1.25 (m, 3H), 1.30 - 1.44 (m, 5H), 1.52 (s, 3H), 1.92 - 2.03 (m, 1H), 2.03 - 2.17 (m, 1H), 2.23 - 2.39 (m, 4H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 4H), 3.33 - 3.46 (m, 2H), 3.52 (t, J = 7.3 Hz, 2H), 3.86 ( s, 3H), 3.95 - 4.17 (m, 7H), 4.45 (dd, J = 12 .0, 2.9 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5. 44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.39 - 6.64 (m, 3H), 6.81 (s, 1H), 6.86 (s, 1H), 6.92 (s , 2H), 7.11 (d, J = 1.7 Hz, 1H), 7.89 (d, J = 7.4 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 8.17 ( d, J = 6.7 Hz, 1H), 8.28 (t, J = 6.3 Hz, 1H), 8.43 (s, 1H).<00033​​​​​​​​​​​​​​​​​Mal-C5-L-Ala-D-Ala-L-Ala-Imm-C6-May: The reaction of L-Ala-D-Ala-L-Ala-CH2-S-(CH2)5-CO-MayNMA (compound I-1a) (25 mg, 0.024 mmol) with 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (7.54 mg, 0.024 mmol) gave Mal-C5-L-Ala-D-Ala-L-Ala-Imm-C6-May (compound I-2a) (20.8 mg, 0.017 mmol, 70.0% yield). LRMS (M+H) + Calculated value 1215.52, measured value 1216.4. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.05 (d, J = 6.4 Hz, 3H), 1.07 - 1.1 4 (m, 14H), 1.15 - 1.25 (m, 3H), 1.39 (t, J = 9.2 Hz, 10H), 1.52 (s, 3H), 2.01 (t, J = 7.6 Hz, 3H), 2.26 (t, J = 1.9 Hz, 1H), 2.28 - 2.3 8 (m, 2H), 2.57 - 2.62 (m, 1H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.14 (d, J = 12.5 Hz, 1H), 3.18 (s, 3H), 3.29 (t, J = 7.1 Hz, 2H), 3.36 (d, J= 12.5 Hz, 1H), 3. 42 (d, J = 9.0 Hz, 1H), 3.86 (s, 3H), 3.96 - 4.05 (m, 1H), 4.04 - 4.15 (m, 4H), 4.41 - 4.48 (m, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.46 - 5.54 (m, 1H), 5.82 - 5.88 (m, 1H), 6.47 - 6.50 ( m, 2H), 6.54 (t, J = 11.4 Hz, 2H), 6.82 (s, 1 H), 6.92 (s, 2H), 7.11 (d, J = 1.8 Hz, 1H), 7 .86 - 7.93 (m, 2H), 7.95 (s, 1H), 8.05 (d, J= 7.4 Hz, 1H), 8.24 (t, J = 6.2 Hz, 1H).

[0399] Mal-(CH2)2-PEG2-CO-L-Ala-D-Ala-L-ALa-NH- CH2-S-(CH2)5-CO-MayNMA [ka] Mal-(CH2)2-PEG2-CO-L-Ala-D-Ala-L-ALa-NH-CH2-S-(CH2)5-CO-MayNMA: The reaction of L-Ala-D-Ala-L-Ala-NH-CH2-S-(CH2)5-CO-MayNMA (compound I-la) (25 mg, 0.024 mmol) with Mal-amide-PEG2-NHS (10.40 mg, 0.024 mmol) gave Mal-(CH2)2-PEG2-CO2-L-Ala-D-Ala-L-ALa-NH-CH2-S-(CH2)5-CO-MayNMA (compound I-3a) (14.1 mg, 10.58 μmol, 43.3% yield). LRMS(M+H) + Calculated value 1332.58, measured value 1332.95. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 4H), 1.05 (d, J = 6.3 Hz, 4H), 1.07 - 1.14 (m, 15H), 1.18 (d, J = 9.0 Hz, 2H), 1.37 (d, J = 11.8 Hz, 6H), 1.52 (s, 3H), 2.23 - 2.38 (m, 5H), 2.63 (s, 4H), 2.72 (d, J = 9.7 Hz, 1H), 3.02 (s, 3H), 3.07 (q, J = 5.7 Hz, 2H), 3.18 (s, 3H), 3.39 (s, 4 H), 3.41 (d, J = 9.9 Hz, 2H), 3.47 - 3.56 (m, 4H), 3.86 (s, 4H), 3.95 - 4.08 (m, 2H), 4.08 - 4.19 (m, 3H), 4.41 - 4.51 (m, 1H), 5.23 - 5 .31 (m, 1H), 5.44 - 5.54 (m, 1H), 5.85 (s, 1H), 6.46 - 6.50 (m, 2H), 6.54 (t, J = 11.3 Hz, 2H), 6.83 (s, 1H), 6.93 (s, 2H), 7.12 (s, 1H ), 7.88 - 8.00 (m, 2H), 8.01 - 8.08 (m, 2H), 8.27 (t, J = 6.2 Hz, 1H).

[0400] Mal-(CH2)2-PEG4-CO-L-Ala-D-Ala-L-ALa-NH-CH2-S-(CH2)5-CO-MayNMA

Chem.

[0401] Example 3. Synthesis of Metabolites DM-CO-(CH)-SH (24a): To the DM-H stock solution (1.5 mL, 0.100 mmol), EDC (29 mg, 0.150 mmol) and DIPEA (17.5 μL, 0.100 mmol) were added over 10 min at room temperature with magnetic stirring. 6-Mercaptohexanoic acid (13.8 μL, 0.100 mmol) was then added. After 30 min, the crude material was purified by semi-preparative HPLC using an XDB-C18, 21.2 × 5 mm, 5 μm column, eluting with deionized water containing 0.1% formic acid and a linear gradient of 5% to 95% acetonitrile over 30 min at 20 mL / min. Fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 12 mg (15% yield) of a white solid. HRMS (M+H) + Calculated value 780.3291, measured value 780.3281. 1 H NMR (400 MHz, DMSO-d6) δ 0.79 (s, 3H), 1.06 - 1.21 (m, 5H), 1.21 - 1.57 (m, 6H), 1. 60 (s, 2H), 2.00 - 2.12 (m, 2H), 2.12 - 2.27 (m, 2H), 2.27 - 2.37 (m, 2H), 2.50 (s, 4H), 2.70 (s, 3H), 2.74 - 2.91 (m, 2H), 2.91 - 3.09 (m, 1H), 3.10 (s, 2H), 3.19 - 3.24 (m, 2H), 3.26 (s, 3H), 3.39 - 3.53 (m, 2H), 3.94 (s, 3H), 4.03 - 4.11 (m, 1H), 4.52 (dd, J = 12.0, 2.9 Hz, 1H), 5.35 (q, J = 6.8 Hz, 1H), 5.52 - 5.62 (m, 1H), 5.93 (d, J = 1.3 Hz, 1H), 6.49 - 6.67 (m, 3H), 6.89 (s, 1H), 7.20 (d, J = 1. 8 Hz, 1H).

[0402] DM-CO-(CH)-SMe (25a): DM-CO-(CH)-SH (12 mg, 0.015 mmol) was dissolved in DMF (2 mL) and treated with DIPEA (24 μL, 0.139 mmol) and iodomethane (2.88 μL, 0.046 mmol). The mixture was allowed to proceed under argon at room temperature for 2 h. The crude material was purified by semi-preparative HPLC using an XDB-C18, 21.2 × 5 mm, 5 μm column at a flow rate of 20 mL / min, eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% at 20 mL / min over 30 min. Fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 2 mg of a white solid (16% yield). HRMS (M+H) + Calculated value 794.3448, measured value 794.3440. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.01 - 1.1 3 (m, 6H), 1.13 - 1.27 (m, 3H), 1.27 - 1.50 ( m, 6H), 1.53 (s, 3H), 1.87 (s, 2H), 1.93 - 2.04 (m, 2H), 2.04 - 2.15 (m, 1H), 2.15 - 2.27 (m, 2H), 2.27 - 2.41 (m, 1H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.10 - 3.22 (m, 5H), 3.33 - 3.49 (m, 2H), 3.86 (s, 3H), 3.94 - 4.06 (m, 1H), 4.45 (dd, J = 12.1, 2.8 Hz, 1H), 5.28 (q, J = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.42 - 6.62 (m, 3H), 6.81 (s, 1H), 7.13 (d, J = 1.7 Hz, 1H).

[0403] DM-CO-(CH)-SSPy (26): DM-CO-(CH)-SSPy (3267-50-R1): SPDB (30.1 mg, 0.092 mmol) was added to the DM-H stock solution (0.81 mL, 0.046 mmol) at room temperature with magnetic stirring. After 30 min, the solution was purified on an XDB-C18, 21.2 × 5 mm, 5 μm column at a flow rate of 20 mL / min using deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% at 20 mL / min over 30 min. Fractions containing the desired product were combined, frozen, and then lyophilized to give 6 mg of a white solid (15% yield). HRMS (M+H) + Calculated value: 861.2964, measured value: 861.2963. 1 H NMR (400 MHz, DMSO-d6) δ 0.70 (s, 3H), 1.02 - 1. 12 (m, 7H), 1.13 - 1.21 (m, 1H), 1.31 - 1.45 (m, 3H), 1.52 (s, 3H), 1.70 - 1.90 (m, 2H), 1.96 (dd, J = 14.2, 2.9 Hz, 1H), 2.19 - 2.31 ( m, 1H), 2.62 (s, 3H), 2.68 - 2.81 (m, 4H), 3.00 (s, 2H), 3.18 (s, 4H), 3.34 (d, J = 12.4 H z, 1H), 3.41 (d, J = 9.1 Hz, 1H), 3.87 (s, 3H ), 4.00 (t, J = 11.2 Hz, 1H), 4.44 (dd, J = 12.0, 2.9 Hz, 1H), 5.25 (q, J = 6.8 Hz, 1H), 5. 41 - 5.52 (m, 1H), 5.85 (s, 1H), 6.43 - 6.53 (m, 3H), 6.81 (s, 1H), 7.09 (d, J = 1.8 Hz, 1 H), 7.12 - 7.19 (m, 1H), 7.50 - 7.61 (m, 1H), 7.66 - 7.76 (m, 1H), 8.31 - 8.39 (m, 1H).

[0404] DM-CO-(CH)-SH (24b): DM-CO-(CH)-SSPy (6 mg, 6.96 μmol) was added to a solution of DTT (1.1 mg, 6.96 μmol) in 2:1 DMSO:potassium phosphate, 2 mM EDTA (pH 7.5) buffer (0.5 mL) and magnetically stirred at room temperature for 20 min. The crude solution was purified on an XDB-C18, 21.2 × 5 mm, 5 μm column at a flow rate of 20 mL / min using deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% at 20 mL / min over 30 min. Fractions containing the desired product were combined, frozen, and then lyophilized to give 5 mg of a white solid (95% yield). HRMS (M+H) + Calculated value 752.2978, measured value 752.2962. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (d, J = 1.9 Hz, 3H), 1.08 (dd, J = 18.6, 6.6 Hz, 7H), 1.18 (d, J = 12.4 Hz, 2H), 1.27 - 1.49 (m, 3H), 1.52 (d, J = 2.7 Hz, 4H), 1.55 - 1.68 (m, 1H), 1.77 (J = 14.2, 8.5, 6.5 Hz, 1H), 1.91 - 2.05 (m, 1H), 2.15 (t, J = 7.9 Hz, 1H), 2.38 (s, 2H), 2.44 - 2.60 (m, 1H), 2.64 (s, 2H), 2.66 - 2.84 (m, 1H), 3.03 (d, J = 12. 6 Hz, 3H), 3.09 - 3.18 (m, 1H), 3.18 (s, 3H), 3.36 (d, J = 12.2 Hz, 1H), 3.42 (d, J = 9.0 H z, 1H), 3.86 (s, 2H), 3.93 - 4.08 (m, 1H), 4.45 (dd, J = 12.0, 2.8 Hz, 1H), 5.27 (q, 1H), 5.42 - 5.58 (m, 1H), 5.85 (d, J = 1.3 Hz, 1H) , 6.40 - 6.61 (m, 4H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H).

[0405] DM-CO-(CH)-SMe (25b): DM-CO-(CH)-SH (5 mg, 6.65 μmol) was dissolved in anhydrous DMF (0.3 mL) and DIPEA (3.57 μL, 0.020 mmol) and iodomethane (1.2 μL, 0.020 mmol) were added at room temperature with magnetic stirring. After 1 h, the crude solution was purified on an XDB-C18, 21.2 × 5 mm, 5 μm column at a flow rate of 20 mL / min using deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% at 20 mL / min over 30 min. Fractions containing the desired product were combined, frozen, and then lyophilized to give 1 mg (19% yield) of a white solid. HRMS (M+H) + Calculated value 766.3135, measured value 766.3121. 1 H NMR (400 MHz, DMSO-d6) δ 0.71 (s, 3H), 1.08 (dd, J = 18.1, 6.6 Hz, 7H), 1.18 (d, J = 12.9 Hz, 1H), 1.30 - 1.47 (m, 2H), 1.53 (s, 3H), 1.56 - 1.68 (m, 1H), 1.68 - 1.78 (m, 1H), 1.78 (s, 3H), 1.91 - 2.05 (m, 1H), 2.18 - 2.31 (m, 1H), 2.33 - 2.41 (m, 2H), 2.63 (s, 3H), 2.73 (d, J = 9.7 Hz, 1H), 3.0 5 (s, 3H), 3.18 (s, 4H), 3.33 - 3.48 (m, 2H), 3.86 (s, 3H), 4.00 (t, J = 11.5 Hz, 1H), 4.4 5 (dd, J = 12.1, 2.8 Hz, 1H), 5.28 (q, J = 6.7 Hz, 1H), 5.43 - 5.57 (m, 1H), 5.85 (s, 1H), 6.41 - 6.62 (m, 3H), 6.81 (s, 1H), 7.12 (d, J = 1.8 Hz, 1H), 8.47 (s, 1H).

[0406] Example 4. Preparation of ADCs (16a-16i, 17a-17i, 18a-18i). Preparation of maytansinoid solutions for preparing ADCs (16a–16i, 17a–17i, 18a–18i) Sulfo-GMBS and one of the thiol-bearing compounds (14a-14j) were dissolved in a 3:7 (50 mM sodium succinate, pH 5.0:DMA) solution to give concentrations of 1.5 mM and 1.9 mM, respectively. The solution was gently stirred at room temperature for 30 min, and then excess thiol was quenched by bringing the solution to 0.5 mM in N-ethylmaleimide (NEM) with gentle stirring for 10 min.

[0407] Preparation of ADCs (16a–16i, 17a–17i, 18a–18i) To a solution of antibody (2.5 mg / mL) in 60 mM EPPS containing 15% by volume of N,N-dimethylacetamide (DMA), pH 8.0, 6.5 molar equivalents of maytansinoid solution were added. After 16 hours, the reaction mixture was purified using a NAP-G25 column pre-equilibrated with 10 mM sodium succinate, pH 5.5, 250 mM glycine, 0.5% sucrose, and 0.01% Tween-20 buffer. The purified conjugate was analyzed to determine the maytansinoid per antibody ratio (MAR), percent aggregated conjugate, free maytansinoid level, and endotoxin units (EU), as previously described in Widdison W. et al. J Med Chem (2006) 49, 4392-408. All conjugates had protein aggregate levels below 3%, free maytansinoid levels below 1%, and endotoxin levels below 0.2 EU / mg.

[0408] ADCs 1a to 1d and 4a to 4c were used as comparative substances to evaluate the conjugates of the present invention. ADCs 1a to 1d and 4a to 4c were prepared as described in Widdison W., et al. .al., Bioconjugate Chem., (2015), 26, 2261-2278.

[0409] Preparation of C242-sGMBS-LDL-DM (ADC18c) Prior to conjugation, sGMBS-LDL-DM was prepared by mixing a stock solution of sulfo-GMBS (compound 15 in Figure 4) in N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (compound 14c in Figure 3) in DMA in the presence of succinate buffer pH 5.0 to obtain a 60 / 40 organic / aqueous solution and a final concentration of 1.5 mM sulfo-GMBS and 1.95 mM LDL-DM. The reaction was incubated for 10 minutes at 25 °C. The crude sGMBS-LDL-DM mixture was added to a solution containing C242 antibody in phosphate-buffered saline (PBS) pH 7.4 spiked with a 5x solution of 300 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) pH 8.0 and 15% DMA (v / v) to a final ratio of 7.5 mol sulfo-GMBS-LDL-DM to 1 mol C242 antibody. The reaction was incubated overnight at 25°C.

[0410] The reaction was purified using a NAP desalting column (GE Healthcare) in 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 formulation buffer and filtered through a syringe filter with a 0.22 μm PVDF membrane.

[0411] The purified conjugate was found to have 3.8 mol LDL-DM / mol antibody by UV-Vis, 95% monomer by SEC, and less than 1% free drug by HPLC Hisep column analysis. The SEC / MS spectrum of C242-sGMBS-LDL-DM is shown in Figure 20.

[0412] Preparation of ML66-sGMBS-LDL-DM(ADC16c) Prior to conjugation, sGMBS-LDL-DM was prepared by mixing a stock solution of sulfo-GMBS (compound 15 in Figure 4) in N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (compound 14c in Figure 3) in DMA in the presence of succinate buffer, pH 5.0, to obtain a 60 / 40 organic / aqueous solution and a final concentration of 1.5 mM sulfo-GMBS and 1.95 mM LDL-DM. The reaction was incubated for 10 minutes at 25 °C. The crude sGMBS-LDL-DM mixture was added to a solution containing ML66 antibody in phosphate-buffered saline (PBS), pH 7.4, spiked with a 5x solution of 300 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), pH 8.0, and 15% DMA (v / v), to a final ratio of 8.0 mol sulfo-GMBS-LDL-DM to 1 mol ML66 antibody. The reaction was incubated overnight at 25°C.

[0413] The reaction was purified using a NAP desalting column (GE Healthcare) in 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 formulation buffer and filtered through a syringe filter with a 0.22 μm PVDF membrane.

[0414] The purified conjugate was found to have 3.7 mol LDL-DM / mol antibody by UV-Vis, 98% monomer by SEC, and less than 1% free drug by HPLC Hisep column analysis. The SEC / MS spectrum of C242-sGMBS-LDL-DM is shown in Figure 21.

[0415] Preparation of M9346A-sGMBS-LDL-DM (conjugate 17c) Prior to conjugation, sGMBS-LDL-DM (compound 15 in Figure 4) was prepared by mixing a stock solution of sulfo-GMBS in N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (compound 14c in Figure 3) in DMA in the presence of succinate buffer pH 5.0 to obtain a 60 / 40 organic / aqueous solution and a final concentration of 3 mM sulfo-GMBS and 3.9 mM LDL-DM. The reaction was incubated for 2 hours at 25 °C. The crude sGMBS-LDL-DM mixture was added to a solution containing M9346A antibody in phosphate-buffered saline (PBS) pH 7.4 spiked with a 5x solution of 300 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) pH 8.5 and 10% DMA (v / v) to a final ratio of 9.5 mol sulfo-GMBS-LDL-DM to 1 mol M9346A antibody. The reaction was incubated overnight at 25°C.

[0416] The reaction was purified using a NAP desalting column (GE Healthcare) in 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 formulation buffer and filtered through a syringe filter with a 0.22 μm PVDF membrane.

[0417] The purified conjugate was found to have 3.7 mol LDL-DM / mol antibody by UV-Vis, 99% monomer by SEC, and less than 1% free drug by SEC / reverse-phase HPLC dual-column analysis. The SEC / MS spectrum for C242-sGMBS-LDL-DM is shown in Figure 22.

[0418] Preparation of M9346A-C442-MalC5-LDL-DM (conjugate 26c) M9346A-C442 (an anti-folate antibody incorporating Cys at position 442) in phosphate-buffered saline (PBS) pH 7.4 (Life Technologies) was treated with 50 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich) and incubated for 1 hour at 37°C. TCEP was removed using a NAP desalting column (GE Healthcare), and 100 molar equivalents of dehydroascorbic acid (Sigma-Aldrich) was added to purified, reduced M9346A-C442 in PBS pH 7.4, 2 mM EDTA (Sigma-Aldrich) and incubated for 90 minutes to 4 hours at 25°C. The reduced and reoxidized antibody solution was immediately used for conjugation with MalC5-LDL-DM (compound I-2a shown above).

[0419] Reoxidized M9346A-C442 antibody was spiked with 2 mM EDTA in PBS pH 6.0, and conjugation was carried out in a 90% aqueous solution containing 10% N-N-dimethylacetamide (DMA, SAFC) and 5 equivalents of MalC5-LDL-DM. The reaction was incubated overnight at 25°C.

[0420] After the reaction, the conjugate was purified using a NAP desalting column (GE Healthcare) in 10 mM acetate, 9% sucrose, 0.01% Tween 20, pH 5.0 formulation buffer and filtered through a syringe filter with a 0.22 μm PVDF membrane.

[0421] The purified conjugate was found to have 2 mol LDL-DM / mol antibody by UV-Vis, 97% monomer by SEC, and less than 3% free drug by SEC / reverse-phase HPLC dual-column analysis. The SEC / MS spectrum for C242-sGMBS-LDL-DM is shown in Figure 23.

[0422] Example 5. Cell Binding Assay Binding of naked antibodies or ADCs to antigen-positive cells was assessed by indirect immunofluorescence assay using flow cytometry. Cells (5 × 10 per well) were plated in round-bottom 96-well plates and incubated with serial dilutions of test articles in 0.2 mL of alpha-MEM supplemented with 2% (v / v) normal goat serum (Sigma, St. Louis, MO) for 3 hours at 4°C. Each sample was assayed in triplicate. Control wells contained no test article. Cells were then washed with 0.2 mL of cold (4°C) medium and stained with fluorescein-labeled goat anti-human immunoglobulin G (IgG) antibody for 1 hour at 4°C. Cells were washed again with medium, fixed in 1% formaldehyde / PBS solution, and analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA).

[0423] As shown in Figure 7, conjugation only modestly affected the binding affinity of the naked antibody.

[0424] Example 6. In vitro cytotoxicity assay with ADC and metabolites Assays were performed in flat-bottom 96-well plates, with each data point performed in triplicate. Test articles were initially diluted in complete cell culture medium using a 5-fold dilution series, and 100 μL was added to each well. Final concentrations were typically 3 × 10 -8 M~8×10 -14The concentrations ranged from 1,500 to 3,000 M. Target cells were then added to the test samples at 1,500–3,000 cells per well in 100 μL of complete culture medium. The mixture was incubated at 37°C in a humidified 5% CO2 incubator for 5 days. The viability of the remaining cells was determined by a WST-8 (tetrazolium salt-8; 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium)-based colorimetric assay using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Rockville, MD). WST-8 is reduced in viable cells by dehydrogenase to yield a yellow-colored formazan product that is soluble in tissue culture medium. The amount of formazan dye is directly proportional to the number of viable cells. WST-8 was added to 10% of the final volume, and the plates were incubated for an additional 4 hours in a humidified 5% CO2 incubator at 37°C. The WST-8 signal was then measured at an optical density of 450 nm using a microplate reader. Viability was calculated by dividing the value of each treated sample by the mean value of the untreated controls and plotted for each treatment on a semi-logarithmic plot against the test article concentration. IC 50 Values ​​were determined using nonlinear regression (curve fit) with the GraphPad Prism v5 program (GraphPad Software, La Jolla, CA).

[0425] As shown in Figure 8, the conjugates of the present invention are highly potent against KB cells, and the in vitro cytotoxicity is antigen-specific, as the addition of naked antibody significantly reduced the cytotoxicity of the conjugates.

[0426] The in vitro cytotoxicity of the conjugates of the present invention was compared with that of Ab-sSPDB-DM4 conjugates and peptide-anilino maytansinoid conjugates, both of which have cleavable disulfide linkers (Table 1). As shown in Table 1, the conjugates of the present invention were generally more cytotoxic than Ab-sSPDB-DM4 conjugates. In addition, the length of the alkyl chain in the spacer L1 group had little effect on cytotoxicity against antigen-positive cells. [Table 1b]

[0427] The in vitro cytotoxicity of the conjugates of the present invention was also tested against CA922 cells. As shown in Figure 14, D-Ala in the peptide linker of the conjugate is linked to -NH-CR of the conjugate of formula (I). 1 R 2 When directly attached to the sacrificial nitrogen in the -S- moiety, it is detrimental to cytotoxicity.

[0428] The in vitro cytotoxicity of the main ADC metabolites was tested against Colo720E, H1703, H1975, and COLO704 cells, and the data are shown in Figure 15 and Table 2 below. The data suggest that increasing the hydrophobicity of the metabolites (longer alkyl chains in the L1 spacer) increased the cytotoxicity of the metabolites. [Table 2]

[0429] Example 7. In vivo efficacy studies The in vivo efficacy of the ADC was evaluated using established xenografts (H1703 250mm 3 ), HT-29 (100mm 3 ) or NCI-H2110(100mm 3) were evaluated in mice bearing the ADC. Female SCID mice were inoculated subcutaneously in the right flank with the desired cell type in serum-free medium / Matrigel. Tumors were allowed to grow to the designated size. Animals were then randomized into groups (6 animals per group). Control mice were treated with phosphate-buffered saline. Mice were administered the ADC at the mg / kg level indicated in the study. All doses in the xenograft models were based on the weight of the antibody component of the conjugate. All treatments were administered by intravenous tail vein injection. Tumor size was measured twice weekly in three dimensions using calipers, and tumor volume was measured in mm 3 and calculated using the formula V = 1 / 2 (length x width x height). Body weight was also measured twice a week.

[0430] As shown in Figures 10, 11A, 11B and 12, the conjugates of the present invention are In in vivo mouse models, it is highly active against H1703 (Figure 10), HT-29 (Figures 11A and 11B), and NCI-H2110 (Figure 12) xenograft tumors. Conjugate 17c is well tolerated compared to peptide-anilino maytansinoid conjugates (see Figure 13).

[0431] A mouse tolerability study was also conducted on the conjugates of the present invention having different peptide linkers (i.e., A in Formula (I)). The body weights of mice administered with the conjugates were measured. As shown in Figure 18, when the peptide linker contains consecutive L-alanines, mouse tolerance is reduced.

[0432] Example 8. In vitro bystander killing assay As previously described, bystander killing assays were performed in which the number of antigen-negative cells was held constant in the presence of varying numbers of antigen-positive cells, using the ratio of antigen-positive to antigen-negative cells specified in the relevant figures. This assay, in which the antigen-negative and antigen-positive cells were held constant, was varied as follows: 3,000 EGFR+ Ca9-22 cells were mixed with 2,000 EGFR- MCF7 cells, and the cell mixture was incubated with 0.66 nM of the indicated ADC for 4 days. Viable cells were quantified using a WST-8 assay. The same assay also assessed the cytotoxic potency of the ADCs against Ca9-22 or MCF7 cells; all ADCs killed EGFR+ Ca9-22 cells at similar levels but had no effect on EGFR- MCF7 cells unless antigen-positive cells were added.

[0433] In another experiment, various ratios of FRα(+) / FRα(-) cells were mixed in low-attachment U-bottom wells and exposed to 2 nM of a conjugate of the present invention, which was not toxic to FRα(-) cells (Namalwa, 1000 cells seeded) but killed all FRα(+) cells (JEG-3, 150K FRα ABC). The viability of FRα(-) cells was measured after 4 days using the Cell Titer Glo assay (Promega). The data are shown in Figure 9C. In particular, bystander killing with a conjugate of the present invention was compared to a peptide aniline maytansinoid conjugate, and the data are shown in Table 3 below. [Table 3]

[0434] As shown in Figures 9A, 9B, and 9C, the conjugates of the present invention have higher bystander killing efficacy than the Ab-sSPDB-DM4 conjugate and peptide-anilino maytansinoid conjugates, which have cleavable disulfide linkers. Additionally, when other factors are held constant, increasing the hydrophobicity of a metabolite increases its cytotoxicity, which in turn increases the bystander killing of the corresponding conjugate. Furthermore, this data appears to indicate that the conjugates of the present invention differ from the Ab-sSPDB-DM4 conjugate and peptide-anilino maytansinoid conjugates in the types and release efficiencies of metabolites released. The bystander killing of the conjugates of the present invention is greater than that of the peptide-anilino maytansinoid conjugate, which in turn is greater than that of Ab-sSPDB-DM4 (17g > 4b > 1b).

[0435] As shown in Figure 9D, D-Ala in the peptide linker of the conjugate is linked to -NH-CR of the conjugate of formula (I). 1 R 2 When directly attached to the sacrificial nitrogen in the -S- moiety, it is detrimental to bystander killing.

[0436] Example 9. In vitro metabolism studies FRα-expressing KB cells were treated with saturating amounts of the 17c conjugate for 24 hours. The catabolite-containing medium was incubated with 5 mM NEM to cap any free thiols present and then captured by pre-bound protein A-anti-maytansinoid antibody conjugate. Catabolites were released by acetone extraction and analyzed by UHPLC / HRMS.

[0437] The detected metabolites and proposed cleavage sites are shown in Figure 16. The major efflux catabolites identified in the cell culture medium were DM-SMe (25a) and DM-SH species.

[0438] In a separate experiment, antibodies were assayed using a previously described method (Erickson HK, Park PU, Widdison WC, Kovtun YV, Garrett LM, Hoffman K, et al. Antibody-maytansinoid conjugates). Conjugate 18c (anti-CanAg-LDL-DM) was incubated with COLO205 cells, followed by cell lysis, reduction of all disulfide bonds, and capping of the resulting thiols with N-ethylmaleimide. An untreated control was also performed in which COLO205 cells were lysed without treatment with the conjugate, reduction of disulfide bonds, and then capping of the resulting thiols with N-ethylmaleimide. Both samples were analyzed by UPLC / MS using a Thermo Q-Exactive mass spectrometer configured for Pos, Neg, and DDA Top-10 MS / MS detection in series with a Dionex UltiMate 3000 UPLC equipped with a Waters UPLC BEH C8, 1.8 micrometer, 100 x 2.1 mm column. The column compartment was set to 30 °C, and the UV detector was set to 252 nm. The injection volume was 40 μL. The column was eluted with deionized water containing 0.1% formic acid, a linear gradient from 20% to 100% acetonitrile containing 0.1% formic acid over 20 min at a flow rate of 0.35 mL / min, followed by a 10-min flush with 100% acetonitrile containing 0.1% formic acid. As shown in Figure 19, the top UPLC trace is of DTT- and NEM-treated cell lysate from COLO205 cells that were not exposed to either conjugate. The bottom UPLC trace is of a DTT- and NEM-treated cell lysate from COLO205 cells treated with conjugate 18c. The peak at 12.73 min had the same retention time and mass spectrum as the laboratory-made CH3S(CH2)5CO-DM compound (compound 25a).

[0439] In a similar experiment, 100 nM of C242-sGMBS-LDL-DM (18c) was added to Colo205 cell cultures and incubated at 37°C for 24 hours. Cells and media were separated, and catabolites were extracted by affinity capture and reconstituted in 20% acetonitrile. Catabolites were analyzed by UHPLC / HRMS. The major catabolite species identified in the cell media included DM-SMe, oxidized DM-SMe, and the acid form of the free drug (see structures below). [ka]

[0440] Example 10. In vivo efficacy in the OV-90 ovarian model The in vivo efficacy of the conjugates of the invention was evaluated in mice bearing OV-90 xenografts using a procedure similar to that described in Example 7.

[0441] As shown in Figures 17A and 17B and Table 4, the conjugates of the present invention exhibit enhanced activity against a heterologous ovarian tumor xenograft model with relatively low FRα expression levels (H-score 35) compared to peptide-anilino maytansinoid conjugates. [Table 4a]

[0442] Example 11. Mouse Tolerance Study a. The tolerability of 1200 μg / kg huML66-GMBS-LAlaLAlaLAla-Immol-DM (16a), huML66-GMBS-DAlaLAlaLAla-Immol-DM (16b), huML66-GMBS-LAlaDAlaLAla-Immol-DM (16c), huML66-GMBS-LAlaLAlaDAla-Immol-DM (16d), and huML66-sSPDB-DM4(la) was tested in female CD-1 mice. [Table 4b]

[0443] Groups and treatments: (2 mice / group) 1. Vehicle control 2.huML66-GMBS-LAlaLAlaLAla-Immol-DM(16a), 1200μg / kg 3.huML66-GMBS-DAlaLAlaLAla-Immol-DM(16b), 1200μg / kg 4.huML66-GMBS-LAlaDAlaLAla-Immol-DM(16c), 1200μg / kg 5.huML66-GMBS-LAlaLAlaDAla-Immol-DM(16d), 1200μg / kg 6.huML66-sSPDB-DM4(la), 1200μg / kg

[0444] Specific study design: Thirty mice were randomized by body weight into six groups (two mice per group). Body weights ranged from 24.4 to 27.5 grams (26.2 ± 0.96, mean ± SD). Mice in each group were identified by colored markings on their fur. Treatment began 15 days after arrival. Mice were administered conjugates based on their individual body weights. All administrations of conjugates or PBS were administered intravenously with a 1.0 ml syringe equipped with a 27-gauge, 1 / 2 inch needle. The injections were given intravenously. Depending on the given dose, the treatment was divided into two injections, 2 hours apart. Mice could not receive more than 350 μl per injection.

[0445] The body weights of the mice administered the conjugate were measured and are shown in Figures 24A and 24B.

[0446] b. The tolerability of 1000 μg / kg and 1250 μg / kg Movl9vl.6-GMBS-lAladAlalAla-Immol-DM (17c) and 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl (4b) was tested in female CD-1 mice. [Table 4c]

[0447] Groups and treatments: (8 mice / group) 1. Vehicle control 2.Movl9vl.6-GMBS-lAladAlalAla-Immol-DM, 1250μg / kg 3.Movl9vl.6-GMBS-lAladAlalAla-Immol-DM, 1000μg / kg 4.Movl9vl.6-GMBS-dAlalAla-PAB-DMl, 1250μg / kg

[0448] Specific research design: Thirty-two mice were randomized by weight into four groups (8 mice per group). Body weights ranged from 23.4 to 27.3 grams (25.5±1.02, mean±SD). Mice in each group were identified by ear notching. Treatment began 8 days after arrival. Mice were administered conjugates based on their individual body weights. All conjugate or PBS administrations were performed intravenously with a 1.0 ml syringe equipped with a 27-gauge, ½-inch needle. Depending on the given dose, treatment was divided into two injections, 2 hours apart. Mice were not allowed to receive more than 350 μl per injection.

[0449] The body weights of the mice administered the conjugates were measured and are shown in Figures 25A-25D.

[0450] Example 12. Pharmacokinetic studies 1. Conjugate 17c The pharmacokinetics of M-LDL-IMM-DM (conjugate 17c) and M-SPDB-DM4 were evaluated in female CD-1 mice. Mice were randomly distributed based on body weight into two groups of six mice. Mice in group A received a single intravenous injection of 10 mg / kg M-LDL-IMM-DM via the tail vein. Mice in group B received a single intravenous injection of 10 mg / kg M-LDL-IMM-DM via the tail vein. A single intravenous injection of 1000 mg of M-SPDB-DM4 was given. Blood was collected at 2 minutes, 6, 24, 48, 72, 168, 336, 504, 672, and 840 hours. Mice were then bled sequentially to ensure that no mice were bled more than twice within a 24-hour period. Serum was separated from the blood, and samples were frozen at -80°C until analysis by ELISA. Total antibody and ADC ELISAs were performed on the samples, and concentration versus time plots are shown in Figure 26. The total antibody ELISA quantifies antibodies bearing at least one maytansinoid, as well as antibodies without bound maytansinoid. Concentration is determined by capture with an anti-human IgG antibody, followed by quantitation using an enzyme-labeled anti-human IgG antibody. The ADC ELISA involves capturing a conjugate bearing at least one bound maytansinoid using an anti-maytansinoid antibody, followed by capture of the antibody component of the conjugate and detection with an enzyme-labeled anti-human FC antibody. To be detected, the conjugate must contain at least one covalently bound maytansinoid.

[0451] PK parameters were obtained using standard algorithms in the noncompartmental pharmacokinetic analysis program Phoenix WinNonlin, Professional v 6.1 (Certara, Princeton, NJ) and are shown in Table 5. [Table 5]

[0452] 2. Conjugate 26c CD-1 mice were injected with a single 10 mg / kg dose of 26c or M9346A-C442-mal-SPDB-DM4. Blood was collected at 2 minutes, 24 hours, and 72 hours post-injection. The ADC was purified from plasma using affinity capture with a folate receptor α-Fc fusion protein, and samples were analyzed by size-exclusion chromatography (SEC) and mass spectrometry (MS). Loss of DM or DM4 was measured as normalized percent degradation versus time and is plotted in Figure 27. The in vivo stability of 26c was higher than that of M9346A-C442-mal-SPDB-DM4, as demonstrated by the reduced degradation observed at 2 minutes (-0.2 vs. 7.3%) and 24 hours (5.2 vs. 16.5%). The 72-hour sample concentration of M9346A-C442-mal-SPDB-DM4 was too dilute to obtain normalized percent degradation values. [1] Below formula: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, CB is a cell binding agent; L2 is absent or a spacer; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20. [2] R 1 and R 2 The conjugate according to [1], wherein at least one of [3] R 1 and R 2The conjugate according to [1], wherein each of the following is independently H or Me. [4] R 1 and R 2 The conjugate according to [3], wherein one of the groups is H and the other is Me. [5] R 1 and R 2 The conjugate according to [1], wherein both of [6] The conjugate according to any one of [1] to [5], wherein L1 is -L1'-C(=O)-; and L1' is alkylene or cycloalkylene, wherein the -C(=O)- moiety in L1 is connected to D. [7] L1' is C 1~10 The conjugate according to [6], wherein the conjugate is alkylene. [8] L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me. [9] L1 is -CR 3 R 4 -(CH2) 3~5 The conjugate according to [8], wherein -C(=O)-.

[10] R 3 and R 4 The conjugate according to [8] or [9], wherein both of are Me.

[11] L1 is -(CH2) 4~6 The conjugate according to [6], wherein —C(═O)—.

[12] L2 has the following structural formula: [ka] The conjugate according to any one of [1] to

[11] , represented by [In the formula, R A is alkylene, cycloalkylalkylene, or arylene; R B and R C are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W does not exist or [ka] and where s2' is V, R A or J CB s3' indicates the site connected to R B , V', R C or J A indicates the part connected to; J CB is -C(=O)-, [ka] where s1 represents the site attached to the cell-binding agent CB and s2 represents the site attached to R A indicates the part connected to; R a , R b , R c , and R e is, independently for each occurrence, H or alkyl; J A is -C(=O)-].

[13] L2 has the following structural formula: [ka] is represented by R A is alkylene, cycloalkylalkylene, or arylene; R B and RC are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W is not present, or [ka] and; J CB But -C(=O)-, [ka] where s1 represents the site attached to the cell-binding agent CB, and s2 represents R A indicates the part connected to; J A The conjugate according to any one of [1] to

[12] , wherein is —C(═O)—.

[14] p is 0 and R C The conjugate according to

[12] or

[13] , wherein no J CB is -C(=O)- or [ka] The conjugate according to any one of

[12] to

[14] , wherein

[16] L2 has the following structural formula: [ka] The conjugate according to any one of

[12] to

[15] , which is represented by [In the formula, R x , R y , R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + or -OH, halogen, -SO3H or -NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; l and k each independently represent an integer of 1 to 10; s1 indicates the site attached to the cell-binding agent CB, and s3 indicates the site attached to the A group.

[17] R x , R y , R x’ and R y’ The conjugate according to

[16] , wherein all of

[18] The conjugate according to

[16] or

[17] , wherein l and k each independently represent an integer of 2 to 6.

[19] L2 has the following structural formula: [ka] The conjugate according to any one of

[12] to

[15] , which is represented by [In the formula, R x and R y are both H; l and l1 are each an integer of 2 to 6; k1 is an integer from 1 to 5.

[20] The conjugate according to any one of [1] to

[19] , wherein A is a peptide that can be cleaved by a protease. [twenty one] The conjugate according to

[20] , wherein A is a peptide that can be cleaved by a protease expressed in tumor tissue. [twenty two] A is independently selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, each independently as an L or D isomer; -NH-CR 1 R 2 The conjugate according to any one of [1] to

[21] , which is a peptide having an amino acid covalently bonded to -S-L1-D. [twenty three] -NH-CR 1 R 2 The conjugate according to any one of [1] to

[22] , wherein the amino acid connected to -S-L1-D is an L-amino acid. [twenty four] A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala- D-Ala-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val -D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, A selected from the group consisting of la-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, where each The first amino acid in each peptide is connected to the L2 group, and the last amino acid in each peptide is -NH-CR 1 R 2 The conjugate according to any one of [1] to

[19] , wherein the conjugate is connected to -S-L1-D. [twenty five] The conjugate according to

[24] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[26] The conjugate according to any one of [1] to

[25] , wherein D is a maytansinoid.

[27] D is the following formula: [ka] The conjugate according to

[26] , represented by

[28] D is the following formula: [ka] The conjugate according to

[27] , represented by

[29] Below formula: [ka] The conjugate according to any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, represented by [In the formula, [ka] is the cell-binding agent attached to the L2 group via a Lys amine group; [ka] is the cell-binding agent attached to the L2 group via a Cys thiol group; R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1, and t1 each independently represent an integer of 1 to 6; m2, n2, r2, s2, and t2 each independently represent an integer of 1 to 7; t3 is an integer from 1 to 12; D1 is expressed by the following formula: [ka] [represented by]

[30] D1 is expressed as follows: [ka] The conjugate according to

[29] , represented by

[31] The conjugate according to

[29] or

[30] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[32] The conjugate according to any one of

[29] to

[31] , wherein m1, r1, n1, and m3 are each independently an integer of 2 to 4; and m2, n2, and r2 are each independently an integer of 3 to 5.

[33] Below formula: [ka] The conjugate according to any one of

[29] to

[31] , which is represented by [In the formula, r1 and t1 are each an integer of 2 to 6; r2 and t2 are each an integer of 2 to 5; t3 is an integer between 2 and 12].

[34] R 3 and R 4 The conjugate according to any one of

[29] to

[33] , wherein both of are Me.

[35] R 3 and R 4 The conjugate according to any one of

[29] to

[33] , wherein both of

[36] Below formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] The conjugate according to

[29] , or a pharmaceutically acceptable salt thereof, [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; D1 is expressed by the following formula: [ka] [represented by]

[37] Below formula: [ka] [ka] [ka] The conjugate according to

[36] , represented by [Wherein D1 is a compound represented by the following formula: [ka] [represented by]

[38] The conjugate of any one of [1] to

[37] , wherein the cell-binding agent is an antibody or antigen-binding fragment thereof, a single-chain antibody, a single-chain antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a lymphokine, a hormone, a vitamin, a growth factor, a colony-stimulating factor, a nutrient transport molecule, a Bicycles® peptide, or a pentaline.

[39] The conjugate according to

[38] , wherein the cell-binding agent is an antibody or an antigen-binding fragment thereof.

[40] The conjugate according to

[38] , wherein the cell-binding agent is a resurfaced antibody or a resurfaced antibody fragment thereof.

[41] The conjugate according to

[38] , wherein the cell-binding agent is a monoclonal antibody or a monoclonal antibody fragment thereof.

[42] The conjugate according to

[38] , wherein the cell-binding agent is a humanized antibody or a humanized antibody fragment thereof.

[43] The conjugate according to

[38] , wherein the cell binding agent is a chimeric antibody or a chimeric antibody fragment thereof.

[44] The conjugate according to

[38] , wherein the cell-binding agent is an anti-folate receptor antibody or an antibody fragment thereof, an anti-EGFR antibody or an antibody fragment thereof, an anti-CD33 antibody or an antibody fragment thereof, an anti-CD19 antibody or an antibody fragment thereof, an anti-Muc1 antibody or an antibody fragment thereof, or an anti-CD37 antibody or an antibody fragment thereof.

[45] The conjugate according to

[38] , wherein the cell-binding agent is an anti-CD123 antibody or an antibody fragment thereof.

[46] The anti-CD123 antibody or antibody fragment thereof comprises: a) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 33, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 34, and a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 35; and b) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 37. and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 38.

[47] The conjugate according to

[45] , wherein the anti-CD123 antibody or antibody fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40.

[48] The conjugate according to

[47] , wherein X (or Xaa), the second residue from the N-terminus of SEQ ID NO: 39, is Phe (F).

[49] The conjugate according to

[47] , wherein X (or Xaa), the second residue from the N-terminus of SEQ ID NO: 39, is Val (V).

[50] The conjugate according to

[45] , wherein the anti-CD123 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 42 and a light chain having the amino acid sequence of SEQ ID NO: 43.

[51] The conjugate according to

[45] , wherein the anti-CD123 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 44 and a light chain having the amino acid sequence of SEQ ID NO: 43.

[52] The conjugate according to

[50] or

[51] , wherein X (or Xaa), the second residue from the N-terminus of SEQ ID NO: 42 or SEQ ID NO: 44, is Val.

[53] Below formula: [ka] The conjugate of claim 1, represented by [wherein Ab is an anti-folate receptor antibody; and q is an integer from 1 to 10].

[54] Below formula: [ka] The conjugate according to [1], represented by [wherein Ab is an anti-folate receptor antibody; and q is an integer from 1 to 10].

[55] The anti-folate receptor antibody comprises: (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 6. and (b) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 7; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 10; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 9. The conjugate of

[53] or

[54] ,

[56] The conjugate according to

[53] or

[54] , wherein the anti-folate receptor antibody comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[57] The conjugate according to

[53] or

[54] , wherein the anti-folate receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13.

[58] The conjugate according to

[53] or

[54] , wherein the anti-folate receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 13.

[59] The conjugate according to

[53] or

[54] , wherein the anti-folate receptor antibody comprises a heavy chain encoded by a plasmid DNA having ATCC deposit number PTA-10772 and a light chain encoded by a plasmid DNA having ATCC deposit number PTA-10774.

[60] Below formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, L2 ’ is absent or is a spacer having a reactive moiety capable of forming a covalent bond with the cell-binding agent; A is a peptide containing 1 amino acid or 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[61] R 1 and R 2

[60] The compound according to

[60] , wherein at least one of

[62] R 1 and R 2 are each independently H or Me.

[63] R 1 and R 2

[60] The compound according to

[60] , wherein one of

[64] R 1 and R 2 The compound according to

[60] , wherein both are H.

[65] The compound according to any one of

[60] to

[64] , wherein L1 is -L1'-C(=O)-; and L1' is alkylene or cycloalkylene.

[66] L1' is C 1~10 The compound according to

[65] , wherein the alkylene is alkylene.

[67] L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R3 and R 4 But,

[65] The compound according to

[65] , wherein each of the is independently H or Me.

[68] L1 is -CR 3 R 4 -(CH2) 3~5 The compound according to

[67] , wherein —C(═O)—.

[69] R 3 and R 4 The compound according to

[67] or

[68] , wherein both of are Me.

[70] L1 is -(CH2) 4~6 The compound according to

[66] , wherein —C(═O)—.

[71] L2' has the following structural formula: J CB '-R A -VWR B -V'-R C -J A - The compound according to any one of

[60] to

[70] , represented by [In the formula, R A is alkylene, cycloalkylalkylene, or arylene; R B and R C are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W does not exist or [ka] and where s2' is V, R A or J CB ' indicates the site connecting to R, and s3' indicates the site connecting to R. B , V', RC or J A indicates the part that connects to; J CB 'teeth, [ka] and; R a , R b , R c , and R e is, independently for each occurrence, H or alkyl; X 1 is -Cl, -Br or -I; COE is a reactive ester; J A is -C(=O)-].

[72] L2' has the following structural formula: J CB '-R A -VWR B -V'-R C -J A - The compound according to any one of

[60] to

[70] , represented by [In the formula, R A is alkylene, cycloalkylalkylene, or arylene; R B and R C are each independently absent, alkylene, cycloalkylene, or arylene; V and V' are each independently -(O-CH-CH) P - or -(CH2-CH2-O) P - and; p is 0 or an integer from 1 to 10; W is absent or [ka] and; J CB 'teeth [ka] and; COE is a reactive ester; J A is -C(=O)-].

[73] p is 0 and R C The compound according to

[71] or

[72] , wherein

[74] J CB ' is -C(=O)OH, -COE or [ka] The compound according to any one of

[71] to

[73] , wherein

[75] L2' has the following structural formula: [ka] The compound according to any one of

[71] to

[74] , represented by [In the formula, R x , R y , R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + or -OH, halogen, -SO3H or -NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; l and k are integers from 1 to 10; J CB ' is -C(=O)OH or -COE].

[76] Rx , R y , R x’ and R y’ The compound according to

[75] , wherein all of

[77] The compound according to

[75] or

[76] , wherein l and k each independently represent an integer of 2 to 6.

[78] L2' has the following structural formula: [ka] The compound according to any one of

[71] to

[74] , represented by [In the formula, R x and R y are both H; l and l1 are each an integer of 2 to 6; k1 is an integer between 1 and 12.

[79] The compound according to any one of

[60] to

[78] , wherein A is a peptide that can be cleaved by a protease.

[80] The compound according to

[79] , wherein A is a peptide cleavable by a protease expressed in tumor tissue.

[81] A is independently selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, each independently as an L or D isomer; -NH-CR 1 R 2 The compound according to any one of

[60] to

[80] , which is a peptide having an amino acid covalently bonded to -S-L1-D.

[82] -NH-CR 1 R 2 The compound according to any one of

[60] to

[81] , wherein the amino acid connected to -S-L1-D is an L-amino acid.

[83] A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is connected to the L2 group and the last amino acid in each peptide is connected to the L2 group. , -NH-CR 1 R 2 The compound according to any one of

[60] to

[78] , wherein -S-L1-D is connected to

[84] The compound according to

[83] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[85] The compound according to any one of

[60] to

[84] , wherein D is a maytansinoid.

[86] D is the following formula: [ka] 86. The compound of claim 85, represented by:

[87] D is the following formula: [ka] The compound according to

[86] , represented by

[88] Below formula: [ka] or a pharmaceutically acceptable salt thereof, [In the formula, R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1, and t1 each independently represent an integer of 1 to 6; m2, n2, r2, s2, and t2 each independently represent an integer of 1 to 7; t3 is an integer from 1 to 12; J CB ' is -C(=O)OH or -COE; D1 is expressed by the following formula: [ka] [represented by]

[89] The compound according to

[88] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[90] The compound according to

[88] or

[89] , wherein m1, r1, n1, and m3 are each independently an integer of 2 to 4; and m2, n2, and r2 are each independently an integer of 3 to 5.

[91] Below formula: [ka] The compound according to

[88] , [In the formula, r1 and t1 are each an integer of 2 to 6; r2 and t2 are each an integer of 2 to 5; t3 is an integer between 2 and 12].

[92] R 3 and R 4 The compound according to any one of

[88] to

[91] , wherein both of are Me.

[93] R 3 and R 4 The compound according to any one of

[88] to

[91] , wherein both are H.

[94] Below formula: [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; J CB ' is -C(=O)OH or -COE; D1 is expressed by the following formula: [ka] [represented by]

[95] Below formula: [ka] The compound according to

[94] , [Wherein D1 is a compound represented by the following formula: [ka] [represented by]

[96] The compound according to any one of

[60] to

[95] , wherein -COE is a reactive ester selected from N-hydroxysuccinimide ester, N-hydroxysulfosuccinimide ester, nitrophenyl (e.g., 2- or 4-nitrophenyl) ester, dinitrophenyl (e.g., 2,4-dinitrophenyl) ester, sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) ester, and pentafluorophenyl ester.

[97] The compound according to any one of

[60] to

[95] , wherein -COE is an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

[98] Below formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, A' is a peptide containing 1 amino acid or 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[99] R 1 and R2

[98] The compound according to

[98] , wherein at least one of

[0100] R 1 and R 2 are each independently H or Me.

[0101] R 1 and R 2 The compound according to

[0100] , wherein one of the groups is H and the other is Me.

[0102] R 1 and R 2 The compound according to

[98] , wherein both are H.

[0103] The compound according to any one of

[98] to

[0102] , wherein L1 is -L1'-C(=O)-; and L1' is alkylene or cycloalkylene.

[0104] L1' is C 1~10 The compound according to

[0103] , wherein the alkylene is alkylene.

[0105] L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me.

[0106] L1 is -CR 3 R 4 -(CH2) 3~5 The compound according to

[0105] , wherein -C(=O)-.

[0107] R 3 and R 4 The compound according to

[0105] or

[0106] , wherein both are Me. L1 is -(CH2) 4~6 The compound according to

[0103] , wherein -C(=O)-.

[0109] The compound according to any one of

[98] to

[0108] , wherein A' is a peptide that can be cleaved by a protease.

[0110] The compound according to

[0109] , wherein A' is a peptide that can be cleaved by a protease expressed in tumor tissue.

[0111] A' is independently selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, each independently as an L or D isomer; -NH-CR 1 R 2 The compound according to any one of

[98] to

[0110] , which is a peptide having an amino acid covalently bonded to -S-L1-D.

[0112] -NH-CR 1 R 2 The conjugate according to any one of

[98] to

[0111] , wherein the amino acid connected to -S-L1-D is an L-amino acid.

[0113] A' is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Al a-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-A and D-Ala-tBu-Gly, D-Val-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is connected to the L2 group and the last amino acid in each peptide is connected to the L2 group. 1 R 2 The compound according to any one of

[98] to

[0108] , wherein -S-L1-D is connected to

[0114] The compound according to

[0113] , wherein A' is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0115] The compound according to any one of

[98] to

[0114] , wherein D is a maytansinoid. .

[0116] D is the following formula: [ka] The compound according to

[0115] , represented by:

[0117] D is the following formula: [ka] The compound according to

[0116] , represented by:

[0118] Below formula: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, R 3 and R 4 are each independently H or Me; m2 is an integer from 1 to 7; D1 is expressed by the following formula: [ka] [represented by]

[0119] D1 is expressed as follows: [ka] The compound according to

[0118] , represented by:

[0120] A compound according to

[0118] or

[0119] , wherein A' is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0121] The compound according to any one of

[0118] to

[0120] , wherein m2 is an integer of 3 to 5.

[0122] R 3 and R 4 The compound according to any one of

[0118] to

[0121] , wherein both are Me.

[0123] R 3 and R 4 The compound according to any one of

[0118] to

[0121] , wherein both are H.

[0124] Below formula: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, A' is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; D1 is expressed by the following formula: [ka] [represented by]

[0125] D1 is expressed as follows: [ka] The compound according to

[0124] , represented by:

[0126] Below formula: [ka] or a pharmaceutically acceptable salt thereof [In the formula, L3 is given by the following formula: [ka] Represented by; R x’ and R y’ is independently for each occurrence H, -OH, halogen, -O-(C 1~4 alkyl), -SO3H, -NR 40 R 41 R 42 + or -OH, halogen, -SO3H or -NR 40 R 41 R 42 + C optionally replaced with 1~4 alkyl, where R40 , R 41 and R 42 are each independently H or C 1~4 is alkyl; k is an integer from 1 to 10; A is a peptide containing 1 amino acid residue or 2 to 20 amino acid residues; R 1 and R 2 are each independently H or C 1~3 is alkyl; L1 is a spacer; D-L1-SH is a cytotoxic drug; and q is an integer from 1 to 20.

[0127] R 1 and R 2 The compound according to

[0126] , wherein at least one of the following is H.

[0128] R 1 and R 2 are each independently H or Me.

[0129] R 1 and R 2 The compound according to

[0128] , wherein one of the groups is H and the other is Me.

[0130] R 1 and R 2 The compound according to

[0126] , wherein both are H.

[0131] The compound according to any one of

[0126] to

[0130] , wherein L1 is -L1'-C(=O)-; and L1' is alkylene or cycloalkylene.

[0132] L1' is C 1~10 The compound according to

[0131] , wherein the alkylene is alkylene.

[0133] L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4are each independently H or Me.

[0134] L1 is -CR 3 R 4 -(CH2) 3~5 The compound according to

[0133] , wherein -C(=O)-.

[0135] R 3 and R 4 The compound according to

[0133] or

[0134] , wherein both are Me. L1 is -(CH2) 4~6 The compound according to

[0131] , wherein -C(=O)-.

[0137] R x’ and R y’ The compound according to any one of

[0126] to

[0136] , wherein both are H.

[0138] The compound according to

[0137] , wherein k is an integer of 2 to 6.

[0139] The compound according to

[0137] , wherein k is 3.

[0140] A compound according to any one of

[0126] to

[0139] , wherein A is a peptide that can be cleaved by a protease.

[0141] A compound according to

[0140] , wherein A is a peptide that can be cleaved by a protease expressed in tumor tissue.

[0142] A is independently selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, serino-Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, each independently as an L or D isomer; -NH-CR 1 R 2 A compound according to any one of

[0126] to

[0141] , which is a peptide having an amino acid covalently bonded to -S-L1-D.

[0143] -NH-CR 1 R 2The compound according to any one of

[0126] to

[0142] , wherein the amino acid connected to -S-L1-D is an L-amino acid.

[0144] A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is connected to the L2 group and the last amino acid in each peptide is connected to the L2 group. , -NH-CR 1 R 2 -S-L1-D is connected to the compound according to any one of

[0126] to

[0139] .

[0145] The compound according to

[0144] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0146] 146. The compound of any one of claims 126 to 145, wherein D is a maytansinoid.

[0147] D is the following formula: [ka] The compound according to

[0146] , represented by:

[0148] D is the following formula: [ka] The compound according to

[0147] , represented by:

[0149] Below formula: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, R 3 and R 4 are each independently H or Me; m3 is an integer from 1 to 6; m2 is an integer from 1 to 7; D1 is expressed by the following formula: [ka] [represented by]

[0150] The compound according to

[0149] , wherein A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

[0151] The compound according to

[0149] or

[0150] , wherein m3 is an integer of 2 to 4; and m2 is an integer of 3 to 5.

[0152] R 3 and R 4The compound according to any one of

[0149] to

[0151] , wherein both are Me.

[0153] R 3 and R 4 The compound according to any one of

[0149] to

[0151] , wherein both are H.

[0154] Below formula: [ka] or a pharmaceutically acceptable salt thereof. [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly; D1 is expressed by the following formula: [ka] [represented by]

[0155] D1 is expressed as follows: [ka] The compound according to

[0154] , represented by:

[0156] Below formula: D-L1-SZ 0 (V) A compound represented by [In the formula, L1 is a spacer; Z 0 is H or Me, except that Z 0 is H, L1 is -C(=O)-(CH2) q - or is not -C(=O)-CH2-CH2-C(CH3)2-, where q is an integer from 1 to 3; Z 0 is Me, then L1 is not -C(=O)-(CH2)2- or -C(=O)-CH2-CH2-C(CH3)2-; D-L1-SH is a cytotoxic drug].

[0157] A compound according to

[0156] , wherein L1 is -L1'-C(=O)-; and L1' is alkylene or cycloalkylene, wherein the -C(=O)- moiety in L1 is bonded to D.

[0158] L1' is C 1~10 The compound according to

[0157] , wherein the alkylene is alkylene.

[0159] L1 is -CR 3 R 4 -(CH2) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me.

[0160] L1 is -CR 3 R 4 -(CH2) 3~5 The compound according to

[0159] , wherein -C(=O)-.

[0161] R 3 and R 4 The compound according to

[0159] or

[0160] , wherein both are Me. L1 is -(CH2) 4~6 The compound according to

[0157] , wherein -C(=O)-.

[0163] D is the following formula: [ka] The compound according to any one of

[0156] to

[0162] , represented by:

[0164] D is the following formula: [ka] The compound according to

[0163] , represented by:

[0165] A pharmaceutical composition comprising the conjugate according to any one of [1] to

[59] and a pharmaceutically acceptable carrier.

[0166] A method for inhibiting abnormal cell proliferation or treating a proliferative disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the conjugate described in any one of [1] to

[59] .

[0167] A method according to

[0166] for treating cancer.

[0168] The cancer may be kidney cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, brain cancer, prostate cancer, endometrial cancer, cervical cancer, renal cancer, pancreatic cancer, ovarian cancer (e.g., epithelial ovarian cancer), head and neck cancer, melanoma, colorectal cancer, gastric cancer, squamous cell carcinoma, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), testicular cancer, choriocarcinoma, Merkel cell carcinoma, sarcoma (e.g., osteosarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma), glioblastoma, neuroblastoma, 168. The method of claim 167, wherein the cancer is selected from blastoma, lymphoma (e.g., non-Hodgkin's lymphoma), myelodysplastic syndrome (MDS), peritoneal cancer, fallopian tube cancer, uterine cancer, or leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (e.g., B-ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML)).

[0169] The method described in

[0168] , wherein the cancer is breast cancer, ovarian cancer or renal cancer.

[0170] The cancer is cervical cancer, ovarian cancer, uterine cancer, endometrial cancer, or fallopian tube cancer. The method described in

[0168] .

Claims

1. Below formula: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof. [In the formula, CB is a cell binding agent, said cell binding agent being an antibody or an antigen-binding fragment thereof; L ２ has the following structural formula: 【Chemistry 2】 is represented by [In the formula, R ｘ , R ｙ , R ｘ’ and R ｙ’ are all H; l and k are each independently an integer from 1 to 10; s1 represents the site connected to the cell-binding agent CB, and s3 represents the site connected to the A group. A is a peptide containing 2 to 20 amino acid residues and cleavable by a protease; R １ and R ２ are each independently H or C １～３ is alkyl; L １ Is, -L １ '-C(=O)-, L １ ' is alkylene or cycloalkylene; １ the -C(=O)- group in is connected to D; q is an integer from 1 to 20; and D is of the following formula: 【Chemistry 3】 [represented by]

2. R １ and R ２ one of which is H and the other is Me; or R １ and R ２ The conjugate of claim 1 , wherein both are H.

3. L １ But -CR ３ R ４ - (CH ２ ) １～８ -C(=O)-; R ３ and R ４ The conjugate of claim 1 , wherein each is independently H or Me.

4. The conjugate of any one of claims 1 to 3, wherein l and k are each independently an integer from 2 to 6.

5. The conjugate according to any one of claims 1 to 4, wherein A is a peptide cleavable by a protease expressed in tumor tissue.

6. A is Gly-Gly-Gly, Ala-Val, Val-Ala, D-Val-Ala, Val-Cit, D-Val-Cit, Val-L ys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N ９ -Tosyl-Arg, Phe-N ９ -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu , Ile-Ala-Leu, Val-Ala-Val, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Al a-Ala, Ala-Ala-D-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, V and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is selected from the group consisting of L, G, Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly, wherein the first amino acid in each peptide is selected from the group consisting of L, G, Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, D-Ala-Pro, and D-Ala-tBu-Gly. ２ The last amino acid in each peptide is connected to a -NH-CR １ R ２ -S-L １ The conjugate according to any one of claims 1 to 5, wherein the conjugate is connected to -D.

7. D is of the following formula: 【Chemistry 4】 The conjugate according to any one of claims 1 to 6, represented by:

8. Below formula: 【Chemistry 5】 The conjugate according to any one of claims 1 to 7, represented by: [In the formula, 【Chemistry 6】 is linked to the L via the Lys amine group. ２ the cell-binding agent connected to a group; R ３ and R ４ are each independently H or Me; m1, m3, and s1 each independently represent an integer from 1 to 6; m2, n2, and s2 each independently represent an integer from 1 to 7; D １ is expressed by the following formula: 【Chemistry 7】 [represented by]

9. Below formula: 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof. [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

10. Below formula: 【Chemistry 12】 The conjugate of claim 9, represented by [In the formula, D １ is expressed by the following formula: 【Chemistry 13】 [represented by]

11. 11. The conjugate of any one of claims 1 to 10, wherein the cell binding agent is a single chain antibody, a single chain antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a probody, or a nanobody.

12. The conjugate of claim 11, wherein the cell binding agent is a resurfaced antibody or a resurfaced antibody fragment thereof, a monoclonal antibody or a monoclonal antibody fragment thereof, a humanized antibody or a humanized antibody fragment thereof, or a chimeric antibody or a chimeric antibody fragment thereof.

13. The conjugate of claim 11, wherein the cell binding agent is an anti-folate receptor antibody or antibody fragment thereof, an anti-EGFR antibody or antibody fragment thereof, an anti-CD33 antibody or antibody fragment thereof, an anti-CD19 antibody or antibody fragment thereof, an anti-Mucl antibody or antibody fragment thereof, an anti-CD37 antibody or antibody fragment thereof, or an anti-CD123 antibody or antibody fragment thereof.

14. Below formula: 【Chemistry 14】 The conjugate of claim 1, represented by [wherein Ab is an anti-folate receptor antibody; q is an integer from 1 to 10; and D １ is expressed as follows: 【Chemistry 15】 [represented by]

15. The anti-folate receptor antibody (i) (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5; and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:6; and (b) a light chain CDR1 having the amino acid sequence of SEQ ID NO:7; a light chain CDR2 having the amino acid sequence of SEQ ID NO:10; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:9; (ii) a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 14, and a light chain variable domain having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16; or (iii) a heavy chain having the amino acid sequence of SEQ ID NO:11 and a light chain having the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:13; or (iv) comprising a heavy chain encoded by plasmid DNA having ATCC deposit number PTA-10772 and a light chain encoded by plasmid DNA having ATCC deposit number PTA-10774; The conjugate of claim 14.

16. Below formula: 【Chemistry 16】 or a pharma- ceutically acceptable salt thereof [In the formula, R ３ and R ４ are each independently H or Me; m1, m3, n1, and s1 each independently represent an integer from 1 to 6; m2, n2, and s2 each independently represent an integer from 1 to 7; J ＣＢ ' is -C(=O)OH or -COE, where COE is an N-hydroxysuccinimide ester, N-hydroxysulfosuccinimide ester, nitrophenyl ester, dinitrophenyl ester, sulfo-tetrafluorophenyl ester, or pentafluorophenyl ester; A is a peptide containing 2-20 amino acids and cleavable by a protease; D １ is expressed by the following formula: 【Chemistry 17】 [represented by]

17. Below formula: 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 or a pharma- ceutically acceptable salt thereof. [In the formula, A is Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.

18. Below formula: 【Chemistry 21】 The compound of claim 17 represented by [In the formula, D １ is expressed by the following formula: 【Chemical 22】 [represented by]

19. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 or a compound according to any one of claims 16 to 18 and a pharma- ceutically acceptable carrier.

20. 19. A pharmaceutical composition for use in a method of inhibiting abnormal cell proliferation or treating a proliferative disorder in a mammal, said pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 or a compound according to any one of claims 16 to 18.

21. 21. The pharmaceutical composition of claim 20, wherein the method is for treating cancer, (i) the cancer is selected from the group consisting of kidney cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), colon cancer, brain cancer, prostate cancer, endometrial cancer, cervical cancer, renal cancer, pancreatic cancer, ovarian cancer (e.g., epithelial ovarian cancer), head and neck cancer, melanoma, colorectal cancer, gastric cancer, squamous cell carcinoma, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), testicular cancer, choriocarcinoma, Merkel cell carcinoma, sarcoma (e.g., osteosarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma), neurosarcoma, and / or sarcoma. selected from glioblastoma, neuroblastoma, lymphoma (e.g., non-Hodgkin's lymphoma), myelodysplastic syndrome (MDS), peritoneal cancer, fallopian tube cancer, uterine cancer, or leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (e.g., B-ALL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML)); (ii) the cancer is breast cancer, ovarian cancer or renal cancer; or (iii) The pharmaceutical composition, wherein the cancer is cervical cancer, ovarian cancer, uterine cancer, endometrial cancer, or fallopian tube cancer.

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