Maytansinoid derivative having self-sacrificial peptide linker, and conjugate of the same

Abstract

To provide an antibody-drug conjugate having a linker constituent of a new class.SOLUTION: Provided is a novel cell-binding agent-maytansinoid conjugate having a self-sacrificial peptide linker, more specifically, a conjugate of formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, CB denotes a cell-binding agent; L2 is absent or a spacer; A denotes a peptide containing a single amino acid residue or 2 to 20 amino acid residues; R1 and R2 denote each independently H or C1-3 alkyl; L1 denotes a spacer; D-L1-SH denotes a cytotoxic drug; and q denotes an integer of 1 to 20]SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] As a class of potent antitumor agents that are effective across a broad range of cancers, antibody-drug conjugates (ADCs) have emerged. An ADC generally consists of three distinct elements: a cell-binding agent; a linker; and a cytotoxic drug. The linker component of an ADC is an important element in the development of optimized therapeutic windows, i.e., targeted anticancer drugs with high activity at low non-toxic doses.

[0003] Accordingly, there is a need for ADCs having a new class of linker components.

Summary of the Invention

[0004] The present invention is directed to a cell-binding agent-cytotoxic drug conjugate represented by the following formula:

Chemical Formula

[0005] The present invention also relates to a compound of formula (II):

Chemical formula

[0006] A compound of formula (III):

Chemical formula

[0007] The present invention also relates to a compound of formula (IV): [Chemical formula] or a pharmaceutically acceptable salt thereof [wherein, L 3 is represented by the following formula: [Chemical formula] ; R x’ and R y’ are each independently, for each occurrence, H, -OH, halogen, -O-(C 1~4 alkyl), -SO 3 H, -NR 40 R 41 R 42 + , or C 3 alkyl optionally substituted with -OH, halogen, SO 40 H or NR 41 R 42 + wherein R 1~4 , R 40 , and R 41 and R 42 are each independently H or C 1~4 alkyl; k is an integer from 1 to 10; A is one 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 alkyl; L 1 is a spacer; D-L 1 -SH is a cytotoxic drug; q is an integer from 1 to 20].

[0008] The present invention also relates to a composition (e.g., a pharmaceutical composition) 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 a composition (e.g., a pharmaceutical composition) 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), and further comprising a second therapeutic agent. The composition is useful for inhibiting abnormal cell proliferation or treating a proliferative disorder in a mammal (e.g., a human). The composition is useful for treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), transplant rejection, lupus, myositis, infection, immunodeficiency such as AIDS, and inflammatory diseases in a mammal (e.g., a human).

[0009] The present invention also relates to a method of inhibiting abnormal cell proliferation or treating a proliferative disorder in a mammal (e.g., a human), the method comprising administering to the abnormal cell or the mammal a therapeutically effective amount of a conjugate (e.g., a conjugate of formula (I)) or a 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 DESCRIPTION OF THE DRAWINGS

[0010]

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BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Here, some embodiments of the present invention will be referred to in detail and their examples will be described with the accompanying structures and formulas. While the present invention is described in conjunction with the recited embodiments, it will be understood that the present invention is not intended to be limited to those embodiments. In contrast, the present invention is intended to cover all alternative forms, modifications, and equivalents, which may be included within the scope of the present invention as defined in the claims. Those skilled in the art will recognize numerous methods and materials similar or equivalent to those described herein and can use them in the practice of the present invention.

[0012] Any of the embodiments described herein, including those described in different aspects of the present invention (e.g., compounds, compound-linker molecules, conjugates, compositions, methods of making and using) and different parts of this specification (including embodiments described only in the examples), can be combined with one or more other embodiments of the present invention, unless expressly negated or inappropriate. Combinations of embodiments are not limited to the specific combinations claimed by a plurality of dependent claims.

[0013] Definitions As used herein, the term "treating" or "treatment" includes reversing, alleviating, or arresting the symptoms, clinical signs, and underlying pathology of a condition so as to improve or stabilize the condition of a subject. As used herein, "treatment" is a method for obtaining a beneficial or desired result, including clinical results, as is well understood in the art. Beneficial or desired clinical results include, but are not limited to, alleviation, remission, or deceleration of progression of one or more symptoms or conditions associated with a condition, such as cancer, whether detectable or undetectable, reduction in the size of a disease, stabilization of a disease state (i.e., not worsening), delay or deceleration of disease progression, improvement or remission of a pathological condition, and amelioration (partial or total). "Treatment" can also mean an extension of survival time as 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 subsequent described circumstance may or may not occur, and thus 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 thus its application includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0015] Unless specifically stated as "unsubstituted", references to chemical moieties herein are understood to include substituted variants. For example, references to an "alkyl" group or moiety implicitly include both substituted and unsubstituted variants. Examples of substituents on chemical moieties 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, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aryl or heteroaryl moieties.

[0016] As used herein, the term "cell binding agent" or "CBA" preferably specifically refers to a compound that can bind to a cell (e.g., on a cell surface ligand), or accompany or be in proximity to a ligand that binds to a cell. In certain embodiments, the binding to a cell or a ligand on or near a cell is specific. CBAs can include peptides and non-peptides.

[0017] As used herein, "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon radical. In preferred embodiments, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its main chain (e.g., C 1 ~C 30 for straight-chain and C 3 ~C 30 for branched-chain), more preferably 20 or fewer. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH 2 CH(CH 3 ) 2) 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-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, etc. are included. Further, the term "alkyl" used throughout this specification, the examples, and the claims is intended to include both "unsubstituted alkyl" and "substituted alkyl", where the latter refers to an alkyl moiety having substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. In certain embodiments, the straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its main chain (e.g., straight-chain C 1 ~C 30 , branched-chain C 3 ~C 30 ). In preferred embodiments, the chain has 10 or fewer carbon (C 1 ~C 10 ) atoms in its main chain. In other embodiments, the chain has 6 or fewer carbon (C 1 ~C 6 ) atoms in its main chain. In another embodiment, the chain has 3 or fewer carbon (C 1 ~C 3 ) atoms in its main chain.

[0018] "Alkylene" as used herein refers to a saturated straight-chain or branched-chain divalent hydrocarbon radical. In preferred embodiments, the straight-chain or branched-chain alkylene has 30 or fewer carbon atoms in its main chain (e.g., straight-chain C 1 ~C 30 , branched-chain C 3 ~C 30)、more preferably having 20 or fewer. Further, the term "alkylene" as used throughout this specification, the examples, and the claims is intended to include both "unsubstituted alkylene" and "substituted alkylene", the latter referring to an alkylene moiety having substituents replacing the hydrogen on one or more carbons of the hydrocarbon backbone. In certain embodiments, the straight-chain or branched-chain alkylene has 30 or fewer carbon atoms in its main chain (e.g., straight-chain C 1 ~C 30 , branched-chain C 3 ~C 30 ). In preferred embodiments, the chain has 10 or fewer carbon (C 1 ~C 10 ) atoms in its main chain. In other embodiments, the chain has 6 or fewer carbon (C 1 ~C 6 ) atoms in its main chain. In another embodiment, the chain has 3 or fewer carbon (C 1 ~C 3 ) atoms in its main chain.

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

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

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

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

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

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

[0025] The term "cycloalkylene" refers to a divalent radical of a saturated carbocyclic ring. In preferred embodiments, cycloalkylene has 3 to 10 carbon atoms in their ring structure, more preferably 5 to 7 carbon atoms in the ring structure. In some embodiments, two cyclic rings may share two or more atoms, for example, the rings are "fused rings". Suitable cycloalkylenes include cycloheptylene, cyclohexylene, cyclopentylene, cyclobutylene, and cyclopropylene. In some embodiments, cycloalkylene is a monocyclic group. In some embodiments, cycloalkylene is a bicyclic group. In some embodiments, 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 where 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, etc. The term "aryl" also includes "polysicyclic", "polycyclic", and "polycyclic" ring systems having two or more rings where 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, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl. In some preferred embodiments, the polycycle has 2 to 3 rings. In certain preferred embodiments, the polycyclic ring system has two cyclic rings where both rings are aromatic. Each of the rings of the polycycle may be substituted or unsubstituted. In certain embodiments, each ring of the polycycle 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, and further benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. In some embodiments, aryl is a monocyclic aromatic group. In some embodiments, aryl is a bicyclic aromatic group. In some embodiments, aryl is a tricyclic aromatic group.

[0029] As used herein, "arylene" is the divalent radical of the above aryl group.

[0030] As used herein, the terms "heterocyclic ring", "heterocyclyl", and "heterocyclic ring system" refer to a 3- to 18-membered ring, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, having a ring structure that is a substituted or unsubstituted non-aromatic ring structure containing at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 to 2 heteroatoms. In certain embodiments, the ring structure may have two cyclic rings. In some embodiments, the two cyclic rings may share two or more atoms, e.g., the rings are "fused rings". Examples of heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like. Heterocyclic rings are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly, Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, since 1950), particularly, Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Examples of heterocyclic ring systems include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, 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, dihydrothiophene, 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 the ring atoms are substituted with an oxo (=O) moiety are pyrimidi none and 1,1-dioxo-thiomorpholine.

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

[0032] As used herein, the term "heteroaryl" refers to a substituted or unsubstituted aromatic monocyclic structure 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, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring. When two or more heteroatoms are present in the heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "policyclic", "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., the rings are "fused rings", where at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclyl. In some preferred embodiments, preferred polycycles have 2 to 3 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, in the ring. 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, etc. 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 the above heteroaryl group.

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

[0035] By way of example and not limitation, a nitrogen-linked complex ring or heteroaryl is 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 oxidation states such as NO, SO, and SO 2 and the like.

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

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

[0039] The term "substituted" refers to a moiety having a substituent replacing hydrogen on one or more carbons of the backbone of a compound. It is understood that "substituted" or "substituted with" includes the implicit conditions that such substitution is in accordance with the valences of the atoms being substituted and the substituents, and that the substitution results in a stable compound that does not undergo spontaneous transformation, such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all acceptable substituents of organic compounds. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Acceptable substituents may be one or more and may be the same or different for a suitable organic compound. For the purposes of the present invention, a heteroatom such as nitrogen may have any acceptable substituent of the organic compounds described herein that satisfies the valences of the hydrogen substituents and / or the heteroatom. 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 thiophormate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. By way of illustration, monofluoroalkyl is alkyl substituted with a fluoro substituent, and difluoroalkyl is alkyl substituted with two fluoro substituents. It should be understood that when there is more than one substitution on a substituent, each non-hydrogen substituent may be the same or different (unless otherwise stated). "Optional" or "optionally" means that the subsequently described situation may or may not occur, so that its application includes cases where the situation occurs and cases where the situation 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 means including structures with and without non-hydrogen substituents. When it is described that a carbon of a substituent is optionally substituted with one or more of a list of substituents, one or more (if any) of the hydrogens on that carbon may be replaced separately and / or together with an optionally selected substituent independently selected. When it is described that a nitrogen of a substituent is optionally substituted with one or more of a list of substituents, one or more (if any) of the hydrogens on that nitrogen may each be replaced with an optionally selected substituent independently selected. One exemplary substituent can be represented as -NR’R”, where R’ and R” together with the nitrogen atom to which they are attached may form a heterocyclic ring. The heterocyclic ring formed by R’ and R” together with the nitrogen atom to which they are attached may be partially or fully saturated. In some embodiments, the heterocyclic ring consists of 3 to 7 atoms. In another embodiment, the heterocyclic ring is selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl, and thiazolyl.

[0040] In this specification, the terms "substituent" and "group" are used interchangeably.

[0041] When a group of substituents is collectively described as being optionally substituted with one or more of a list of substituents, the group may include (1) a non-substitutable substituent, (2) a substitutable substituent not substituted with an optionally selected substituent, and / or (3) a substitutable substituent substituted with one or more of the optionally selected substituents.

[0042] When a substituent is described as being optionally substituted with up to a specific number of non-hydrogen substituents, the substituent may be either (1) unsubstituted or (2) substituted by either up to that specific number of non-hydrogen substituents or up to the lesser of the maximum number of substitutable sites on the substituent. Thus, for example, when a substituent is described as a heteroaryl optionally substituted with up to 3 non-hydrogen substituents, a heteroaryl having less than 3 substitutable positions may be optionally substituted with non-hydrogen substituents up to the same number as the heteroaryl has substitutable positions. Such substituents include, by 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 2 , -OR 100 、NR 101 R 102 、-NO 2 、-NR 101 COR 102 、-SR 100 、-SOR 101 represented by sulfoxide, -SO 2 R 101 represented by sulfone, sulfonate -SO 3 M, sulfate -OSO 3 M, -SO 2 NR 101 R 102 represented by sulfonamide, cyano, azide, -COR 101 、-OCOR 101 、-OCONR 101 R 102 and polyethylene glycol units (-CH 2 CH 2 O) n R 101 (wherein M is H or a cation (Na + or K + etc.); R 101 、R 102and R are each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms), polyethylene glycol unit (-CH 2 CH 2 O) n -R 104 (wherein n is an integer from 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 may be selected from H or linear or branched alkyl having 1 to 4 carbon atoms), wherein R 100 , R 101 , R 102 , R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl in the groups represented by are independently selected from one or more (e.g., 2, 3, 4, 5, 6 or more) substituents selected from halogen, -OH, -CN, -NO 2 and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms and are optionally substituted. Preferably, the substituents for the above-mentioned optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclic, aryl, heterocyclyl and heteroaryl include halogen, -CN, -NR 102 R 103 , -CF 3 , -OR 101 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO 2 R 101 and -SO 3 M are included.

[0043] The terms "compound" or "cytotoxic compound" are used interchangeably. These are intended to include compounds whose structure or formula or any derivatives thereof are disclosed in the present invention or are incorporated by reference. This term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, as well as prodrug salts of all compounds of all formulas disclosed in the present invention. This term also includes any solvates, hydrates, and polymorphs of any of the above. Specific references to "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salts", "prodrugs", "prodrug salts", "conjugates", "conjugate salts", "solvates", "hydrates", or "polymorphs" in specific embodiments of the invention described in this application are not to be construed as intended to exclude these forms in other embodiments of the invention where the term "compound" is used without description of these other forms.

[0044] As used herein, the term "conjugate" refers to a derivative thereof linked to a compound or cell-binding agent described herein.

[0045] As used herein, the term "linkable to a cell-binding agent" means that a compound or derivative thereof described herein contains at least one linking group or precursor thereof suitable for binding these compounds or derivatives thereof to a cell-binding agent.

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

[0047] The term "linked to a cell-binding agent" means that a conjugate molecule contains at least one of a compound or derivative thereof described herein that is linked to a cell-binding agent via a suitable linking group or precursor thereof.

[0048] The term "chiral" refers to a molecule having the property that it cannot be superimposed on its mirror partner, and the term "achiral" refers to a molecule that can be superimposed on its mirror partner.

[0049] The term "stereoisomer" refers to a compound having the same chemical composition and connectivity but differing in the arrangement of their atoms in space and not being interconvertible by rotation about single bonds.

[0050] "Diastereomer" refers to stereoisomers that have two or more chirality centers and are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. A mixture of diastereomers can be separated by high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography.

[0051] "Enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0052] The definitions and rules of stereochemistry used in this specification generally follow 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 can therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, and 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. When describing an optically active compound, the prefixes D and L, or R and S, are used to describe the absolute configuration of the molecule with respect to its chiral center(s). The prefixes d and l or (+) and (-) are used to designate the sign of rotation of the plane of plane-polarized light by the compound, and (-) or l means that 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 each other. A particular stereoisomer may be referred to as an enantiomer, 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 that is not optically active.

[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) involve interconversion by the movement of a proton, such as keto-enol and imine-enamine isomerization. Valence tautomers involve interconversion by the rearrangement of some bonding electrons.

[0054] As used herein, the term "prodrug" refers to a precursor or derivative form of a compound of the invention that can be enzymatically or hydrolytically activated or converted 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 Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs of the 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 that can be converted to a more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized to a prodrug form for use in the present invention include, but are not limited to, compounds of the invention as described above and chemotherapeutic agents.

[0055] The term "prodrug" is also meant to include derivatives of compounds that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to afford the compounds of the present invention. Prodrugs may become active only upon such reaction under biological conditions or may be active in their unreacted reactive forms. Examples of prodrugs contemplated in the present invention include, but are not limited to, analogs or derivatives of any one of the compounds of the formulas disclosed herein that contain biodegradable moieties such as biodegradable amides, biodegradable esters, biodegradable carbamates, biodegradable carbonates, biodegradable ureides, and biodegradable phosphate analogs. Other examples of prodrugs include derivatives of any one of the compounds of the formulas disclosed herein that contain -NO, -NO 2 , -ONO, or -ONO 2 moieties. 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 salts" refers to pharmaceutically acceptable organic or inorganic salts of the compounds 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, gentisinate, fumarate, gluconate, glucuronate Salts, saccharinates, formates, benzoates, glutamates, methanesulfonates (''mesylates''), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoates)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts are included. Pharmaceutically acceptable salts may include the intervention of another molecule such as an acetate ion, a succinate ion or other counterions. The counterion may be any organic or inorganic moiety that stabilizes the charge of the parent compound. Further, pharmaceutically acceptable salts may have two or more charged atoms within their structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions can be present. Thus, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counterions.

[0057] When the compound of the invention is a base, the desired pharmaceutically acceptable salt can be prepared by treatment with any suitable method available in the art, e.g., a 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, 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] When the compound of the present invention is an acid, the desired pharmaceutically acceptable salt can 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, an alkali metal hydroxide or an 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" means a compound further comprising 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., which are bound by non-covalent intermolecular forces. Solvates or hydrates of compounds are readily prepared by adding to the compound at least 1 molar equivalent of a hydroxyl solvent such as methanol, ethanol, 1-propanol, 2-propanol or water to effect solvation or hydration of the imine moiety.

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

[0061] The terms "cancer" and "cancerous" refer to or describe a physiological state in mammals that is typically characterized by uncontrolled cell growth. A "tumor" contains one or more 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 compound useful in the treatment of cancer.

[0064] A "metabolite" is a product formed when a designated compound, its derivative, or its conjugate, or a salt thereof, is metabolized in the body. The metabolites of a compound, its derivative, or its conjugate can be identified using routine techniques known in the art, and their activities can be determined using tests such as those described herein. Such products can result, for example, from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes compounds, derivatives thereof, or conjugates thereof, including metabolites of the compounds, derivatives thereof, or conjugates thereof, produced by a process that includes contacting a compound, derivative thereof, or conjugate thereof of the present invention with a mammal for a time sufficient to obtain the metabolite.

[0065] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other components 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 other functional groups on a compound, its derivatives, or its conjugates are being reacted. For example, an "amine protecting group" or "amino protecting moiety" is a substituent that is attached to an amino group and blocks or protects the amino functional group in a compound. Such groups are well known in the art (see, e.g., 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 carbamate, FMOC, substituted ethyl carbamate, carbamates cleaved by 1,6-β-elimination (also referred to as "self-sacrificing"), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For an overview 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 group of 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, halogen, ester, alkoxy, hydroxyl, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, thioether and diazonium compounds.

[0068] The terms "bifunctional crosslinker", "bifunctional linker" or "crosslinker" refer to a modifier having two reactive groups, one of which can react with a cell binding agent and the other of which can react with a cytotoxic compound to link these two moieties together. Such bifunctional crosslinkers are well known in the art (see, for example, Isalm and Dent in Bioconjugation chapter 5, p218-363, Groves Dictionaries Inc., New York, 1999). For example, bifunctional crosslinkers that enable linkage via thioether bonds include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) for introducing maleimide groups, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) for introducing iodoacetyl groups. Other bifunctional crosslinkers for introducing 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, available from Pierce Biotechnology Inc., P.O. Box 117, Rockland, IL 61105, USA), and 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" analog 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-maleimidopropionamide) hexanoate (SMPH), Succinimidyl-(4-vinylsulfonyl) benzoate (SVSB), Dithiobis-maleimide ethane (DTME), 1,4-Bis-maleimidobutane (BMB), 1,4-Bismaleimidyl-2,3-dihydroxybutane (BMDB), Bis-maleimidohexane (BMH), Bis-maleimidoethane (BMOE), Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexanecarboxylate (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) are included.

[0069] A heterobifunctional crosslinker is a bifunctional crosslinker having 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 a cell-binding agent (e.g., an antibody). 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 enable the linkage of a cell-binding agent and a cytotoxic compound via a disulfide bond are known in the art and include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succ inimidyl-4-(2-pyridyldithio)butanoate (SPDB), and N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SPDB). Other bifunctional crosslinkers that can be used to introduce a disulfide group are known in the art and are disclosed in U.S. Pat. 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 a thiol group, such as 2-iminothiolane, homocysteine thiolactone, or S-acetylthioglycolic anhydride, can also 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 another embodiment, the reactive moiety is an aldehyde-reactive group that can form a covalent bond with an aldehyde group located on the CBA. In yet another embodiment, 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 together, such as a cell binding agent and a cytotoxic compound. Typically, the linker is substantially inert under conditions where the two groups being connected are linked. A bifunctional crosslinker can have one reactive group that first reacts with the cytotoxic compound to yield a compound with a linker moiety, and then the second reactive group can react with the cell binding agent, with one reactive group at each end of the linker moiety. Alternatively, one end of the bifunctional crosslinker can first react with the cell binding agent to yield a cell binding agent with a linker moiety, and then the second reactive group can react with the cytotoxic compound. The crosslinking moiety may contain a chemical bond that allows for the 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, photosensitive 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 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 hereby expressly incorporated herein by reference.

[0073] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, selenocysteine, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., compounds having a hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium compounds. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. One amino acid that can be used is citrulline, which is a derivative of arginine and 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 in a manner similar to naturally occurring amino acids. The term "unnatural amino acid" is intended to denote the "D" stereochemical form of the above-mentioned 20 naturally occurring amino acids. Furthermore, it is understood that the term unnatural amino acid includes homologs of natural amino acids or their D-isomers, and synthetic modified forms of natural amino acids. Synthetic modified forms include, but are not limited to, amino acids having side chains shortened or extended by up to two carbon atoms, amino acids containing an optionally substituted aryl group, and amino acids containing a halogenated group, preferably a halogenated alkyl and aryl group, and also N-substituted amino acids, such as N-methyl-alanine. An amino acid or peptide can be bound to a linker / spacer or a cell binder via the terminal amine or terminal carboxylic acid of the amino acid or peptide. An amino acid can also be bound to a linker / spacer or a cell binder via a side chain reactive group such as the thiol group of cysteine, the epsilon amine of lysine or the side chain hydroxyl of serine or threonine. retains the same basic chemical structure as naturally occurring amino acids. One amino acid that can be used is citrulline, which is a derivative of arginine and 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 in a manner similar to naturally occurring amino acids. The term "unnatural amino acid" is intended to denote the "D" stereochemical form of the above-mentioned 20 naturally occurring amino acids. Furthermore, it is understood that the term unnatural amino acid includes homologs of natural amino acids or their D-isomers, and synthetic modified forms of natural amino acids. Synthetic modified forms include, but are not limited to, amino acids having side chains shortened or extended by up to two carbon atoms, amino acids containing an optionally substituted aryl group, and amino acids containing a halogenated group, preferably a halogenated alkyl and aryl group, and also N-substituted amino acids, such as N-methyl-alanine. An amino acid or peptide can be bound to a linker / spacer or a cell binder via the terminal amine or terminal carboxylic acid of the amino acid or peptide. An amino acid can also be bound to a linker / spacer or a cell binder via a side chain reactive group such as the thiol group of cysteine, the epsilon amine of lysine or the side chain hydroxyl of serine or threonine.

[0074] In some embodiments, the amino acid is NH 2 -C(R aa’ R aa )-C(=O)OH (wherein R aa and R aa’ are each independently H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl, aryl, heteroaryl or heterocyclyl having 1 to 10 carbon atoms, or R aa and the nitrogen atom at the N-terminus may together form a heterocyclic ring (e.g., as in proline). The term "amino acid residue" refers to a corresponding residue in which one hydrogen atom has been removed from the amine and / or the hydroxyl group has been removed from the carboxy terminus of the amino acid, such as -NH-C(R aa’ R aa )-C(=O)O-.

[0075] As used herein, the amino acid may be the L or D isomer. Unless otherwise specified, when an amino acid is referred to, it may be the 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 the L or D isomer unless otherwise specified. If one of the amino acids in the peptide is designated as the D isomer, the other amino acid(s) are the L isomer unless otherwise specified. For example, the peptide D-Ala-Ala means D-Ala-L-Ala.

[0076] Amino acids and peptides may be protected by a blocking group. A blocking group is an atom or chemical moiety that protects the N-terminus of an amino acid or peptide from unwanted reactions and can be used during the synthesis of a drug-ligand conjugate. This 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 completion of the synthesis of the drug conjugate. 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 ester, tert-butyl ester, 9-fluorenylmethyl carbamate (Fmoc), and carbobenzoxy (Cbz).

[0077] The term "peptide cleavable by a protease" refers to a peptide containing a cleavage recognition sequence for a protease. As used herein, a protease is an enzyme capable of cleaving a peptide bond. A cleavage recognition sequence for a protease is a specific amino acid sequence that is recognized by a protease during proteolytic cleavage. Many protease cleavage sites are known in the art and these, and other cleavage sites, may be included in the linker portion. For example, Matayoshi et al. Science 247:954 (1990); Dunn et al. Meth. Enzymol. 241:254 (1994); Seidah et al. Meth. Enzymol. 244:175 (199 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 IN DEVELOPMENT, AGING, AND ALZHEIMER’S DISEASE, ed. Masters et al. pp. 190-198 (1994). See also.

[0078] The peptide sequences are selected based on their ability to be cleaved by proteases, non-limiting examples of which include cathepsin B, C, D, H, L, and S, as well as furin. Preferably, the peptide sequences 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 sequences are selected based on their ability to be cleaved by lysosomal proteases. Lysosomal proteases are proteases mainly located in lysosomes, but can also be located in endosomes. Examples of lysosomal proteases include, but are not limited to, cathepsin B, C, D, H, L, and S, as well as furin.

[0080] In another embodiment, the peptide sequences are selected based on their 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 such proteases 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 the enzymes described in the following references (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 having a positive charge. Cations can be monovalent (e.g., Na + , K + , etc.), divalent (e.g., Ca 2+ , Mg2+ etc.) or polyvalent (e.g., Al 3+ etc.). In some embodiments, the cation is monovalent.

[0082] The term "therapeutically effective amount" means the amount of an active compound or conjugate that elicits a desired biological response in a subject. Such responses include alleviation of symptoms of a disease or disorder being treated, prevention, inhibition or delay of recurrence of symptoms of the disease or the disease itself, increase in the lifespan of the subject compared to not being treated, or prevention, inhibition or delay of progression of symptoms of the disease or the disease itself. Determination of an effective amount is well within the ability of one of ordinary skill in the art, particularly in view 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 the compounds or conjugates or other therapeutic agents of the invention to be administered to a subject depends on the stage, category and condition of multiple myeloma, as well as characteristics of the subject such as general health, age, sex, weight and drug tolerance. The effective amount of the compounds or conjugates or other therapeutic agents of the invention to be administered also depends on the route of administration and dosage form. Dosage amount and interval may be adjusted individually to obtain plasma levels of the active compound sufficient to achieve the desired therapeutic effect.

[0083] Cell-binding agent-cytotoxic drug conjugate In a first aspect, the 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 formula:

Chemical formula

[0085] In one embodiment, L 1 is -L 1 ’-C(=O)-; L 1 ’ is alkylene, alkenylene, alkynylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, where the -C(=O)- group in L 1 is connected to D.

[0086] In another embodiment, at least one of R 1 and R 2 is H. In a more specific embodiment, one of R 1 and R 2 is H and the other is Me.

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

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

[0089] In the third specific embodiment, for the conjugate of formula (I), L 1 is -CR 3 R 4 -(CH 2 ) 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 the first specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me.

[0090] In the fourth specific embodiment, for the conjugate of formula (I), L 1 is -CR 3 R 4 -(CH 2 ) 2~5 -C(=O)- or -CR 3 R 4 -(CH 2 ) 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 the first specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0091] In the fifth specific embodiment, for the conjugate of formula (I), L 1 is -(CH 2 ) 4~6 -C(=O)-; the remaining variables are as described above in the first embodiment or the first specific embodiment.

[0092] In the sixth specific embodiment, for the conjugate of formula (I), L 2 is the following structural formula:

Chemical formula

Chemical formula

Chemical formula

[0093] In a more specific embodiment of the sixth specific embodiment, R A is alkylene, cycloalkylalkylene, or arylene; W is absent or

Chemical formula

Chemical formula

[0094] In a more specific embodiment, p is 0 and R C is absent; the remaining variables are as described above in the sixth specific embodiment.

[0095] In another more specific embodiment, J CB is -C(=O)- or

Chemical formula

[0096] In the seventh embodiment, for the conjugate in formula (I), L 2 has the following structural formula: [Chemical formula] represented by [wherein, R x 、R y 、R x’ and R y’ are, for each occurrence independently, H, -OH, halogen, -O-(C 1~4 alkyl), -SO 3 H, -NR 40 R 41 R 42 + 、or -OH, halogen, -SO 3 H, -NR 40 R 41 R 42 + optionally substituted C 1~4 alkyl, where R 40 、R 41 and R 42 are each independently H or C 1~4 alkyl; l and k are each independently an integer from 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 embodiments.

[0097] In a more specific embodiment, 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; the remaining variables are as described above in the seventh specific embodiment.

[0100] In another more specific embodiment, L 2 has the following structural formula:

Chemical formula

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

[0102] In the 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 the ninth specific embodiment, for the conjugate in formula (I), 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 as the L or D isomer, -NH-CR 1 R 2 -S-L 1A peptide having an amino acid covalently bonded to -D; 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, -NH-CR 1 R 2 -S-L 1 The amino acid connected to -D is an L-amino acid.

[0104] In the tenth specific embodiment, for the conjugate in formula (I), 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, Al a-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 L 2 group-connected, and the last amino acid in each peptide is -NH-CR 1 R 2 -S-L 1-D is connected; 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 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 the eleventh specific embodiment, for the conjugate in formula (I), D is a maytansinoid; 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 the following formula:

Chemical formula

[0106] In a more specific embodiment, D is the following formula:

Chemical formula

[0107] In the twelfth specific embodiment, the conjugate of the present invention is the following formula:

Chemical formula

Chemical formula

Chem.

Chem.

[0108] In a more specific embodiment, 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 a more specific embodiment, D 1 is the following formula:

Chem.

[0110] 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 (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 12th specific embodiment.

[0111] In another more specific embodiment, m1, r1, n1, p1, 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.

[0112] In a more specific embodiment, m2, n2, p2, r2, and t2 are each independently an integer from 2 to 10. In a more specific embodiment, m2, n2, p2, r2, and t2 are each independently an integer from 3 to 6. In a more specific embodiment, 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 from 2 to 10, and m2, n2, p2, r2, and t2 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, and m2, n2, p2, r2, and t2 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, and m2, n2, p2, r2, and t2 are each independently an integer from 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 a more specific embodiment, r2 and r1 are each independently an integer from 2 to 6. In a more specific embodiment, r2 is an integer from 3 to 5 and r1 is an integer from 2 to 6. In a more specific embodiment, r2 is an integer from 3 to 5 and r1 is an integer from 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.

[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 formula:

Chemical formula

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

[0119] In a 13th specific embodiment, the conjugate of the present invention has the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0120] In a more specific embodiment, D 1 is represented by the formula:

Chemical formula

[0121] Also, in a 13th specific embodiment, the conjugate of the present invention has the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

[0122] In a more specific embodiment, D 1 is of the formula:

Chem.

[0123] In another more specific embodiment, the conjugate is of the formula:

Chem.

Chem.

Chem.

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

[0125] In some embodiments, for the conjugate of the present invention described above (e.g., the conjugate described in the first embodiment or the specific embodiments of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth), the cell binding agent (CBA) binds to target cells selected from tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; cells expressing CA6, CAK1, CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, 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 cells expressing insulin growth factor receptor, epidermal growth factor receptor, nectin-4, mesothelin, GD3, prolactin receptor, and folate receptor.

[0126] In some embodiments, for the conjugate of the present invention described above (e.g., the conjugate described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiment), the cell binding agent is an antibody or an 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 pentarin.

[0127] In some embodiments, for the conjugate of the present invention described above (e.g., the conjugate described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiment), 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 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 present invention has the following formula:

Chemical formula

[0129] In another embodiment, the conjugate of the present invention is represented by the following formula:

Chemical formula

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

[0131] In some embodiments, for a composition (e.g., a pharmaceutical composition) comprising the conjugate of the present invention described above (e.g., the conjugate described in the first embodiment or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth specific embodiment), the average number of cytotoxic drugs per cell binding agent (CBA, e.g., an antibody), also known as the drug - antibody ratio (DAR) in the composition, is in the range of 1.0 to 8.0. In some embodiments, the DAR is in 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] The compound of the present invention In a second aspect, the present invention provides a maytansinoid derivative as described herein.

[0133] In a second embodiment, the compound of the present invention has the formula (II), (III) or (IV): L 2 ’-A-NH-CR 1 R 2 -S-L 1 -D (II), A’-NH-CR 1 R 2 -S-L 1 -D (III), or L 3 -A-NH-CR 1 R 2 -S-L1 -D(IV) or represented by a pharmaceutically acceptable salt thereof [wherein, L 2 ’ is either absent or a spacer having a reactive moiety capable of forming a covalent bond with a cell-binding agent; A is an amino acid residue or a peptide containing 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); L 1 is a spacer; D-L 1 -SH is a cytotoxic drug; q is an integer from 1 to 20; A’ is an amino acid residue or a peptide containing 2 to 20 amino acid residues; L 3 is of the formula:

Chemical formula

[0134] In one embodiment, L 1 is -L 1 ’-C(=O)-; and L 1 ’ is alkylene, alkenylene, alkynylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene, where the -C(=O)- group in L 1 is connected to D.

[0135] In another embodiment, at least one of R 1 and R 2 is H. In a more specific embodiment, one of R 1 and R 2 is H and the other is Me.

[0136] In the 14th specific embodiment, for the compounds of formulas (II), (III), and (IV), R 1 and R 2 are each independently H or Me; and the remaining variables are as described above in the second embodiment. In a more specific embodiment, both R 1 and R 2 are H.

[0137] In the 15th specific embodiment, for the compounds of formulas (II), (III), and (IV), L 1 is -L 1 ’-C(=O)-; L 1 ’ is alkylene or cycloalkylene, where the -C(=O)- group in L 1 is connected to D; and the remaining variables are as described above in the second embodiment or the 14th specific embodiment. In a more specific embodiment, L 1 ’ is C 1~10 alkylene. In another more specific embodiment, L 1 ’ is C 1~20 alkylene.

[0138] In the 16th specific embodiment, for the compounds of formula (II), (III) and (IV), L 1 is -CR 3 R 4 -(CH 2 ) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me; the remaining variables are as described above in the 2nd embodiment or the 14th specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me.

[0139] In the 17th specific embodiment, for the compounds of formula (II), (III) and (IV), L 1 is -CR 3 R 4 -(CH 2 ) 2~5 -C(=O)- or -CR 3 R 4 -(CH 2 ) 3~5 -C(=O)-; R 3 and R 4 are each independently H or Me; the remaining variables are as described above in the 2nd embodiment or the 14th specific embodiment. In a more specific embodiment, R 3 and R 4 are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0140] In the 18th specific embodiment, for the compounds of formula (II), (III) and (IV), L 1 is -(CH 2 ) 4~6 -C(=O)-; the remaining variables are as described above in the 2nd embodiment or the 14th specific embodiment.

[0141] In the 19th specific embodiment, for the compounds of formulas (II), (III) and (IV), A or A' is a peptide cleavable by a protease; the remaining variables are as described above in the 2nd 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 the 20th specific embodiment, for the compounds of formulas (II), (III) and (IV), A or A' is independently a peptide having an amino acid covalently bonded to -NH-CR 1 R 2 -S-L 1 -D, 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 as L or D isomers; the remaining variables are as described above in the 2nd embodiment or the 14th, 15th, 16th, 17th or 18th specific embodiments. In a more specific embodiment, the amino acid connected to -NH-CR 1 R 2 -S-L 1 -D is an L-amino acid.

[0143] In the 21st specific embodiment, for the compounds of formulas (II), (III) and (IV), A or 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, 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 L 2 group and is connected, and the last amino acid in each peptide is -NH-CR 1 R 2 -S-L 1 -D and is connected; 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 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] For A described herein, when a specific amino acid or peptide sequence is mentioned, this means that a hydrogen atom is L 2Removed from the amino terminus of the amino acid connected to the base and having a hydroxyl group of -NH-CR 1 R 2 -S-L 1 -D means an amino acid residue or a peptide containing an amino acid residue removed from the carboxy terminus of the amino acid connected thereto. For example, when A is represented by Ala-Ala-Ala, this refers to -NH-CH(CH 3 )-C(=O)-NH-CH(CH 3 )-C(=O)-NH-CH(CH 3 )-C(=O)-.

[0145] Regarding A' described herein, when a specific amino acid or peptide sequence is mentioned, this means an amino acid residue or a peptide containing an amino acid residue removed from the carboxy terminus of the amino acid having a hydroxyl group of -NH-CR 1 R 2 -S-L 1 -D connected thereto. For example, when A' is represented by Ala-Ala-Ala, this refers to NH 2 -CH(CH 3 )-C(=O)-NH-CH(CH 3 )-C(=O)-NH-CH(CH 3 )-C(=O)-.

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

Chemical formula

[0147] In another more specific embodiment, D is of the following formula:

Chemical formula

[0148] In the 23rd specific embodiment, for the compound of formula (II), L 2 ’ is the following structural formula:

Chemical formula

Chemical formula

Chemical formula

[0149] In a more specific embodiment, for the compound of the 23rd specific embodiment, R A is alkylene, cycloalkylalkylene, or arylene; W is absent or [Chemical formula] and J CB ’ is -C(=O)OH, -COE, [Chemical formula] and the remaining variables are as described above in the 23rd specific embodiment.

[0150] In a more specific embodiment, p is 0 and R c is absent; the remaining variables are as described above in the 23rd specific embodiment.

[0151] In other more specific embodiments, J CB ’ is -C(=O)OH, -COE or [Chemical formula] and the remaining variables are as described above in the 23rd specific embodiment.

[0152] In the 24th embodiment, for the compound of formula (II), L 2 ’ is the following structural formula: [Chemical formula] [Chemical formula] is represented by [wherein, R x , R y , R x’ and R y’ are each independently, for each occurrence, H, -OH, halogen, -O-(C 1~4 alkyl), -SO 3 H, -NR 40 R 41 R 42 + , or -OH, halogen, -SO 3 H, NR 40 R 41 R 42 + optionally substituted C 1~4 alkyl, where R 40 , R 41 and R 42 are each independently H or C 1~4 alkyl; l and k are each independently an integer from 1 to 10; J CB is -C(=O)OH or -COE; the remaining variables are as described above in the 2nd or 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd or 23rd specific embodiment.

[0153] In a more specific embodiment, R x , R y , R x’ and R y’All are H; the remaining variables are as described in the 24th 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 in the 24th specific embodiment.

[0155] 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; the remaining variables are as described in the 24th specific embodiment.

[0156] In another more specific embodiment, L 2 ’ is the following structural formula:

Chemical formula

[0157] In an even more specific embodiment, l and l1 are each an integer from 2 to 6.

[0158] In the 25th specific embodiment, for the compound of formula (IV), R x’ and R y’ are both H; the remaining variables are as described in the 2nd embodiment or the 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st or 22nd specific embodiment.

[0159] In a more specific embodiment, k is an integer from 2 to 6; the remaining variables are as described in the 25th specific embodiment.

[0160] In another more specific embodiment, k is 3; the remaining variables are as described above in the 25th specific embodiment.

[0161] In the 26th specific embodiment, the compound of formula (II) is of the formula:

Chemical formula

Chemical formula

[0162] 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.

[0163] In a more specific embodiment, 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 a more specific embodiment, m2, n2, p2, r2 and t2 are each independently an integer from 2 to 10. In a more specific embodiment, m2, n2, p2, r2 and t2 are each independently an integer from 3 to 6. In a more specific embodiment, 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 from 2 to 10, and m2, n2, p2, r2 and t2 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, and m2, n2, p2, r2 and t2 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, and m2, n2, p2, r2 and t2 are each independently an integer from 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 from 2 to 6. In a more specific embodiment, r2 is an integer from 3 to 5 and r1 is an integer from 2 to 6. In a more specific embodiment, r2 is an integer from 3 to 5 and r1 is an integer from 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 formula: [Chemical formula] represented by [wherein, r1 and t1 are each an integer from 2 to 10; r2 and t2 are each an integer from 2 to 19; t3 is an integer from 2 to 12 (for example, t3 is 2, 4, 6, 8, 10 or 12).

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

[0172] In a 27th specific embodiment, the compound of formula (II) has the formula: [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] or a pharmaceutically acceptable salt thereof, represented by [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; D 1 is of the formula:

Chemical formula

[0173] In an even more specific embodiment, D 1 is of the formula:

Chemical formula

[0174] In the 27th specific embodiment as well, the compound of formula (II) is of the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0175] In a more specific embodiment, D 1 is the following formula:

Chemical formula

[0176] In another more specific embodiment, the compound is the following formula:

Chemical formula

Chemical formula

[0177] In some embodiments, -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. More specifically, -COE is N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester.

[0178] In the 28th specific embodiment, the compound of formula (III) has the following formula:

Chemical formula

Chemical formula

[0179] In a more specific embodiment, D 1 has the following formula:

Chemical formula

[0180] In a more specific embodiment, A is Ala-Ala-Ala, Ala-D-Ala-A la, 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 a 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 the 29th specific embodiment, the compound of formula (III) is of the following formula:

Chemical formula

Chemical formula

[0184] In a more specific embodiment, D 1 is the following formula:

Chemical formula

[0185] Also, in the 29th specific embodiment, the compound of formula (III) is the following formula:

Chemical formula

Chemical formula

[0186] In a more specific embodiment, D 1 is the following formula:

Chemical formula

[0187] In the 30th specific embodiment, the compound of formula (IV) is the following formula:

Chemical formula

Chemical formula

[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 the 31st specific embodiment, the compound of formula (IV) has the following formula:

Chemical formula

Chemical formula

[0191] In a more specific embodiment, D 1 has the following formula:

Chemical formula

[0192] Also, in the 31st specific embodiment, the compound of formula (IV) has the following formula:

Chemical formula

Chem.

[0193] In a more specific embodiment, D 1 is of the formula:

Chem.

[0194] Metabolite In certain embodiments, the conjugate of the invention is cleaved at the peptide moiety represented by variable A, followed by release of a free cytotoxic drug (e.g., maytansinoid) via self-sacrifice of the -NH-CR 1 R 2 -S- moiety to have a thiol group that may be further methylated.

Chem.

[0195] Thus, in a third embodiment, the invention is directed to a compound of formula (V): D-L 1 -SZ 0 (V) directed to [wherein, L 1 is a spacer; Z0 is H or Me, provided that Z 0 is H, L 1 is -C(=O)-(CH 2 ) q - or -C(=O)-CH 2 -CH 2 -C(CH3) 2 - instead, where q is an integer from 1 to 3; Z 0 is Me, L 1 is -C(=O)-(CH 2 ) 2 - or -C(=O)-CH 2 -CH 2 -C(CH3) 2 - instead; D-L 1 -SH is a cytotoxic drug].

[0196] In the 32nd specific embodiment, L 1 is -L 1 ’-C(=O)-; L 1 ’ is alkylene or cycloalkylene. More specifically, L 1 ’ is C 1~10 alkyl ylene. In another more specific embodiment, L 1 ’ is C 1~20 alkylene.

[0197] In the 33rd specific embodiment, L 1 is -CR 3 R 4 -(CH 2 ) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me.

[0198] In the 34th specific embodiment, L 1 is -CR 3 R 4 -(CH 2 ) 2~5 -C(=O)- or -CR 3 R 4 -(CH 2 )3~5 -C(=O)-. In a more specific embodiment, R 3 and R 4 are both Me. In another more specific embodiment, R 3 and R 4 are both H.

[0199] In the 35th specific embodiment, L 1 is -(CH 2 ) 2~10 -C(=O)-. In a more specific embodiment, L 1 is -(CH 2 ) 4~6 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 5 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 6 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 7 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 8 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 10 -C(=O)-. In another more specific embodiment, L 1 is -(CH 2 ) 15 -C(=O)-.

[0200] In the 36th specific embodiment, for the compound of formula (V), D is the following formula:

Chemical formula

[0201] More specifically, D is represented by the following formula [Chemical formula] as represented by

[0202] In the 37th specific embodiment, the compound of formula (V) is represented by the following formula: [Chemical formula] [Chemical formula] as represented by

[0203] Cell binding agent The effectiveness of the conjugate of the present invention as a therapeutic agent depends on the careful selection of an appropriate cell binding agent. The cell binding agent may be of any type known or to be discovered, including peptides and non-peptides. Generally, these may be antibodies (polyclonal and monoclonal antibodies, especially monoclonal antibodies, etc.), lymphokines, hormones, growth factors, vitamins (such as folic acid that can bind to its cell surface receptor, e.g., 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 specific cell population targeted. However, in many (but not all) cases, it is advisable to select a human monoclonal antibody if one is available. For example, the monoclonal antibody MY9 is a mouse IgG 1 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 can express CD33, as in the case of the disease acute myeloid leukemia (AML).

[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 to the STRA6 receptor with high affinity, increasing the uptake of vitamin A. In another example, folic acid / folic acid compound / vitamin B 9 binds with high affinity to the cell surface folic acid receptor (FR), e.g., FRα. Folic acid or an antibody that binds to FRα can be used to target folic acid receptors expressed on ovaries 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 comprising a protein or polypeptide, including an antibody, a non-antibody protein, or a polypeptide. Preferably, the protein or polypeptide comprises one or more Lys residues having a side chain -NH 2 group. The Lys side chain -NH 2 group can covalently bind to a bifunctional cross-linking linker, which in turn binds to the dimer compound of the present invention to conjugate the cell binding agent to the dimer compound of the present invention. Each protein-based cell binding agent may contain multiple Lys side chain -NH 2 groups available for linking the compound of the present invention via a bifunctional cross-linking linker.

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

[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 antibody mimetic such as an ankyrin repeat protein, centyrin, or adnectin / monobody.

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

[0211] In certain embodiments, the cell-binding agent is a humanized antibody, humanized single-chain antibody, or humanized anti- It is a body fragment (or "antigen-binding portion"). In a specific embodiment, the humanized antibody is huMy9-6 or another related antibody, which are described in U.S. Patent Nos. 7,342,110 and 7,557,189. In another specific embodiment, the humanized antibody is the anti-folate receptor antibody described in WO2011 / 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 these applications are hereby incorporated 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, an avibody, a diabody, a tribody, a tetrabody, a nanobody, a probody, a domain antibody, or a 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; see O’Keefe et al. (1985) J. Biol. Chem. 260:932-937, which is incorporated herein by reference), centyrin (a scaffold 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, which are incorporated herein by reference), ankyrin repeat proteins (e.g., engineered ankyrin repeat proteins known as DARPins; see U.S. Patent Application Publication Nos. 2004 / 0132028, 2009 / 0082274, 2011 / 0118146, and 2011 / 0224100, which are 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, H.K., Amstutz, P. & Pluckthun, A., see also Nature Biotechnology (2005) 23:1257-1268 (incorporated herein by reference), ankyrin-like repeat proteins or synthetic peptides (e.g., see US Patent Application Publication No. 2007 / 0238667, US Patent No. 7,101,675, WO2007 / 147213, and WO2007 / 062466 (incorporated herein by reference)), adnectin (a fibronectin domain scaffold protein; see US Patent Application Publication Nos. 2007 / 0082365 and 2008 / 0139791 (incorporated herein by reference)), avibodies (including diabodies, tribodies, and tetrabodies; see US Application Publication Nos. 2008 / 0152586 and 2012 / 0171115), dual-receptor retargeted (DART) molecules (P.A. 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-penetrating supercharged proteins (Metho. ds in Enzymol. 502,293-319 (2012), and other cell-binding molecules or substances may be included.

[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 fragment that binds to a growth factor or a growth factor receptor; or a fragment that binds to a cytokine or 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 an antigen-binding portion thereof (including antibody derivatives), or a specific antibody mimetic, the CBA can bind to a ligand on a target cell, such as a cell surface ligand including a cell surface receptor.

[0217] Specific exemplary antigens or ligands include renin; growth hormone (e.g., human growth hormone and bovine growth hormone); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors (e.g., vmc factor, factor IX, tissue factor, and von Willebrand factor); anticoagulation factors (e.g., protein C); atrial natriuretic factor; pulmonary surfactant; plasminogen activator (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., a substance that regulates the activation of normal T cell expression and secretion); human macrophage inflammatory protein-1-alpha; serum albumin (human serum albumin); Müllerian duct inhibitory factor; relaxin A chain; relaxin B chain; prolactin; mouse gonadotropin-related peptide; microbial proteins (beta-lactamase); DNA degrading enzymes; IgE; cytotoxic T lymphocyte-associated antigen (e.g., CTLA-4); inhibin; activin; vascular endothelial cell growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors (e.g., bone-derived neurotrophic factor, neurotrophin-3, -4, -5, or -6) or nerve growth factor (e.g., NGF-beta); platelet-derived growth factor; fibroblast growth factor (e.g., aFGF and bFGF); fibroblast growth factor receptor 2; epidermal growth factor; transforming growth factor (e.g., TGF-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, and TGF-beta5), insulin-like growth factor I and II, des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins, melanotransferrin; CA6, CAK1, CAEEA, CAECAM5, GD3; FET3; PSMA; PSCA; MUC1; STEAP; CEA; TENB2; EphA receptor; EphB receptor; folate receptor; FOLR1; mesothelin; crypt; alpha vbeta 6 ; Integrin; 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, CD134, 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 hereby incorporated by reference in their entirety); erythropoietin; osteogenic factor; immunotoxin; bone morphogenetic protein; interferon (e.g., interferon-alpha, -beta, and gamma); colony-stimulating factor (e.g., M-CSF, GM-CSF, and G-CSF); interleukin (e.g., IL-1 to IL-10); superoxide Sodismutase; T cell receptor; surface membrane protein; decay-accelerating factor; viral antigen (e.g., a part of the envelope of HIV); transport protein; homing receptor; addressin; regulatory protein; integrin (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 may be included.

[0218] As used herein, the term "antibody" includes immunoglobulin (Ig) molecules. In certain embodiments, an antibody is a full-length antibody that includes four polypeptide chains interconnected by disulfide bonds, namely, two heavy chains (HC) and two light chains (LC). 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, and the light chain constant region consists of one domain CL. The VH region and the VL region can be further subdivided into hypervariable regions called complementarity determining regions (CDR). Such regions are interspersed with more conserved framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, and they are arranged in the following order from the amino terminus to the carboxy terminus: 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 IgA1 or IgA2.

[0220] In certain embodiments, the cell binding agent is the "antigen-binding portion" of a monoclonal antibody, which portion shares with the antibody the sequences essential for antigen binding (such as huMy9-6 or related antibodies described in U.S. Patent Nos. 7,342,110 and 7,557,189; huMovl9 or related antibodies 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 (which, in some cases, is also interchangeably referred to as "antibody fragment" synonymously) 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 specific fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include the following: (i) Fab fragment, i.e., a monovalent fragment consisting of VL, VH, CL, and CH1 domains (e.g., when an antibody is digested with papain, three fragments are obtained, i.e., two antigen-binding Fab fragments and one Fc fragment that does not bind to the antigen); (ii) F(ab’) 2 fragment, i.e., a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region (e.g., when an antibody is digested with pepsin, two fragments are obtained, i.e., a divalent antigen-binding F(ab’) 2 fragment and a pFc’ fragment that does not bind to the antigen) and related F(ab’) monovalent units; (iii) Fd fragment consisting of VH domain and CH1 domain (i.e., the heavy-chain portion contained in Fab); (iv) Fv fragment consisting of VL domain and VH domain of a single arm of an antibody and related disulfide-linked Fv; (v) dAb (domain antibody) or sdAb (single-domain antibody) fragment (Ward et al., Nature 341:544-546, 1989) (this fragment consists of a VH domain), and (vi) isolated complementarity-determining regions (CDRs) (including, but not limited to, these). In certain embodiments, the antigen-binding portion is an sdAb (single-domain antibody).

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

[0223] For example, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined into one by a synthetic linker that allows them to be made into a single protein chain using standard recombinant methods, in which the VL region and the VH region can pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. Science 242:423-426, 1988, and Huston et al., Proc. Nat1. Acad. Sci. USA 85:5879-5883, 1988).

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

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

[0226] In certain embodiments, the scFv may be dimerized to form a diabody by linking via a linker peptide that is too short (about five amino acids) for the two variable regions to fold together (see, for example, Holliger et al., Proc. Nat1. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994). The diabody may be bispecific or monospecific. The diabody has been shown to have a dissociation constant up to 40-fold lower than the corresponding scFv, i.e., a higher affinity for the target.

[0227] Even shorter linkers (one or two amino acids) result in the formation of trimers, so-called tribodies or triabodies. Tetrabodies are also similarly produced. These exhibit even higher affinity for the target than diabodies. Diabodies, tribodies, and tetrabodies are collectively sometimes referred to as "AVIBODY (trademark)" cell-binding agents (or simply "AVIBODY"). That is, an AVIBODY having two, three, or four target-binding regions (TBRs) is generally known as a diabody, tribody, and tetrabody. For example, for details, see U.S. Patent Application Publication Nos. 2008 / 0152586 and 2012 / 0171115. The entire teachings of these are incorporated herein by reference.

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

[0229] In certain embodiments, each scFv of the tandem scFv or diabody / tribody / tetrab ody may have the same or different binding properties and may 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 the human IgG Fc portion to have IgG-like properties, yet they remain encoded by a single gene. By transiently producing such scFv-Fc proteins in mammals, milligram amounts can be easily obtained, so this derivative antibody form is particularly suitable for many research applications.

[0231] Fcab is an antibody fragment engineered from the Fc constant region of an antibody. Fcab can be expressed as a soluble protein or can be engineered back into a full-length antibody such as IgG to generate mAb2. mAb2 is a full-length antibody that has Fcab instead of the normal Fc region. Using these additional binding sites, mAb2 bispecific monoclonal antibodies can bind to two different targets simultaneously.

[0232] In certain embodiments, engineered antibody derivatives are generated by removing domains that are not thought to be essential for function, resulting in a reduction in the size of the antigen-binding Ig-derived recombinant protein ( "miniaturization" of the full-length mAb). One of the best examples of this is SMIP.

[0233] Small modular immunopharmaceuticals, or SMIPs, are artificial proteins mainly constructed from the antibody portion (immunoglobulin) and are intended for use as pharmaceuticals. SMIPs have a biological half-life similar to that of antibodies but are smaller than antibodies and thus may have better tissue penetration properties. SMIPs are single-chain proteins containing one binding region, one hinge region as a linker, and one effector domain. The binding region contains a modified single-chain variable fragment (scFv), and the rest of the protein can be constructed from the Fc of an antibody (such as CH2 and CH3 as the effector domain) and a hinge region, for example, from IgG1. Genetically modified cells produce an SMIP that is approximately 30% smaller than the actual antibody as an antibody-like dimer.

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

[0235] Single domain antibodies (sdAbs, including but not limited to those called nanobodies by Ablynx) are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, single domain antibodies can bind selectively to specific antigens, but are even smaller because of their molecular weight of only 12-15 kDa. In certain embodiments, single domain antibodies are engineered from heavy chain antibodies (hcIgGs). The first such sdAb was engineered based on hcIgG found in camels and is called the V H H fragment. In certain embodiments, single domain antibodies are engineered using the V NAR fragment from IgNAR (see below, "Immunoglobulin New Antigen Receptor"). Cartilaginous fish (such as sharks) have such heavy chain IgNAR antibodies. In certain embodiments, sdAbs are engineered by splitting the dimeric variable domains of common immunoglobulin G (IgG), such as those from humans or mice, into monomers. In certain embodiments, nanobodies are derived from the heavy chain variable domain. In certain embodiments, nanobodies are derived from the light chain variable domain. In certain embodiments, sdAbs are obtained by screening a library of single domain heavy chain sequences (e.g., human single domain HC) for binders to a target antigen.

[0236] Single variable new antigen receptor domain antibody fragments (V NAR , or V NAR domains) are derived from cartilaginous fish (e.g., shark) immunoglobulin new antigen receptor antibodies (IgNAR). Such single domain proteins exhibit desirable size and potential epitope recognition properties because they are among the smallest of known immunoglobulin framework proteins. Mature IgNAR antibodies consist of one variable new antigen receptor (V NAR ) domain and five constant new antigen receptor (C NAR) It consists of a homodimer of the domain. This molecule has high stability and efficient binding properties. Its inherent stability is likely due to both (i) the Ig scaffold providing a significant number of charged hydrophilic surface-exposed residues compared to the conventional antibody VH and VL domains found in mouse antibodies, and (ii) the structural stabilization properties in the complementarity-determining region (CDR) loops, including the pattern of inter-loop disulfide bridges and intra-loop hydrogen bonds.

[0237] A minibody is an engineered antibody fragment that includes an scFv linked to a CH domain, such as CH3γ1 (the CH3 domain of IgG1) or CH4ε (the CH4 domain of IgE). For example, an scFv specific for carcinoembryonic antigen (CEA) is linked to CH3γ1 to generate a minibody, which has already been demonstrated to have excellent tumor-targeting ability in addition to rapid clearance in vivo (Hu et al., Cancer Res. 56:3055 - 3061, 1996). The scFv may have an N-terminal VH or VL. The linkage may be a short peptide (e.g., a linker of two amino acids such as ValGlu) that results in a non-covalent hinge-less minibody. Alternatively, the linkage may be an IgG1 hinge and a GlySer linker peptide that generates 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 having two or more different antigen-binding domains, with each antigen-binding domain having a different target specificity. Bispecific antibodies can be generated by fusing two antibody-producing cells having different specificities. These "quadromas" produce multiple molecular species because the two distinct light chains and two distinct heavy chains freely recombine in various arrangements within the quadroma. Since then, bispecific Fabs, scFvs, and full-size mAbs have been produced using various techniques (see above).

[0239] Bispecific 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, the DVD-Ig protein is unique in that each arm of the molecule contains two variable domains (VDs). The VDs within the arm are tandemly linked and can have different binding specificities.

[0240] Trispecific antibody derivative molecules can also be produced, for example, by expressing a bispecific antibody having 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 thus is thought to enhance the simultaneous stimulation of immune cells and antitumor function.

[0241] A probody is a fully recombinant masked monoclonal antibody that remains inactive in healthy tissue but is activated, particularly in the disease microenvironment (e.g., by protease cleavage by proteases abundant or specific to the disease microenvironment). See Desnoyers et al., Sci Transl. Med., 5:207,144, 2013. Similar masking techniques can be used for any antibody or antigen-binding portion thereof described herein.

[0242] ​An intracellular antibody is an antibody that is modified to localize intracellularly in order to act intracellularly and bind to an intracellular antigen. The intracellular antibody may remain in the cytoplasm, or may have a nuclear localization signal, or may have a KDEL sequence for ER targeting. The intracellular antibody may be a single-chain antibody (scFv), a modified immunoglobulin VL domain with enhanced stability, a selected antibody resistant to the more reducing intracellular environment, or may be expressed as a fusion protein with maltose-binding protein or other stable intracellular proteins. Such optimizations improve the stability and structure of the intracellular antibody and are generally applicable to any antibody or antigen-binding portion thereof described herein.

[0243] The antigen-binding portion or derivative antibody of the present invention may have substantially the same, or identical, (1) light chain and / or heavy chain CDR3 region; (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 / generated. The sequences within these regions may contain conservative amino acid substitutions, such substitutions including those within the CDR regions. In certain embodiments, there are 1, 2, 3, 4, or 5 or fewer conservative substitutions. Alternatively, the antigen-binding portion or derivative antibody may have a light chain region and / or heavy chain region that is at least about 90%, 95%, 99%, or 100% identical to the antibody from which they are derived / generated. 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 d and / or k off values are within a range of 10-fold (higher or lower), 5-fold (higher or lower), 3-fold (higher or lower), or 2-fold (higher or lower) of those of the antibodies described herein.

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

[0245] Monoclonal antibody technology can be used to generate highly specific cell-binding agents in the form of specific monoclonal antibodies. Well-known in the art is the technique of producing monoclonal antibodies by immunizing mice, rats, hamsters, or any other mammal 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 of producing monoclonal antibodies is the use of scFv (single-chain variable region), particularly phage libraries of human scFv (see, for example, Griffiths et al., U.S. Pat. Nos. 5,885,793 and 5,969,108, McCafferty et al., WO92 / 01047, Liming et al., WO99 / 06587). In addition, the resurfaces antibodies disclosed in U.S. Pat. No. 5,639,641 can also be used as chimeric and humanized antibodies.

[0246] The cell-binding agent may be a peptide derived from phage display (see, for example, Wang et al., Proc. Nat1. Acad. Sci. USA (2011) 108(17), 6909-6914) or peptide library technology (see, for example, Dane et al., M ol. Cancer Ther. (2009) 8(5):1312-1318).

[0247] In certain embodiments, the CBAs of the present invention include antibody mimetics such as DARPins, Affibodies, Affilins, Affitins, Anticalins, Avimers, Fynomers, Knotted domain peptides, Monobodies, Nanofitins, Bicycles® peptides such as those described in US Patent Application Publication No. 2014 / 0163201, which is incorporated herein by reference, and Pentins such as those described in Abstract 3674, AACR 106th Annual Meeting 2015; April 18 - 22, 2015; Philadelphia, PA, which is incorporated herein by reference.

[0248] As used herein, the terms “DARPin” and “(designed) ankyrin repeat protein” are used interchangeably and refer to a genetically engineered specific antibody mimetic protein typically exhibiting preferential (and in some cases specific) target binding. The target may be a protein, carbohydrate, or other chemical component, and the binding affinity can be quite high. A DARPin may be derived from a natural ankyrin repeat-containing protein, preferably consisting of at least 3, usually 4 or 5 ankyrin repeat motifs (typically with 33 residues per ankyrin repeat motif). In certain embodiments, a DARPin may contain about 4 or 5 repeats and may have a molecular weight of about 14 or 18 kDa each. Various techniques such as ribosome display or signal recognition particle (SRP) phage display are used to select DARPins that bind to a desired target with picomolar affinity and specificity (e.g., acting as receptor agonists or antagonists, inverse agonists, enzyme inhibitors, or simple target protein binders). 12A library of DARPins with randomized interaction residues with a target candidate having a diversity of mutants exceeding can be created at the DNA level. For example, for DARPin preparation, see US 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, H.K., Amstutz, P. & Pluckthun, A. (2005) Nature Biotechnology, 23:1257-1268 (all of which are incorporated herein by reference). Also, for related ankyrin-like repeat proteins or synthetic peptides, see US Patent Application Publication No. 2007 / 0238667, US Patent No. 7,101,675, WO2007 / 147213, and WO2007 / 062466 (the entire teachings of which are incorporated herein by reference).

[0249] Affibody molecules are small proteins engineered to mimic monoclonal antibodies that bind with high affinity to a number of target proteins or peptides. Affibodies consist of three alpha helices with 58 amino acids and have a molecular weight of approximately 6 kDa. Affibodies have been shown to withstand high temperatures (90 °C) or acidic and alkaline conditions (pH 2.5 or pH 11), and binders with affinities reduced to the sub-nanomolar range have been obtained from naïve library selections, and binders with picomolar affinity have been obtained after affinity maturation. In certain embodiments, the Affibody is conjugated to a weak electrophile for covalent binding to the target.

[0250] A monobody (also known as Adnectin) is a genetically engineered antibody mimetic protein that can bind to an antigen. In certain embodiments, the mon obody 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 particularly the tenth extracellular type III domain of human fibronectin, which has a structure similar to an antibody variable domain, has seven beta sheets that form a barrel, and has three exposed loops on each side corresponding to 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 engineered.

[0251] A tribody is a self-assembling antibody mimetic designed based on the C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) that self-assembles into a parallel trimeric complex. A tribody is a highly stable trimeric target-directed ligand created by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion protein can efficiently self-assemble into a well-defined, highly stable parallel homotrimer. Surface plasmon resonance (SPR) analysis of the trimeric target-directed ligand demonstrated a significant improvement in target-binding strength compared to the corresponding monomer. Cell-binding studies confirmed that such tribodies have excellent binding strength for their respective receptors.

[0252] Centyrin is another antibody mimetic that can be obtained using a library constructed in the framework of a 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 to select for high-affinity centyrin variants 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 an 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 native human FOLR1 unless otherwise indicated. Thus, all of these terms can refer to either the protein or nucleic acid sequence shown herein. The term "FOLR1" encompasses "full-length" unprocessed FOLR1, as well as any form of FOLR1 obtained by intracellular processing. The FOLR1 antibody has (a) a heavy chain CDR1 containing GYFMN (SEQ ID NO: 4); RIHPYDGDTFYNQXaa 1 FXaa 2 Xaa 3 (SEQ ID NO: 5) containing heavy chain CDR2; and YDGSRAMDY (SEQ ID NO: 6) containing heavy chain CDR3; and (b) KASQSVSFAGTSLMH (SEQ ID NO: 7) containing light chain CDR1; RASNLEA (SEQ ID NO: 8) containing light chain CDR2; and QQSREYPYT (SEQ ID NO: 9) containing light chain CDR3 (wherein Xaa 1 is selected from K, Q, H, and R, and Xaa 2 is selected from Q, H, N, and R, and Xaa 3 is selected from G, E, T, S, A, and V). Preferably, the heavy chain CDR2 sequence contains RIHPYDGDTFYNQKFQG (SEQ ID NO: 10).

[0254] In another embodiment, the anti-folate receptor antibody is a humanized antibody or an antigen-binding fragment thereof that specifically binds to human folate receptor 1 and has the following amino acid sequence

Chemical formula

[0255] In another embodiment, the anti-folate receptor antibody is a humanized antibody or an antigen-binding fragment thereof encoded by plasmid DNA deposited with the ATCC on April 7, 2010, having ATCC deposit numbers PTA-10772 and PTA-10773 or 10774. In one 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-10773 or 10774. 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-10773. In yet 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 an antigen-binding fragment thereof that specifically binds to human folate receptor 1 and has the following amino acid sequence

Chemical formula

[0257] In another embodiment, the anti-folate receptor antibody is a humanized antibody or an antigen-binding fragment thereof that specifically binds to human folate receptor 1, and includes 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 includes 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) includes 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 an antigen-binding fragment thereof that specifically binds to human folate receptor 1, [Chemical formula] and a heavy chain variable domain that is at least about 90%, 95%, 99% or 100% identical to, [Chemical formula] and a light chain variable domain that is at least about 90%, 95%, 99% or 100% identical to.

[0259] In another embodiment, the anti-folate receptor antibody is huMov19 or M9346A or the M antibody (see, for example, U.S. Patent Nos. 8,709,432, 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).

[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 antibodies 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 specifically, the anti-EGFR antibody is huML66.

[0261] In yet another embodiment, the anti-EGFR antibody includes 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, a double-underlined sequence represents the variable region of the heavy chain or light chain sequence (i.e., the heavy chain variable region or HCVR, and the light chain variable region or LCVR), and a bold sequence represents the CDR region (i.e., CDR1, CDR2, and CDR3 from the N-terminus to the C-terminus of the heavy chain or light chain sequence, respectively).

Table 1a

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

[0263] In another embodiment, the anti-EGFR antibody comprises a heavy chain variable region (HCVR) sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 17 and / or a light chain variable region (LCVR) sequence that is 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 the antibody described in 8,790,649 and WO2012 / 058588, which are incorporated by reference. In some embodiments, the anti-EGFR antibody is the huEGFR-7R antibody.

[0265] In some embodiments, the anti-EGFR antibody is

Chemical Structure

Chemical Structure

Chemical Structure

[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 the heavy chain CDR1-CDR3 of SEQ ID NO: 19 and / or the 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 that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 19 and / or a light chain variable region (LCVR) sequence that is 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 WO2004 / 103272 (incorporated by reference). In some embodiments, the anti-CD19 antibody is

Chemical formula

Chemical formula

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

[0272] In yet another embodiment, the anti-CD19 antibody comprises the heavy chain CDR1-CDR3 of SEQ ID NO: 22 and / or the 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 that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 22, and / or a light chain variable region (LCVR) sequence that is 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 comprises

Chemical formula

Chemical formula

[0275] In another embodiment, the anti-Muc1 antibody is the 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 yet another embodiment, the anti-Muc1 antibody comprises a heavy chain variable region (HCVR) sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 24, and / or a light chain variable region (LCVR) sequence that is 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 a fragment thereof, such as an antibody or a 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 is

Chemical formula

Chemical formula

[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 that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 26, and / or a light chain variable region (LCVR) sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 27, and preferably specifically binds to CD33.

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

[0283] In some embodiments, the anti-CD37 antibody is [Chemical formula] An immunoglobulin light chain region having the amino acid sequence of, and [Chemical formula] an immunoglobulin heavy chain region having the amino acid sequence of, or [Chemical formula] includes an immunoglobulin heavy chain region having the amino acid sequence of.

[0284] In another embodiment, the anti-CD37 antibody includes 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 includes 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 includes heavy chain CDR1-CDR3 of SEQ ID NO: 29 or 30, and / or light chain CDR1-CDR3 of SEQ ID NO: 28, and preferably specifically binds to CD37.

[0287] In yet another embodiment, the anti-CD37 antibody includes a heavy chain variable region (HCVR) sequence that is 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 that is 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 [Chemical formula] An immunoglobulin light chain region having the amino acid sequence of, and

Chemical formula

[0289] In 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 another embodiment, the anti-CD37 antibody comprises a heavy chain variable region (HCVR) sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 32 and / or a light chain variable region (LCVR) sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 31, and preferably specifically binds to CD37.

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

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

[0293] In one embodiment, the anti-CD123 antibody or an 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 is

Chem.

Chem.

[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

Chem.

[0296] In another embodiment, the anti-CD123 antibody is

Chem.

Chem.

[0297] In another embodiment, the anti-CD123 antibody is

Chem.

[0298] In certain embodiments, the cell binding agent (e.g., an antibody) of the present invention has 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 sodium periodate.

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

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

[0302] In certain embodiments, the cell-binding agent (e.g., an antibody) of the present invention has a recombinantly engineered Cys residue, e.g., a Cys residue at position 442 of the EU / OU numbering of the antibody. Thus, the term "cysteine-engineered antibody" includes antibodies having at least one Cys that is not normally present at a given residue of the antibody light or heavy chain. Such Cys, sometimes also referred to as "engineered Cys", may be engineered using any conventional molecular biology or recombinant DNA techniques (e.g., by replacing the coding sequence for a non-Cys residue at the target residue with a coding sequence for Cys). For example, if the original residue is Ser having a coding sequence of 5'-UCU-3', the coding sequence can be mutated to 5'-UGU-3' that encodes Cys (e.g., by site-directed mutagenesis). In certain embodiments, the Cys-engineered antibody of the present invention has the 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 chain (or light chain) sequence can be inserted into a suitable recombinant expression vector to generate an engineered antibody having 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 for their ability to inhibit the growth of various cancer cell lines in vitro. For example, cell lines such as the human choriocarcinoma JEG-3 cells can be used to evaluate the cytotoxicity of these compounds and conjugates. The cells to be evaluated are exposed to the compound or conjugate for 1 to 5 days, and the cell viability can be measured by a direct assay using known methods. Then, from the results of the assay, the IC 50Values can be calculated. Alternatively, or in addition, an in vitro cell line sensitivity screening method, such as the method described by the National Cancer Institute of the United States (see Voskoglou-Nomikos et al., 2003, Clinical Cancer Res. 9:42227-4239, which is incorporated herein by reference), can be used as one of the guidelines for determining the types of cancers that may be sensitive to treatment with the compounds or conjugates of the present invention.

[0304] Examples of the in vitro potency and target specificity of the antibody-cytotoxic drug conjugates of the present invention are described in Example 6. Antigen-negative cell lines remained viable even when exposed to the same conjugates.

[0305] Compositions and Methods of Use The present invention includes compositions (e.g., pharmaceutical compositions) comprising a novel maytansinoid compound, a derivative thereof, or a conjugate thereof (and / or a solvate, hydrate, and / or salt thereof) described herein, and a carrier (a pharmaceutically acceptable carrier). The present invention also includes compositions (e.g., pharmaceutical compositions) comprising a novel maytansinoid compound, a derivative thereof, or a conjugate thereof (and / or a solvate, hydrate, and / or salt thereof) described herein, and a carrier (a pharmaceutically acceptable carrier). The compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human).

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

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

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

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

[0310] Examples of suitable carriers, diluents, and / or additives include: (1) Dulbecco's phosphate buffered saline (pH about 7.4, containing or not containing 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; and they may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

[0311] The method for inducing cell death in a selected cell population can be performed in vitro, in vivo, or ex vivo.

[0312] Examples of in vitro uses include: treatment of autologous bone marrow before transplantation into the same patient to kill diseased or malignant cells; treatment of bone marrow before transplantation to kill competent T cells and prevent graft-versus-host disease (GVHD); treatment of cell cultures to kill all cells other than the desired variant that do not express the target antigen; or killing of variants that express unwanted antigens.

[0313] The conditions for non-clinical in vitro uses can be easily determined by those skilled in the art.

[0314] Examples of clinical ex vivo uses are for removing tumor cells or lymphocytes from bone marrow in cancer treatment or prior to autologous transplantation in the treatment of autoimmune diseases, or for removing T cells and other lymphocytes from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent GVHD. The treatment can be carried out as follows. Bone marrow is collected 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 the cytotoxic drug of the present invention has been added in a concentration range of about 10 μM to 1 pM. The exact conditions for the concentration and time of incubation, i.e., the dose, can be readily determined by those skilled in the art. After incubation, the bone marrow cells are washed with a serum-containing medium and returned to the patient via the vein. If the patient undergoes other treatments, such as a series of excision chemotherapy or total body irradiation, between the time of bone marrow collection and the reinfusion of the treated cells, the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment. Accordingly, the bone marrow cells are washed with a serum-containing medium and returned to the patient via the vein. If the patient undergoes other treatments, such as a series of excision chemotherapy or total body irradiation, between the time of bone marrow collection and the reinfusion of the treated cells, the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0315] For clinical in vivo uses, the cytotoxic drug of the present invention will be supplied as a solution or lyophilized powder that has been tested for sterility and endotoxin levels.

[0316] In some embodiments, the compounds and conjugates of the present invention can be used to treat cancer (e.g., renal 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), glioblastoma, neuroblastoma, lymphoma (e.g., non-Hodgkin 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] Analogues and derivatives Those skilled in the art of cytotoxic drugs will readily understand that each of the cytotoxic drugs described herein can be modified such 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 analogues and derivatives of the compounds described herein.

[0318] All the reference documents cited in this specification and the following examples are hereby incorporated by reference in their entirety.

Example

[0319] Example 1. Preparation of DM-H(7) stock solution

Chemical formula

[0320] Example 2. Synthesis of Thio-Peptide-Metanocinoid FMoc-Peptide-NH-CH 2 -OAc type compounds were prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -OAc.

[0321] FMoc-Peptide-OAc Compound FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -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 thereto under argon with magnetic stirring. When the solid had dissolved, lead tetraacetate (434 mg, 0.979 mmol) was added. The reaction was allowed to proceed at 60 °C for 20 minutes and then purified by eluting with deionized water containing a 5% - 55% linear acetonitrile gradient at a flow rate of 125 mL / min over 26 minutes on a C18, 30 micron 450 g column cartridge. The fractions containing the pure desired product were frozen and lyophilized to obtain 178 mg (yield 34%) of a white solid. HRMS (M+Na) + Calculated value 547.2163; Measured value 547.2160. 11H 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), 4.05 (q, J = 7.3 Hz, 1H), 4.23 (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-CH 2 -OAc(9b):HRMS(M+Na) + Calculated value: 547.2163, measured value: 547.2167. 1 1H 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).

[0323] FMoc-L-Ala-D-Ala-L-Ala-NH-CH 2 -OAc(9c):HRMS(M+Na)+ Calculated value: 547.2163, measured value: 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-CH 2 -OAc(9d): HRMS(M+Na) + Calculated value: 547.2163, measured value: 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-CH 2-OAc(9f): HRMS(M+Na) + Calculated value: 476.1792, Measured value: 476.1786. 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-CH 2 -OAc(9g): HRMS(M+Na) + Calculated value: 476.1792, Measured value: 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-CH 2 -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 compound FMoc-peptide-NH-CH 2 -S-(CH2) n -CO 2 The H-type compound was prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO 2 H.

[0329] FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -C O 2H(10a): Dissolve 6-mercaptohexanoic acid (287 μL, 2.07 mmol) in a 1:4 solution of TFA:dichloromethane (5 mL), then add it to a vial containing FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -OAc (178 mg, 0.339 mmol). The reaction was allowed to proceed for 20 minutes at room temperature under an argon atmosphere with magnetic stirring. The crude material was concentrated under vacuum and redissolved in the minimum volume of DMF, and purified by eluting with deionized water containing 0.1% formic acid and having a linear gradient of 5% - 95% acetonitrile over 13 minutes at 35 mL / min on a C18, 30 micron 30 g cartridge. The fractions containing the pure desired product were frozen and lyophilized to give 200 mg (yield 96%) of a white solid. HRMS (M+H) + Calculated 613.2690; Found 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10b): HRMS (M+Na)+ Calculated value: 635.2510, measured value: 635.2515. 1 H 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10c): HRMS (M+Na) + Calculated value: 635.2510, measured value: 635.2514. 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10d):HRMS(M+Na) + Calculated value 635.2510, measured value 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10g):HRMS(M+H) + Calculated value 542.2319, measured value 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10f): HRMS(M + H) + Calculated value 542.2319, measured value 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-CH 2 -S-(CH 2 ) 5 -CO 2 H(10h): (16.7 mg, 0.031 mmol, yield 70%). HRMS(M+H) + Calculated value 542.2319, measured value 542.2318.

[0336] FMoc-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 3 -CO 2 H(10j): HRMS(M+H) + Calculated value 585.2377, measured value 585.2367. 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-CH 2 -S-(CH) 2 -CO 2 H(10i): HRMS(M+H) + Calculated value 500.1850, measured value 500.1843. 1 H N 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), 4.07 (q, J = 7.0 Hz, 1H), 4.17 - 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-CH 2 -OAc(9c): [Chemical formula] 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 CH2Cl2 (100 ml) and treated with COMU (20.63 g, 48.2 mmol) and DIPEA (11.22 ml, 64.2 mmol). The reaction was carried out under argon at room temperature. After 2 hours, the reaction was shown to be complete by UPLC, diluted with 2-MeTHF (50 ml), and washed with 10% aqueous citric acid solution (2 × 100 mL), water (100 mL), and then brine (100 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated to obtain crude FMoc-L-Ala-D-Ala-OtBu. The estimated yield was 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 was carried out at room temperature under an argon atmosphere. After 4 hours, the reaction was shown to be complete by UPLC. This was diluted with toluene (25 mL) and co-evaporated (3 times) to obtain FMoc-L-Ala-D-Ala. The estimated yield was 100%.

[0340] Step 3: FMoc-L-Ala-Gly-OtBu (9c3) Z-L-Ala-ONHS (10 g, 31.2 mmol) and tert-butyl glycinate (6.28 g, 37.5 mmol) were dissolved in CH2Cl2 (100 ml) and treated with DIPEA (10.91 ml, 62.4 mmol). The reaction was carried out under argon at room temperature. After 2 hours, UPLC showed completion. The reaction mixture 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 obtain Z-L-Ala-Gly-OtBu. The estimated yield was 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 flask of a reduction apparatus, and treated with Pd / C (1.272 g, 11.95 mmol). The flask of the reduction apparatus was placed on a shaker, and while shaking the flask, air was removed by vacuum. The flask was filled with hydrogen up to 30 psi, shaken for 2 minutes, and the hydrogen was removed by vacuum. This was repeated 2 more times. The flask was filled with hydrogen up to 30 psi and shaken. After 4 hours, UPLC showed completion. The reaction mixture was filtered in vacuo through a plug of celite, dissolved in 2-MeTHF, and concentrated to obtain LAla-Gly-OtBu. The estimated yield was 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 CH2Cl2 (10 ml) and treated with COMU (1.181 g, 2.76 mmol) and DIPEA (0.876 ml, 5.02 mmol). The reaction was carried out under argon at room temperature. After 2 hours, the reaction showed completion. The reaction mixture was concentrated to remove CH2Cl2, redissolved in 2 mL of DMF, and purified using a linear gradient by 18 combiflash to give the combined product 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 carried out under argon at room temperature. After 1 hour, the reaction showed completion according to UPLC. It was diluted with toluene (1 mL) and co-evaporated twice with toluene to give FMoc-L-Ala-D-Ala-L-Ala-Gly-OH. The estimated yield was 100%.

[0344] Step 7. FMoc-L-Ala-D-Ala-L-Ala-CH 2 -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 the reagents had dissolved, the reaction mixture was treated with lead tetraacetate (3.45 g, 7.785 mmol). The reaction was allowed to proceed under argon at 60 °C for 30 minutes. The crude reaction mixture was purified by Combiflash Rf 200i using a C18 450 g column with a flow rate of 125 mL / min and deionized water containing 0.1% formic acid and acetonitrile as the solvent, using the following gradient (time (min), acetonitrile percentage): (0, 5), (8, 50), (26, 55). The desired product had a retention time of 11 minutes. The product fractions were 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, found 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-CH 2 -S-(CH 2 ) n -CO2 -DM compounds are prepared as exemplified by FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM.

[0346] FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM (11a): To a DM-H stock solution (8.2 mL, 0.49 mmol) were added FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 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 for 2 hours at room temperature under an argon atmosphere with magnetic stirring. The crude material was concentrated in vacuo by rotary evaporation, the residue was taken up in a minimum volume of DMF and then purified by elution with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 50% over 25 minutes on a C18, 30 micron, 30 g cartridge. 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 1266.5170; Found 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-CH 2 -S-(CH 2 )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, 3H), 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 , 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-CH 2 -S-(CH 2 ) 5 -CO-DM(11c):HRMS(M+Na) + Calculated value 1266.5170, measured value 1266.5170. 11H 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 - 3.89 (m, 4H), 3.92 - 4 .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-CH 2 -S-(CH 2 ) 5 -CO-DM(11d):HRMS(M+Na) + Calculated value: 1266.5170, measured value: 1266.5158. 1 1H 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-CH 2 -S-(CH 2 ) 5 -CO-DM(11g):HRMS(M+H) + Calculated value 1173.4980, measured value 1173.4964. 1 1H 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-CH 2 -S-(CH 2 ) 5 -CO-DM(11f):HRMS(M+H) + Calculated value 1173.4980, measured value 1173.4969.

[0352] FMoc-D-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(11h):HRMS(M+Na) + Calculated value 1195.4907, measured value 1195.4799. 1 1H NMR (400 MHz, DMSO-d 6 ) 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-CH 2 -S-(CH 2 ) 3 -CO-DM(11j):HRMS(M+H) + Calculated value 1216.5038, measured value 1216.4999. 1 H 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-CH 2 -S-(CH 2 ) 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 - metanoid H 2 N - peptide - NH - CH 2 - S - (CH 2 ) n - CO 2 - DM - type compound was prepared as exemplified by H 2 N - L - Ala - L - Ala - L - Ala - NH - CH2 - S - (CH 2 ) 5 - CO - DM.

[0356] H2 N-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(12a): FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM (151 mg, 0.121 mmol) was treated with 20% morpholine (2 mL) in DMF. The reaction was carried out under argon at room temperature for 1 hour with magnetic stirring. The crude material was purified by eluting on a C18, 30 micron, 150 g cartridge column with deionized water containing a linear gradient of acetonitrile from 5% to 50% over 26 minutes with 0.1% formic acid. The fractions containing the desired product were immediately frozen and lyophilized to give 46 mg (yield 37.1%) 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 = 6.3 H 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] H 2 N-D-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 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] H 2 N-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 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] H 2 N-L-Ala-L-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 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] H 2N-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(12g): HRMS(M+H) + Calculated value 951.4299, measured value 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] H 2 N-L-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(12f): HRMS(M+H) + Calculated value 951.4226, measured value 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] H 2 N-D-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 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, 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] H 2 N-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 3 -CO-DM(12j):HRMS(M+H) + Calculated value 994.4357, measured value 994.4330.

[0364] H 2 N-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 2 -CO-DM(12i):HRMS(M+H) + Calculated value 909.3830, measured value 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-Methanoid SPDB-Peptide-NH-CH 2 -S-(CH 2 ) n -CO 2 -DM type compounds were prepared as exemplified by SPDB-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM.

[0366] SPDB-L-Ala-L-Ala-L-Ala-NH-CH2 -S-(CH 2 ) 5 -CO-DM(13a):H 2 N-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM (46 mg, 0.045 mmol) was dissolved in DMF (2 mL), and SPDB (14.7 mg, 0.045 mmol) was added thereto. The reaction was carried out under an argon atmosphere for 1 hour at room temperature with magnetic stirring. The crude material was purified by eluting on a C18, 430 micro, 30 g cartridge with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 35 minutes with 0.1% formic acid. The fractions containing the pure desired product were frozen and lyophilized to obtain 38 mg (yield 68.5%) 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13b):HRMS(M+H) + Calculated value 1233.4796, measured value 1233.4799. 1 1H 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 - 2.16 (m, 1H), 2.26 (d, 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 - 4.50 (m, 1H), 5.27 (d, J = 6.7 Hz, 1H), 5.50 (dd, 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13c):HRMS(M+H) + Calculated value 1233.4796, measured value 1233.4795. 1 1H 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13d):HRMS(M+H) + Calculated value 1233.4796, measured value 1233.4797. 1 1H 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13g):HRMS(M+H) + Calculated value 1162.4425, measured value 1162.4405.1 1H 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13f):HRMS(M+H) + Calculated value: 1162.4399, Measured value: 1162.455. 1 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-CH 2 -S-(CH 2 ) 5 -CO-DM(13h):HRMS(M+H) + Calculated value 1162.4399, measured value 1162.455. 1 1H 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-CH 2 -S-(CH 2 ) 3 -CO-DM(13j):HRMS(M+H) + Calculated value 1203.4337, measured value 1203.4315. 11H 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-CH 2 -S-(CH 2 ) 2 -CO-DM(13i):HRMS(M+H) + Calculated value: 1120.3955, Measured value: 1120.3951. 1 1H 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 - maitansinoid HS-(CH 2 ) 3 CO - peptide - NH - CH 2 -S-(CH 2 ) n -CO 2 -DM type compounds were prepared as exemplified by HS-(CH 2 ) 3 CO - L - Ala - L - Ala - L - Ala - NH - CH 2 -S-(CH 2 ) 5 -CO - DM.

[0376] HS-(CH 2 ) 3 CO-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-( CH 2 ) 5 -CO-DM(14a):SPDB-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 Dissolve CO-DM(38 mg, 0.031 mmol) in DMSO (1 mL), and add a solution of DTT (19 mg, 0.12 mmol) in 100 mM potassium phosphate, 2 mM EDTA (pH 7.5) buffer (1 mL) thereto. The reaction was carried out under argon at room temperature with magnetic stirring for 1 hour. The crude reaction product was purified by eluting on a C18, 30 micron, 30 g cartridge with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 35 minutes with 0.1% formic acid. The fractions containing the desired product were immediately frozen and lyophilized to obtain 18.2 mg (yield 52.5%) of a white solid. HRMS (M+H)+ calculated value 1124.4809; measured value 1124.4798. 1 1H 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-(CH 2 ) 3 CO-D-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(14b):HRMS(M+Na) + Calculated value 1146.4629, measured value 1146.4591. 1 1H 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-(CH 2 ) 3 CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 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-(CH 2 ) 3 CO-L-Ala-L-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(14d):HRMS(M+Na) + Calculated value 1146.4629, measured value 1146.4519. 1 1H 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, 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, 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).

[0380] HS-(CH 2 ) 3 CO-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(14g):HRMS(M+H) + Calculated value: 1053.4438, measured value: 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-(CH 2 ) 3 CO-L-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(14f):HRMS(M+H) + Calculated value 1053.4366, measured value 1053.4438. 1 H 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-(CH 2 ) 3 CO-D-Ala-D-Ala-NH-CH 2 -S-(CH 2 ) 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-(CH 2 ) 3CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 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-(CH 2 ) 3 CO-(CH 2 ) 3-CO-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 2 -CO-DM(14i):HRMS(M+H) + Calculated value 1011.3 969, Measured value 1011.3961. 1 H 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-(CH 2 ) 3 -CO-peptide-NH-CH 2 -S-(CH 2 ) n -CO 2 -DM type compound was prepared as exemplified by HOOC-(CH 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM.

[0386] HOOC-(CH 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(19a): L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM (17.25 mg, 0.017 mmol) was treated with glutaric anhydride (38.5 mg, 0.337 mmol) and reacted overnight at room temperature under argon with magnetic stirring. The crude reaction product was purified by HPLC using an XDB-C18, 21.2×5 mm, 5 micron column, eluting with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 30 minutes at 0.1% formic acid and 20 ml / min. Fractions containing the pure desired product were immediately combined, frozen and lyophilized to give 3 mg (yield 15%) 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 = 11.8 Hz , 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 = 9.1 Hz , 2H), 8.35 (d, J = 11.6 Hz, 1H), 8.62 (s, 1H ).

[0387] HOOC-(CH 2 ) 3 -CO-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(19g):HRMS(M+H) + Calculated value 1136.4987, measured value 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-(CH 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 3 -CO-DM(19i):HRMS(M+H) + Calculated value 1108.4674, measured value 1108.4634. 1 H 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-(CH 2 ) 3 -CO - Peptide - NH - CH 2 -S-(CH 2 ) n -CO 2 The NHS - OOC - (CH 2 ) 3 -CO - D - Ala - L - Ala - NH - CH 2 -S-(CH 2 ) 5 -CO - DM compound was prepared as exemplified by NHS - OOC - (CH

[0390] NHS - OOC - (CH 2 ) 3 -CO - D - Ala - L - Ala - NH - CH 2 -S-(CH 2 ) 5 -CO - DM(20g): HOOC - (CH 2 ) 3 -CO - D - Ala - L - Ala - NH - CH 2 -S-(CH 2 ) 5-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 was carried out at room temperature under an argon atmosphere with magnetic stirring for 2 hours. The crude material was purified by HPLC using an XDB-C18, 21.2×5 mm, 5 μm column and eluting with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 30 minutes at 0.1% formic acid and 20 mL / min. The fractions containing the desired product were combined, immediately frozen, and then lyophilized to obtain 6.5 mg of a white solid (yield 74%). 1 H NMR (400 MHz, 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-(CH 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(20g):HRMS(M+H) + Calculated value 1233.5151, measured value 1233.5135.

[0392] NHS-OOC-(CH 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 3 -CO-DM(20i):HRMS(M+H) + Calculated value 1205.4838, measured value 1205.4808.

[0393] H 2 N-O-CH 2 -CO-peptide-NH-CH 2 -S-(CH 2 ) n -CO 2 -DM type compound was prepared as exemplified by H 2 N-O-CH 2 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM.

[0394] H 2 N-O-CH 2 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(22c): H 2 N-L-Ala-D-Ala-L-Ala-CH 2 -S-(CH 2 ) 5-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 was allowed to proceed for 3 hours at room temperature under an argon atmosphere with magnetic stirring. 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 preparative HPLC using XDB-C18, 21.2×5 mm, 5 μm, eluting with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 30 minutes at 0.1% formic acid and 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 1095.4834, found 1095.4795. 1 H NMR (400 MHz, DMSO-d 6 ) 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] H 2 N - O - CH 2 CO - L - Ala - D - Ala - L - Ala - NH - CH 2 -S-( CH 2 ) 3 -CO - DM(22i):HRMS(M + H) + Calculated value: 1067.4521, Measured value: 1067.4484. 1 H NMR (400 MHz, DMSO - d 6 ) 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 2 ) 3 -CO-L-Ala-D-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -CO-DM(23c):H 2 N-L-Ala-D-Ala-L-Ala-CH 2 -S-(CH 2 ) 5 Dissolve -CO-DM(8 mg, 7.82 μmol) in DMF (2 mL), and treat 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 was allowed to proceed for 2 hours at room temperature under magnetic stirring and an argon atmosphere. The crude material was purified by preparative HPLC using XDB-C18, 21.2×5 mm, 5 μm, eluting with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 30 minutes at 0.1% formic acid and 20 ml / min. The fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 1.8 mg (yield 19.60%) 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).

[0397]

Chem.

Chem.

[0398] Mal-C5-L-Ala-D-Ala-L-Ala-lmm-C6-May:L-Ala-D-Ala-L-Ala-CH 2 -S-(CH 2 ) 5 -CO-MayNMA (Compound I-la) (25 mg, 0.024 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoate (7.54 mg, 0.024 mmol) were reacted to obtain Mal-C5-L-Ala-D-Ala-L-Ala-Imm-C6-May (Compound I-2a) (20.8 mg, 0.017 mmol, yield 70.0%). LRMS (M+H) + Calculated value 1215.52, found 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-(CH 2 ) 2 -PEG 2 -CO-L-Ala-D-Ala-L-ALa-NH- CH2-S-(CH2)5-CO-MayNMA

Chemical formula

[0400] Mal-(CH 2 ) 2 -PEG 4 -CO-L-Ala-D-Ala-L-ALa-NH-CH 2 -S-(CH 2 ) 5 -CO-MayNMA

Chemical Structure

[0401] Example 3. Synthesis of Metabolite DM-CO-(CH 2 ) 5 -SH(24a): To a 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 minutes at room temperature with magnetic stirring. Then, 6-mercaptohexanoic acid (13.8 μL, 0.100 mmol) was added. After 30 minutes, the crude material was purified by preparative HPLC using XDB-C18, 21.2×5 mm, 5 μm, eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 30 minutes at 20 mL / min. The fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 12 mg (yield 15%) 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 2 ) 5 -SMe(25a):DM-CO-(CH 2 ) 5 -SH (12 mg, 0.015 mmol) was dissolved in DMF (2 mL), treated with DIPEA (24 μL, 0.139 mmol) and iodomethane (2.88 μL, 0.046 mmol), and allowed to proceed under argon at room temperature for 2 hours. The crude material was purified by preparative HPLC using an XDB-C18, 21.2×5 mm, 5 μm column, eluting with deionized water containing a linear gradient of acetonitrile from 5% to 95% over 30 minutes at a flow rate of 20 ml / min and 0.1% formic acid at a flow rate of 20 ml / min. The fractions containing the desired product were immediately combined, frozen, and then lyophilized to give 2 mg (yield 16%) of a white solid. HRMS (M+H) + Calculated value 794.3448, measured value 794.3440. 11H 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 2 ) 3 -SSPy(26):DM-CO-(CH 2 ) 3 -SSPy(3267-50-R1): SPDB (30.1 mg, 0.092 mmol) was added to a DM-H stock solution (0.81 mL, 0.046 mmol) at room temperature with magnetic stirring. After 30 minutes, the solution was purified using deionized water containing a linear gradient of acetonitrile from 5% to 95% at a flow rate of 20 ml / min for 30 minutes on an XDB-C18, 21.2×5 mm, 5 μm column at a flow rate of 20 ml / min. The fractions containing the desired product were combined, frozen, and then lyophilized to obtain 6 mg (yield 15%) of a white solid. HRMS (M+H) + Calculated value 861.2964, measured value 861.2963. 11H 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 2 ) 3 -SH(24b):DM-CO-(CH 2 ) 3-SSPy (6 mg, 6.96 μmol) was added to a solution of DTT (1.1 mg, 6.96 μmol) in a 2:1 DMSO:potassium phosphate 2 mM EDTA (pH 7.5) buffer (0.5 mL) and magnetically stirred at room temperature for 20 minutes. The crude solution was purified on an XDB-C18, 21.2×5 mm, 5 μm column using deionized water containing a linear gradient of acetonitrile from 5% to 95% at a flow rate of 20 ml / min for 30 minutes and 0.1% formic acid at a flow rate of 20 ml / min. Fractions containing the desired product were combined, frozen, and then lyophilized to give 5 mg (95% yield) of a white solid. HRMS (M+H) + Calculated value 752.2978, found 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 2 ) 3 -SMe(25b): DM-CO-(CH 2 ) 3 -SH (5 mg, 6.65 μmol) was dissolved in anhydrous DMF (0.3 mL), and to this, 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 hour, the crude solution was purified on an XDB-C18, 21.2×5 mm, 5 μm column with deionized water containing a linear gradient of acetonitrile from 5% to 95% at a flow rate of 20 ml / min for 30 minutes at a flow rate of 20 ml / min. The fractions containing the desired product were combined, frozen, and then lyophilized to obtain 1 mg (yield 19%) 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 the maitansineoid solution for preparing ADCs (16a - 16i, 17a - 17i, 18a - 18i) One of sulfo - GMBS and the thiol - bearing compounds (14a - 14j) was dissolved in a 3:7 solution of (50 mM sodium succinate, pH 5.0:DMA) to obtain concentrations of 1.5 mM and 1.9 mM, respectively. The solution was gently stirred at room temperature for 30 minutes, and then the excess thiol was quenched by making it 0.5 mM in N - ethylmaleimide (NEM) while gently stirring the solution for 10 minutes.

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

[0408] ADC1a - 1d and 4a - 4c were used as comparative substances for evaluating the conjugates of the present invention. ADC1a - 1d and 4a - 4c were prepared as described in Widdison W., et .al., Bioconjugate Chem., (2015), 26, 2261 - 2278.

[0409] Preparation of C242 - sGMBS - LDL - DM (ADC18c) Before conjugation, sGMBS-LDL-DM was prepared by mixing a stock solution of sulfo-GMBS (Compound 15 in Figure 4) in N-N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (Compound 14c in Figure 3) in DMA in the presence of succinic acid buffer pH 5.0 to obtain a final concentration of 60 / 40 organic / aqueous solution and 1.5 mM sulfo-GMBS and 1.95 mM LDL-DM. The reaction was incubated at 25 °C for 10 minutes. The crude sGMBS-LDL-DM mixture was added to a solution containing the C242 antibody in phosphate buffered saline (PBS) pH 7.4 spiked with a 5-fold 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 per 1 mol of 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 succinic acid compound, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 formulation buffer and filtered through a syringe filter equipped with a 0.22 μm PVDF membrane.

[0411] The purified conjugate was found to have 3.8 mol of LDL-DM / antibody mol 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-N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (Compound 14c in Figure 3) in DMA in the presence of succinic acid buffer pH 5.0 to obtain a final concentration of 60 / 40 organic / aqueous solution and 1.5 mM sulfo-GMBS and 1.95 mM LDL-DM. The reaction was incubated at 25 °C for 10 minutes. The crude sGMBS-LDL-DM mixture was added to a solution containing the ML66 antibody in phosphate buffered saline (PBS) pH 7.4 spiked with a 5-fold 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 moles of sulfo-GMBS-LDL-DM per 1 mole of 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 succinic acid compound, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5 formulation buffer and filtered through a syringe filter equipped with a 0.22 μm PVDF membrane.

[0414] The purified conjugate was found to have 3.7 moles of LDL-DM / antibody mole by UV-Vis, 98% monomer by SEC, and less than 1% free drug by HPLC Hisep column analysis. The SEC / MS spectrum for 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-N-dimethylacetamide (DMA, SAFC) with a stock solution of LDL-DM (Compound 14c in Figure 3) in DMA in the presence of succinic acid buffer pH 5.0 to obtain a final concentration of 60 / 40 organic / aqueous solution and 3 mM sulfo-GMBS and 3.9 mM LDL-DM. The reaction was incubated at 25 °C for 2 hours. The crude sGMBS-LDL-DM mixture was added to a solution containing the M9346A antibody in phosphate buffered saline (PBS) pH 7.4 spiked with a 5-fold 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 moles of sulfo-GMBS-LDL-DM per 1 mole of M9346A antibody. The reaction was incubated overnight at 25 °C.

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

[0417] The purified conjugate was found to have 3.7 moles of LDL-DM / antibody mole by UV-Vis, 99% monomer by SEC, and less than 1% free drug by SEC / reversed 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 with a Cys incorporated 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 at 37 °C for 1 hour. TCEP was removed by a NAP desalting column (GE Healthcare), and 100 molar equivalents of dehydroascorbic acid (Sigma-Aldrich) was added to the purified reduced M9346A-C442 in PBS pH 7.4, 2 mM EDTA (Sigma-Aldrich), and incubated at 25 °C for 90 minutes to 4 hours. The reduced and re-oxidized antibody solution was immediately used to conjugate with MalC5-LDL-DM (Compound I-2a shown above).

[0419] The re-oxidized M9346A-C442 antibody was spiked with PBS pH 6.0, 2 mM EDTA, 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 at 25 °C overnight.

[0420] After the reaction, the conjugate was purified using a NAP desalting column (GE Healthcare) in a formulation buffer of 10 mM acetic acid compound, 9% sucrose, 0.01% Tween20, pH 5.0, and filtered through a syringe filter equipped with a 0.22 μm PVDF membrane.

[0421] The purified conjugate was found to have 2 mol of LDL-DM / antibody mol by UV-Vis, 97% monomer by SEC, and less than 3% free drug by SEC / reversed-phase HPLC dual column analysis. The SEC / MS spectrum of 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 evaluated using flow cytometry by indirect immunofluorescence assay. Cells (5×104 per well) were plated in round - bottom 96 - well plates and incubated at 4°C for 3 hours with serial dilutions of the test article in 0.2 mL of alpha - MEM supplemented with 2% (v / v) normal goat serum (Sigma, St. Louis, MO). Each sample was assayed in triplicate. Control wells contained no test article. The cells were then washed with 0.2 mL of cold (4°C) medium and stained at 4°C for 1 hour with fluorescein - labeled goat anti - human immunoglobulin G (IgG) antibody. The cells were washed again with medium and 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 moderately affected the binding affinity of the naked antibody.

[0424] Example 6. In vitro Cytotoxicity Assay with ADCs and Metabolites The assay was performed in flat - bottom 96 - well plates in triplicate for each data point. The test article was first diluted in complete cell culture medium using a 5 - fold dilution series, and 100 μL was added to each well. The final concentrations typically ranged from 3×10 -8 M to 8×10 -14 M. Then, target cells were added to the test article in 100 μL of complete culture medium at 1,500 - 3,000 cells per well. The mixture was incubated at 37°C in a humidified 5% CO 2It was incubated in an incubator for 5 days. The viability of the remaining cells was determined using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Rockville, MD) by a WST-8 (tetrazolium salt-8; 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) based colorimetric assay. WST-8 is reduced by dehydrogenase in live cells to obtain a yellow-colored formazan product that is soluble in tissue culture medium. The amount of formazan dye is directly proportional to the number of live cells. WST-8 was added to a final volume of 10%, and the plates were incubated at 37 °C in a humidified 5% CO 2 It was further incubated in the incubator for 4 hours. Then, the WST-8 signal was measured at an optical density of 450 nm using a microplate plate reader. The viability was calculated by dividing the value of each treated sample by the average value of the untreated control and plotted for each treatment in a semi-logarithmic plot against the test substance concentration. The IC 50 value was determined using a non-linear regression (curve fit) with the GraphPad Prism v5 program (GraphPad Software, La Jolla, CA).

[0425] As shown in Figure 8, the conjugate of the present invention was highly effective against KB cells, and the addition of the naked antibody significantly reduced the cytotoxicity of the conjugate, so the in vitro cytotoxicity was antigen-specific.

[0426] The in vitro cytotoxicity of the conjugate of the present invention was compared with an Ab-sSPDB-DM4 conjugate having a cleavable disulfide linker and a peptide anilinomercaptopurine conjugate (Table 1). As shown in Table 1, the conjugate of the present invention was generally more cytotoxic than the Ab-sSPDB-DM4 conjugate. In addition, spacer L 1The length of the alkyl chain therein has almost no effect on the cytotoxicity against antigen-positive cells.

Table 1b

[0427] The in vitro cytotoxicity of the conjugate of the present invention was also tested against CA922 cells. As shown in FIG. 14, D-Ala in the peptide linker of the conjugate, when directly bonded to the sacrificial nitrogen in the -NH-CR 1 R 2 -S- moiety of the conjugate of formula (I), 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 FIG. 15 and Table 2 below. This data suggests that an increase in the hydrophobicity of the metabolite (L 1 long alkyl chain in the spacer) increased the cytotoxicity of the metabolite cells.

Table 2

[0429] Example 7. In vivo efficacy study The in vivo efficacy of the ADC was tested in established xenografts (H1703 250 mm 3 ), HT-29 (100 mm 3 ) or NCI-H2110 (100 mm 3Evaluations were conducted in mice bearing 3 . The desired cell type in serum-free medium / matrigel was subcutaneously inoculated into the right abdomen of female SCID mice. The tumors were allowed to grow to the specified size. Next, the animals were randomly divided into multiple groups (6 animals per group). Control mice were treated with phosphate-buffered saline. The ADC was administered to the mice at the mg / kg levels indicated in the study. All dosages in the xenograft model were based on the weight of the antibody component of the conjugate. All treatments were administered by intravenous injection into the tail vein. Tumor size was measured three-dimensionally using calipers twice a week, and the tumor volume was expressed in mm

[0430] As shown in FIGS. 10, 11A, 11B and 12, the conjugates of the present invention are highly active against H1703 (FIG. 10), HT-29 (FIGS. 11A and 11B), NCI-H2110 (FIG. 12) xenograft tumors in an in vivo mouse model. Conjugate 17c has good tolerance compared to the peptide anilinomethylthienoindole conjugate (see FIG. 13).

[0431] Mouse tolerance studies were also conducted with conjugates of the present invention having different peptide linkers (i.e., A in formula (I)). The body weights of the mice administered the conjugate were measured. As shown in FIG. 18, when the peptide linker contains consecutive L-alanine, mouse tolerance decreases.

[0432] Example 8. In vitro bystander killing assay As previously described, a bystander killing assay was performed in which the ratio of antigen-positive to antigen-negative cells designated in the associated drawings was used to keep the number of antigen-negative cells constant in the presence of various numbers of antigen-positive cells. This assay in which antigen-negative and antigen-positive cells were kept constant was varied as follows: 3000 EGFR+ Ca9-22 cells were mixed with 2000 EGFR-MCF7 cells and the cell mixture was incubated with the indicated ADC at 0.66 nM for 4 days. The WST-8 assay was used to quantify viable cells. In the same assay, the cytotoxic efficacy of the ADC against Ca9-22 or MCF7 cells was also evaluated; 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 the conjugate of the invention that is not toxic to FRα(−) cells (Namalwa, seeded with 1000 cells) but kills all FRα(+) cells (JEG-3, 150K FRα ABC). The viability of FRα(−) cells was measured after 4 days by the Cell Titer Glo assay (Promega). The data are shown in FIG. 9C. In particular, bystander killing with the conjugate of the invention was compared to a peptide aniline methanoid conjugate and the data are shown in Table 3 below.

Table 3

[0434] As shown in FIGS. 9A, 9B, and 9C, the conjugates of the present invention have a higher bystander killing effect than the Ab-sSPDB-DM4 conjugate and the peptide anilinomatan sinoid conjugate having a cleavable disulfide linker. In addition, when other factors are kept constant, an increase in the hydrophobicity of the metabolite increases the cytotoxicity of the metabolite, which increases the bystander killing of the corresponding conjugate. Furthermore, this data appears to show that the conjugates of the present invention differ from the Ab-sSPDB-DM4 conjugate and the peptide anilinomatan sinoid conjugate in the type and release efficiency of the metabolites released. The bystander killing of the conjugates of the present invention is greater than that of the peptide anilinomatan sinoid conjugate, which in turn is greater than that of Ab-sSPDB-DM4 (17g > 4b > 1b).

[0435] As shown in FIG. 9D, when D-Ala in the peptide linker of the conjugate is directly bonded to the sacrificial nitrogen in the -NH-CR 1 R 2 -S- moiety, it is harmful to bystander killing.

[0436] Example 9. in vitro Metabolism Study FRα-expressing KB cells were treated with a saturating amount of the 17c conjugate for 24 hours. The catabolic metabolite-containing medium was incubated with 5 mM NEM to cap any free thiols present and then captured by a pre-bound protein A-anti-matan sinoid antibody complex. The catabolic metabolites were released by acetone extraction and analyzed by UHPLC / HRMS.

[0437] The detected metabolites and the presented cleavage sites are shown in FIG. 16. The main efflux catabolic metabolites identified in the cell culture medium were DM-SMe (25a) and DM-SH species.

[0438] In another experiment, conjugate 18c (anti-CanAg-LDL-DM) was incubated with COLO205 cells by the previously described method (see Erickson HK, Park PU, Widdison WC, Kovtun YV, Garrett LM, Hoffman K, et al. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res 2006;66(8):4426-33). Subsequently, the cells were lysed, all disulfide bonds were reduced, and the resulting thiols were capped with N-ethylmaleimide. Non-treated controls were also performed, where COLO205 cells were lysed without treatment with the conjugate, disulfide bonds were reduced, and then the resulting thiols were capped with N-ethylmaleimide. Both samples were analyzed by UPLC / MS using a Thermo Q-Exactive mass spectrometer set for Pos and Neg, and DDA Top-10 MS / MS detection was tandem with a Dionex UltiMate 3000 UPLC equipped with a Waters UPLC BEH C8, 1.8 micrometer, 100×2.1 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 at a flow rate of 0.35 mL / min with a linear gradient from 20% to 100% acetonitrile containing 0.1% formic acid over 20 minutes, followed by a 10-minute flush with 100% acetonitrile containing 0.1% formic acid. As shown in Figure 19, the upper UPLC trace is from the DTT and NEM-treated cell lysate from COLO205 cells that were not exposed to any conjugate. The lower UPLC trace is from the DTT and NEM-treated cell lysate from COLO205 cells treated with conjugate 18c. The retention time peak at 12.73 minutes is for CH prepared in the laboratory 3S(CH 2 ) 5 It had the same retention time and mass spectrum as the CO-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 h. The cells and medium were separated, the catabolites were extracted by affinity capture and reconstituted in 20% acetonitrile. The catabolites were analyzed by UHPLC / HRMS. The main catabolite species identified in the cell medium included DM-SMe, oxidized DM-SMe and the acid form of the free drug (see the structural formula below).

Chemical formula

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

[0441] As shown in FIGS. 17A and 17B and Table 4, the conjugates of the present invention show enhanced activity against a xenograft ovarian tumor xenograft model with a relatively low FRα expression level (H score 35) compared to the peptide anilinomeptansinoid conjugate.

Table 4a

[0442] Example 11. Mouse tolerance study a. The tolerability of 1200 μg / kg of 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] Group and treatment: (2 mice / group) 1. Vehicle control 2. huML66-GMBS-LAlaLAlaLAla-Immol-DM (16a), 1200 μg / kg 3. huML66-GMBS-DAlaLAlaLAla-Immol-DM (16b), 1200 μg / kg 4. huML66-GMBS-LAlaDAlaLAla-Immol-DM (16c), 1200 μg / kg 5. huML66-GMBS-LAlaLAlaDAla-Immol-DM (16d), 1200 μg / kg 6. huML66-sSPDB-DM4 (la), 1200 μg / kg

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

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

[0446] b. The tolerances of Movl9vl.6-GMBS-lAladAlalAla-Immol-DM at 1000 μg / kg and 1250 μg / kg and 1250 μg / kg Movl9vl.6-GMBS-dAlalAla-PAB-DMl were tested in female CD-1 mice.

Table 4c

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

[0448] Specific design of the study: Thirty-two mice were randomized by body weight into four groups (8 mice per group). The body weights ranged from 23.4 to 27.3 grams (25.5 ± 1.02, mean ± SD). The mice in each group were identified by ear notching. Treatment was started on day 8 after arrival. The mice were administered the conjugate 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, 1 / 2-inch needle. Depending on the given dose, the treatment was divided into two injections 2 hours apart. The mice could not receive more than 350 μl per injection.

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

[0450] Example 12. Pharmacokinetic Study 1. Conjugate 17c The pharmacokinetics of M-LDL-IMM-DM (Conjugate 17c) and M-SPDB-DM4 were evaluated in female CD-1 mice. Based on body weight, the mice were randomly assigned to two groups of six mice each. Mice in Group A were given a single intravenous injection of 10 mg / kg of M-LDL-IMM-DM via the tail vein. Mice in Group B were given a single intravenous injection of 10 mg / kg of M-SPDB-DM4 via the tail vein. Blood was collected at 2 minutes, 6, 24, 48, 72, 168, 336, 504, 672, and 840 hours. Then, to ensure that no more than two blood collections were made from a mouse within 24 hours, the mice were bled in sequence. Serum was separated from the blood, and the samples were frozen at -80 °C until analysis by ELISA. Total antibody and ADC ELISA were performed on these samples, and the concentration-time plots are shown in Figure 26. In the total antibody ELISA, antibodies carrying at least one maytansinoid, as well as antibodies to which no maytansinoid is bound, are quantified. The concentration is determined by capture with an anti-human IgG antibody and then quantified using an enzyme-labeled anti-human IgG antibody. The ADC ELISA involves capturing conjugates carrying at least one conjugated maytansinoid using an anti-maytansinoid antibody, then capturing the antibody component of the conjugate and detecting it with an enzyme-labeled anti-human FC antibody. As detected, the conjugate must contain at least one covalently bound maytansinoid.

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

[0452] 2. Conjugate 26c CD-1 mice were injected with a single dose of 10 mg / kg of 26c or M9346A-C442-mal-SPDB-DM4. Blood was collected at 2 minutes, 24 hours, and 72 hours after injection. The ADCs were purified from plasma using affinity capture with folate receptor α-Fc fusion protein, and the samples were analyzed by size exclusion chromatography (SEC) and mass spectrometry (MS). Loss of DM or DM4 was measured as the percent normalized degradation over time and plotted in Figure 27. The in vivo stability of 26c was higher than that of M9346A-C442-mal-SPDB-DM4, as demonstrated by the less degradation observed at the 2-minute time point (-0.2 vs 7.3%) and the 24-hour time point (5.2 vs 16.5%). The 72-hour sample concentration of M9346A-C442-mal-SPDB-DM4 was too low to obtain a percent normalized degradation value. [1] The following formula:

Chemical formula

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

[11] L 1 is -(CH 2 ) 4~6 -C(=O)-. The conjugate according to [6].

[12] L 2 is the following structural formula: [Chemical formula] the conjugate according to any one of [1] to

[11] , represented by [wherein, R A is alkylene, cycloalkylalkylene or arylene; R B and R C are each independently absent, alkylene, cycloalkylalkylene, or arylene; V and V' are each independently -(O-CH 2 -CH 2 ) P -, or -(CH 2 -CH 2 -O) P -; p is 0 or an integer from 1 to 10; W is absent or [Chemical formula] and, where s2' indicates the site connected to V, R A or J CB , and s3' indicates the site connected to R B , V', R C or J A ; J CB is -C(=O)-, [Chemical formula] and, where s1 indicates the site connected to the cell binding agent CB, and s2 indicates the site connected to R A ; R a R b R c , and R e are each independently H or alkyl for each occurrence; J Ais -C(=O)-.

[13] L 2 is the following structural formula: [Chemical formula] represented by R A is alkylene, cycloalkylalkylene or arylene; R B and R C each independently either does not exist, or is alkylene, cycloalkylalkylene, or arylene; V and V’ each independently is -(O-CH 2 -CH 2 ) P -, or -(CH 2 -CH 2 -O) P -; p is 0 or an integer from 1 to 10; W either does not exist, or [Chemical formula] is; J CB is -C(=O)-, [Chemical formula] where s1 indicates the site connected to the cell binding agent CB, and s2 indicates the site connected to R A ; J A is -C(=O)-, the conjugate according to any one of [1] to

[12] .

[14] p is 0 and R C does not exist, the conjugate according to

[12] or

[13] .

[15] J CB is -C(=O)- or [Chemical formula] The conjugate according to any one of

[12] to

[14] .

[16] L 2 is represented by the following structural formula:

Chemical formula

[12] to

[15] , represented by [wherein, R x 、R y 、R x’ and R y’ are each independently, for each occurrence, H, -OH, halogen, -O-(C 1~4 alkyl), -SO 3 H, -NR 40 R 41 R 42 + 、or -OH, halogen, -SO 3 H or -NR 40 R 41 R 42 + optionally substituted C 1~4 alkyl, where R 40 、R 41 and R 42 are each independently H or C 1~4 alkyl; 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].

[17] R x 、R y 、R x’ and R y’ are all H, the conjugate according to

[16] .

[18] l and k are each independently an integer from 2 to 6, the conjugate according to

[16] or

[17] .

[19] L 2 is represented by the following structural formula:

Chemical formula

[12] to

[15] , represented by [wherein, R x and R y are both H; l and l1 are each an integer from 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 cleavable by a protease.

[21] The conjugate according to

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

[22] 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 as L or D isomers, -NH-CR 1 R 2 -S-L 1 The conjugate according to any one of [1] to

[21] , which has an amino acid covalently bonded to -D.

[23] -NH-CR 1 R 2 -S-L 1 The conjugate according to any one of [1] to

[22] , wherein the amino acid connected to -D is an L-amino acid.

[24] 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, 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 an L group, and the last amino acid in each peptide is -NH-CR The first amino acid in the peptide is L 2 group, and the last amino acid in each peptide is -NH-CR 1 R 2 -S-L 1 -D, the conjugate according to any one of [1] to

[19] .

[25] 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:

Chemical formula

[26] , represented by

[28] D is the following formula: [Chemical formula] The conjugate described in

[27] , represented by

[29] The following formula: [Chemical formula] The conjugate according to any one of claims 1 to 28, represented by [In the formula, [Chemical formula] is the cell binding agent connected to the L 2 group via a Lys amine group; [Chemical formula] is the cell binding agent connected to the L 2 group via a Cys thiol group; R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1 and t1 are each independently an integer from 1 to 6; m2, n2, r2, s2 and t2 are each independently an integer from 1 to 7; t3 is an integer from 1 to 12; D 1 is the following formula: [Chemical formula] represented by

[30] D 1 is the following formula: [Chemical formula] The conjugate described in

[29] , represented by

[31] The conjugate described in

[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 from 2 to 4; and m2, n2 and r2 are each independently an integer from 3 to 5.

[33] The following formula:

Chemical formula

[29] to

[31] , represented by [wherein, r1 and t1 are each an integer from 2 to 6; r2 and t2 are each an integer from 2 to 5; t3 is an integer from 2 to 12].

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

[29] to

[33] , wherein both are Me.

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

[29] to

[33] , wherein both are H.

[36] The following formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[29] or a pharmaceutically acceptable salt thereof, represented by [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, D 1 is the following formula: [Chemistry] represented by ].

[37] The following formula: [Chemistry] [Chemistry] [Chemistry] The conjugate described in

[36] or a pharmaceutically acceptable salt thereof, represented by [In the formula, D 1 is the following formula: [Chemistry] represented by ].

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

[37] , wherein the cell-binding agent is an antibody or an antigen-binding fragment thereof, a single-chain antibody, a single-chain antibody fragment specifically binding to a target cell, a monoclonal antibody, a single-chain monoclonal antibody, or a monoclonal antibody fragment specifically binding to a target cell, a chimeric antibody, a chimeric antibody fragment specifically binding to a target cell, a domain antibody, a domain antibody fragment specifically binding 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 (registered trademark) peptide, or a pentrin.

[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-Mucl 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, a light chain variable region CDR2 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, the conjugate according to

[45] . and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 38, the conjugate according to

[45] .

[47] 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, the conjugate according to

[45] .

[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 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, the conjugate according to

[45] .

[51] 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, the conjugate according to

[45] .

[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] The following formula:

Chemical formula

[54] Below formula: [ka] The conjugate according to [1], [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 claim 53 or 54,

[56] The conjugate described in

[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 described in

[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 of

[53] or

[54] , wherein the anti-folate receptor antibody comprises 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.

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

[61] R 1 and R 2 At least one of which is H, the compound according to

[60] .

[62] R 1 and R 2 are each independently H or Me, the compound according to

[60] .

[63] R 1 and R 2 One of which is H and the other is Me, the compound according to

[60] .

[64] R 1 and R 2 Both are H, the compound according to

[60] .

[65] L 1 is -L 1 '-C(=O)-; L 1 ' is alkylene or cycloalkylene, the compound according to any one of

[60] to

[64] .

[66] L 1 ' is C 1~10 alkylene, the compound according to

[65] .

[67] L 1 is -CR 3 R 4 -(CH 2 ) 1~8 -C(=O)-; and R 3 and R 4 are each independently H or Me, the compound according to

[65] .

[68]

[68] L 1 is -CR 3 R 4 -(CH 2 ) 3~5 -C(=O)-, the compound according to

[67] .

[69] R 3 and R 4 are both Me, the compound according to

[67] or

[68] .

[70] L 1 is -(CH 2 ) 4~6 -C(=O)-, the compound according to

[66] .

[71] L 2 ’ is represented by the following structural formula: J CB ’-R A -V-W-R B -V’-R C -J A - the compound according to any one of

[60] to

[70] , [wherein, R A is alkylene, cycloalkylalkylene or arylene; R B and R C each independently do not exist, or are alkylene, cycloalkylalkylene, or arylene; V and V’ each independently are -(O-CH 2 -CH 2 ) P -, or -(CH 2 -CH 2 -O) P -; p is an integer of 0 or 1 to 10; W is absent or

Chem.

Chem.

[72] L 2 ’ has the following structural formula: J CB ’-R A -V-W-R B -V’-R C -J A - represented by the compound according to any one of

[60] to

[70] [wherein, R A is alkylene, cycloalkylalkylene or arylene; R B and R C are each independently absent or alkylene, cycloalkylalkylene, or arylene; V and V’ are each independently -(O-CH 2 -CH 2 ) P-, or -(CH 2 -CH 2 -O) P -; p is an integer of 0 or 1 to 10; W is absent or

Chemical formula

Chemical formula

[73] When p is 0 and R C is absent, the compound described in

[71] or

[72] .

[74] J CB ’ is -C(=O)OH, -COE or

Chemical formula

[71] to

[73] .

[75] L 2 ’ has the following structural formula:

Chemical formula

[71] to

[74] [In the formula, R x 、R y 、R x’ and R y’ are each independently, for each occurrence, H, -OH, halogen, -O-(C 1~4 alkyl), -SO 3 H, -NR 40 R 41 R 42 + 、or -OH, halogen, -SO 3H or -NR 40 R 41 R 42 + optionally substituted C 1~4 alkyl, where R 40 、R 41 and R 42 are each independently H or C 1~4 alkyl; l and k are integers from 1 to 10; J CB ’ is -C(=O)OH or -COE].

[76] R x 、R y 、R x’ and R y’ are all H, the compound according to

[75] .

[77] l and k are each independently integers from 2 to 6, the compound according to

[75] or

[76] .

[78] L 2 ’ is the following structural formula:

Chemical formula

[71] to

[74] [wherein, R x and R y are both H; l and l1 are each, integers from 2 to 6; k1 is an integer from 1 to 12].

[79] A is a peptide cleavable by protease, the compound according to any one of

[60] to

[78] .

[80] A is a peptide cleavable by protease expressed in tumor tissue, the compound according to

[79] .

[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 as L or D isomers, -NH-CR 1 R 2 -S-L 1 -D, a peptide having an amino acid covalently bonded thereto, the compound according to any one of

[60] to

[80] .

[82] -NH-CR 1 R 2 -S-L 1 -D, wherein the amino acid connected to is an L-amino acid, the compound according to any one of

[60] to

[81] .

[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-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, 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 L 2 is connected to the group, and the last amino acid in each peptide is 1 R 2 -S-L 1 -D, a compound according to any one of

[60] to

[78] .

[84] 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, a compound according to

[83] .

[85] D is a methanoid, a compound according to any one of

[60] to

[84] .

[86] D is of the formula:

Chemical formula

[87] D is of the formula:

Chemical formula

[86] , represented by

[88] The following formula: [Chemical formula] The compound described in any one of

[60] to

[84] , represented by [In the formula, R 3 and R 4 are each independently H or Me; m1, m3, n1, r1, s1 and t1 are each independently an integer from 1 to 6; m2, n2, r2, s2 and t2 are each independently an integer from 1 to 7; t3 is an integer from 1 to 12; J CB ’ is -C(=O)OH or -COE; D 1 is the following formula: [Chemical formula] [represented by]]

[89] The compound described in

[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 described in

[88] or

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

[91] The following formula: [Chemical formula] The compound described in

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

[92] R 3 and R 4 are both Me, the compound according to any one of

[88] to

[91] .

[93] R 3 and R 4 are both H, the compound according to any one of

[88] to

[91] .

[94] The following formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[88] or a pharmaceutically acceptable salt thereof [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, J CB ’ is -C(=O)OH or -COE; D 1 is the following formula:

Chemical formula

[95] The following formula:

Chemical formula

[94] , represented by [wherein D 1 is the following formula: [Chemical formula] [represented by].

[96] -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, the compound according to any one of

[60] to

[95] .

[97] -COE is N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester, the compound according to any one of

[60] to

[95] .

[98] The following formula: [Chemical formula] The compound represented by or a pharmaceutically acceptable salt thereof [wherein A’ is one amino acid or a peptide containing 2 to 20 amino acids; R 1 and R 2 are each independently H or C 1~3 alkyl; L 1 is a spacer; D-L 1 -SH is a cytotoxic drug; q is an integer from 1 to 20].

[99] R 1 and R 2 at least one of which is H, the compound according to

[98] .

[0100] R1 and R 2 The compound according to

[98] , wherein each of them is independently H or Me.

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

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

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

[98] , wherein both of them are H.

[0103] L 1 is -L 1 ’-C(=O)-; L 1 ’ is alkylene or cycloalkylene, and the compound according to any one of

[98] to

[0102] .

[0104] L 1 ’ is C 1~10 alkylene, and the compound according to

[0103] .

[0105] L 1 is -CR 3 R 4 -(CH 2 ) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me, and the compound according to

[0103] .

[0106] L 1 is -CR 3 R 4 -(CH 2 ) 3~5 -C(=O)-, and the compound according to

[0105] .

[0107] R 3 and R 4 are both Me, and the compound according to

[0105] or

[0106] .

[0108] L 1 is -(CH 2 ) 4~6 -C(=O)-, and the compound according to

[0103] .

[0109] The compound according to any one of

[98] to

[0108] , wherein A’ is a peptide cleavable by a protease.

[0110] The compound according to

[0109] , wherein A’ is a peptide cleavable 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 as L or D isomers, -NH-CR 1 R 2 -S-L 1 The compound according to any one of

[98] to

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

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

[98] to

[0111] , wherein the amino acid connected to -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, 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, 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 L 2 group is connected, and the last amino acid in each peptide is -NH-CR 1 R 2 -S-L 1 -D is connected, a compound according to any one of

[98] to

[0108] .

[0114] 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, a compound according to

[0113] .

[0115] D is a maitansinoid, a compound according to any one of

[98] to

[0114] 。

[0116] D is the following formula:

Chemical formula

[0115] .

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

[0116] , represented by

[0118] The following formula: [Chemical formula] The compound according to any one of

[98] to

[0117] , represented by [wherein, R 3 and R 4 are each independently H or Me; m2 is an integer from 1 to 7; D 1 is the following formula: [Chemical formula] represented by

[0119] D 1 is the following formula: [Chemical formula] The compound according to

[0118] , represented by

[0120] The 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 from 3 to 5.

[0122] R 3 and R 4 are both Me, the compound according to any one of

[0118] to

[0121] .

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

[0118] to

[0121] , wherein both are H.

[0124] The following formula:

Chemical formula

[0118] or a pharmaceutically acceptable salt thereof represented by [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, D 1 is the following formula:

Chemical formula

[0125] D 1 is the following formula:

Chemical formula

[0124] represented by.

[0126] The following formula:

Chemical formula

Chemical formula

[0127] R 1 and R 2 at least one of which is H, the compound according to

[0126] .

[0128] R 1 and R 2 are each independently H or Me, the compound according to

[0126] .

[0129] R 1 and R 2 one of which is H and the other is Me, the compound according to

[0128] .

[0130] R 1 and R 2 both of which are H, the compound according to

[0126] .

[0131] L 1 is -L 1 ’-C(=O)-; L 1 ’ is alkylene or cycloalkylene, the compound according to any one of

[0126] to

[0130] .

[0132] L 1 ’ is C 1~10 an alkylene, the compound according to

[0131] .

[0133] L 1 is -CR 3 R 4 -(CH 2 ) 1~8 -C(=O)-; R 3 and R 4 are each independently H or Me, the compound according to

[0131] .

[0134] L 1 is -CR 3 R 4 -(CH 2 ) 3~5 -C(=O)-, the compound according to

[0133] .

[0135] R 3 and R 4 are both Me, the compound according to

[0133] or

[0134] .

[0136] L 1 is -(CH 2 ) 4~6 -C(=O)-, the compound according to

[0131] .

[0137] R x’ and R y’ are both H, the compound according to any one of

[0126] to

[0136] .

[0138] k is an integer from 2 to 6, the compound according to

[0137] .

[0139] k is 3, the compound according to

[0137] .

[0140] A is a peptide cleavable by a protease, the compound according to any one of

[0126] to

[0139] .

[0141] A is a peptide cleavable by a protease expressed in tumor tissue, the compound according to

[0140] .

[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 as L or D isomers, -NH-CR 1 R 2 -S-L 1 -D, which is a peptide having an amino acid covalently bonded thereto, the compound according to any one of

[0126] to

[0141] .

[0143] -NH-CR 1 R 2 -S-L 1 -D, wherein the amino acid connected thereto is an L-amino acid, the compound according to any one of

[0126] to

[0142] .

[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-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, 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 L 2 is connected to the group, and the last amino acid in each peptide is 1 R 2 -S-L 1 -D, a compound according to any one of

[0126] to

[0139] .

[0145] A 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] A compound according to any one of claims 126 to 145, wherein D is a maytansinoid.

[0147] D is of the formula:

Chemical formula

[0146] .

[0148] D is of the formula: [Chemical formula] The compound according to

[0147] , represented by

[0149] The following formula: [Chemical formula] The compound according to any one of

[0126] to

[0148] , represented by [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; D 1 is the following formula: [Chemical formula] 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 from 2 to 4; and m2 is an integer from 3 to 5.

[0152] R 3 and R 4 are both Me, the compound according to any one of

[0149] to

[0151] .

[0153] R 3 and R 4 are both H, the compound according to any one of

[0149] to

[0151] .

[0154] The following formula: [Chemical formula] The compound according to

[0149] or a pharmaceutically acceptable salt thereof, represented by [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, D 1 is the following formula:

Chemical formula

[0155] D 1 is the following formula:

Chemical formula

[0154] , represented by.

[0156] The following formula: D-L 1 -SZ 0 (V) The compound represented by [wherein, L 1 is a spacer; Z 0 is H or Me, provided that when Z 0 is H, L 1 is -C(=O)-(CH 2 ) q - or -C(=O)-CH 2 -CH 2 -C(CH 3 ) 2 - and not, where q is an integer from 1 to 3; when Z 0 is Me, L 1 is -C(=O)-(CH 2 ) 2 - or -C(=O)-CH 2 -CH 2 -C(CH 3 ) 2 - and not; D-L 1-SH is a cytotoxic drug.

[0157] L 1 is -L 1 ’-C(=O)-; and L 1 ’ is alkylene or cycloalkylene, where the -C(=O)- moiety in L is bonded to D, the compound according to

[0156] . 1

[0158] 1 L’ is C 1~10 alkylene, the compound according to

[0157] .

[0159] 1 Lis -CR 3 4 R-(CH 2 ) 1~8 -C(=O)-; and R 3 and R 4 are each independently H or Me, the compound according to

[0157] .

[0160] 1 Lis -CR 3 4 R-(CH 2 ) 3~5 -C(=O)-, the compound according to

[0159] .

[0161] 3 Rand R 4 are both Me, the compound according to

[0159] or

[0160] .

[0162] 1 Lis -(CH 2 ) 4~6 -C(=O)-, the compound according to

[0157] .

[0163] D is of the formula:

Chemical formula

[0156] ~

[0162] .

[0164] D is of the formula:

Chemical formula

[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 growth or treating a proliferative disorder in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the conjugate according to any one of [1] to

[59] .

[0167] The method according to

[0166] for treating cancer.

[0168] The method according to claim 167, wherein the cancer is selected from renal 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, lymphoma (e.g., non-Hodgkin lymphoma), myelodysplastic syndrome (MDS), peritoneal cancer, fallopian tube cancer, ute...

Claims

[Claim 1] Below formula: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof. [In the formula, CB is a cell binding agent; L ２ 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; L １ is a spacer; D-L １ -SH is a cytotoxic drug; and q is an integer from 1 to 20.

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