WO2017054080A1

WO2017054080A1 - Cytotoxic and anti-mitotic compounds, and methods of using the same

Info

Publication number: WO2017054080A1
Application number: PCT/CA2016/051135
Authority: WO
Inventors: Geoffrey C. Winters; James R. RICH; Alexander L. Mandel; Tom Han Hsiao HSIEH; Graham Albert Edwin GARNETT
Original assignee: Zymeworks Inc.
Priority date: 2015-09-29
Filing date: 2016-09-29
Publication date: 2017-04-06

Abstract

Compounds of general formula (I) having cytotoxic and/or anti-mitotic activity and methods of using such compounds, as well as pharmaceutical compositions comprising such compounds. The compounds may also be conjugated with a targeting moiety, such as an antibody. Such conjugate compositions may be used in the treatment of cancer and other diseases. (I)

Description

CYTOTOXIC AND ANTI MITOTIC COMPOUNDS, AND METHODS OF USING THE

SAME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of the filing date of U.S. Provisional Patent Application Serial No. 62/234,452, filed on September 29, 2015, the disclosure of which application is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to biologically active compounds, compositions comprising the same, and methods of using such biologically active compounds and compositions for the treatment of cancer and other diseases.

BACKGROUND

[0003] Tubulysins are a class of cytostatic compounds originally isolated from strains of Mycobacteria (Sasse, F., et al , (2000) J. Antibiot. (Tokyo) 53:879-885). Tubulysins have a tetrapeptidyl scaffold constructed from one proteinogenic and three non-proteinogenic amino acid subunits as shown in Formula A: N-methylpipecolic acid (Mep), isoleucine (He), tubuvaline (Tuv), and either tubuphenylalanine (Tup, R" is H) or tubutyrosine (Tut, R" is OH). Various tubulysins have been identified. Some examples of naturally occurring tubulysins are shown in Table 1 :

TABLE 1: Certain Naturally Occurring Tubuly

Formula A:

[0004] Kaur et al. ((2006) Biochem. J. 396:235-242) studied the antiproliferative properties of tubulysin A and found that it was more potent than other antimitotic agents such as paclitaxel and vinblastine and was active in xenograft assays against a variety of cancer cell lines. Further, tubulysin A induced apoptosis in cancer cells but not normal cells and showed significant potential anti-angiogenic properties in in vitro assays. The antimitotic properties of other tubulysins have also been evaluated and generally have been found to compare favorably against those of non- tubulysin antimitotic agents (see, e.g., Balasubramanian et al , (2009) J. Med. Chem. 52(2):238- 240; Steinmetz et al, (2004) Angew. Chem. Int. Ed. 43:4888-4892; Wipf et al, (2004) Org. Lett. 6(22):4057-4060). For these reasons, there is considerable interest in the tubulysins as anti-cancer agents (see, e.g. , Domling et al, (2005) Mol. Diversity 9: 141-147; and Hamel et al, (2002) Curr. Med. Chem.-Anti-Cancer Agents 2: 19-53).

[0005] Numerous publications describe efforts directed at the synthesis of tubulysins, including Domling et al, (2006) Ang. Chem. Int. Ed. 45:7235-7239; Hoefle et al, (2003) Pure Appl. Chem. 75(2-3): 167-178; Neri et al, (2006) Chem MedChem 1 : 175-180; Peltier et al, (2006) J. Am. Chem. Soc. 128: 16018-16019; Sani et al , (2007) Angew. Chem. Int. Ed. 46:3526-3529; Sasse et al, (2007) Nature Chem. Biol. 3(2):87-89; Shankar et al , (2009) Synlett 8: 1341-1345; Shibue et al, (2009) Tetrahedron Lett. 50:3845-3848; and Wipf et al , (2004) Org. Lett. 6(22):4057-4060.

[0006] Other publications describe the preparation and evaluation of tubulysin analogs or derivatives including International Patent Publication Nos. WO 2009/012958, WO 2008/138561 and WO 2009/055562; U.S. Patent Publication Nos. US 2011/0021568, US 2013/0204033, US

2014/0227295, US 2014/0227298, US 2005/0239713, US 2006/0128754 and US 2010/0047841;

German Patent Application Nos. DE 10 2004 030 227 and DE 100 08 089; Yang et al, (2013)

Tetrahedron Lett. 54:2986-2988; Raghavan et al, (2008) J. Med. Chem. 51(6): 1530-1533; Balasubramanian et al, (2008) Bioorg. Med. Chem. Lett. 18:2996-2999; Balasubramanian et al,

(2009) J. Med. Chem. 52(2):238-240; Patterson et al , (2007) Chem. Eur. J. 13:9534-9541;

Patterson et al, (2008) J. Org. Chem. 73:4362-4369; Wang et al , (2007) Chem. Biol. Drug. Des.

70:75-86, and Wipf et al , (2007) Org. Lett. 9(8): 1605-1607.

[0007] Tubulysins conjugated to various targeting moieties have also been described, including those described in International Patent Publication Nos. WO 2015/113760, WO 2014/009774, WO

2014/134543, WO 2013/173393, WO 2009/002993 and WO 2009/026177; U.S. Patent Publication

Nos. US 2008/0248052, US 2011/0027274, US 2010/0048490 and US 2008/0279868; U.S. Pat.

Nos. 7,691,962 and 7,776,814; Vlahov et al , (2008) Bioorg. Med. Chem. Lett. 18(16):4558-4561;

Leamon et al , (2008) Cancer Res. 68(23):9839-9844, and Reddy et al , (2009) Mol. Pharmaceutics 6(5): 1518-1525. [0008] It has been reported that deacetylation of the Tuv in tubulysin subunit leads to loss of biological activity (Domling et al, (2006) Ang. Chem. Int. Ed. 45:7235-7239). In a study of tubulysins U and V, which differ in the former being acetylated and the latter being deacetylated, tubulysin V was reported to be less potent by about 200 ^χ to 600 ^χ, depending on the assay (Balasubramanian et al , (2009) J. Med. Chem. 52(2):238-240).

[0009] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention.

SUMMARY

[0010] The present disclosure relates to biologically active compounds, compositions comprising the same, and methods of using such compounds and compositions.

[0011] In one aspect, the present disclosure relates to a compound of general Formula I:

I or a pharmaceutically acceptable salt thereof, or a precursor thereof that comprises more protecting groups, wherein:

R is selected from: amino-Ci-C6 alkyl, amino-aryl, amino-C3-C? cycloalkyl, amino- heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from aryl, aryl-G-C6 alkyl, G-C6 alkyl, G-C6 alkylthio, carboxyl, carboxamide, C3-C7 cycloalkyl, C3-C7 cycloalkyl -G-G; alkyl, guanidino, halo, G-C6 haloalkyl, heterocyclyl, heterocyclyl -G-C6 alkyl, hydroxyl, and thio; or

R is R^NCH R³)-; is selected from: H and G-C6 alkyl; R² is C1-C6 alkyl; and

R³ is selected from: H, R¹⁵ and R⁷-CH(CH₃)₂-; or

R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl;

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-C6 alkoxycarbonyl, G-C6 alkyl, G-C6 alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloacyl, G-C6 haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁷ is selected from: H, aryl, G-G cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: G-G acylthio, G-G alkenyl, G- G alkyl, G-G alkylamino, G-G alkyloxy, amino, amino-G-G alkyl, halo, G-G haloalkyl, hydroxyl, hydroxy-G-G alkyl, and thio, wherein G-G alkenyl, G-G alkylamino and G-G alkyloxy are further optionally substituted with one substituent selected from G-G alkylaryl, hydroxyl, and thio;

R⁸ is selected from: aryl, heteroaryl, and G-G cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl;

R⁹ and R¹⁰ are each independently selected from: H, G-G alkyl, aryl, aryl-G-G alkyl, G- G cycloalkyl-G-G alkyl, and hydroxy-G-G alkyl;

R¹⁵ is G-G alkyl; X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹;

Y is heteroaryldiyl, or Y is absent; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; with the proviso that when R is R^NGr^R³)-; R¹ is H; R² is methyl; R³ is PhCH(CH₃)₂-; R⁵ is methyl; X is -CH=CH(CH3)-; Y is absent and Z is absent, then R⁴ is other than tert- butyl.

In one aspect, the present disclosure relates to a compound of Formula le:

le or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵ and Z are as defined above for general Formula I;

R⁹ and R¹⁰ are each independently H or G-G alkyl, and R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰, with the proviso that when R¹ is H; R² and R³ taken together with the atoms to which they are each bonded form piperidin-2-yl; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; R¹¹ is acetyl; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl. [0013] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a compound as described above and a pharmaceutically acceptable carrier. [0014] In one aspect, the present disclosure relates to a method of inhibiting proliferation of cancer cells comprising contacting the cancer cells with an effective amount of a compound as described above.

[0015] In one aspect, the present disclosure relates to a method of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a compound as described above.

[0016] In one aspect, the present disclosure relates to a method of treating cancer in a subject comprising administering to the subject an effective amount of a compound as described above.

[0017] In one aspect, the present disclosure relates to a conjugate of general Formula VI: T-(L-(D)_m)_n

VI

[0018] wherein: T is a targeting moiety; L is an optional linker; D is a monovalent radical of a compound of any one of claims 1 to 32; m is an integer between 1 and about 10, and n is an integer between 1 and about 20. [0019] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a conjugate as described above and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present disclosure relates to a method of inhibiting proliferation of cancer cells comprising contacting the cancer cells with an effective amount of a conjugate as described above. [0021] In one aspect, the present disclosure relates to a method of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a conjugate as described above.

[0022] In one aspect, the present disclosure relates to a method of treating cancer in a subj comprising administering to the subject an effective amount of a conjugate as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 presents an example of a synthetic scheme that may be used to prepare certain compounds of general Formula I as described in Example 1 herein.

[0024] FIG. 2 presents another example of a synthetic scheme that may be used to prepare certain compounds of general Formula I as described in Examples 2.1 to 2.5.

[0025] FIG. 3 presents another example of a synthetic scheme that may be used to prepare certain compounds of general Formula I as described in Examples 2.6 to 2.9.

[0026] FIG. 4 presents another example of a synthetic scheme that may be used to prepare certain compounds of general Formula I as described in Examples 2.10 and 2.11. [0027] FIG. 5 presents another example of a synthetic scheme that may be used to prepare certain compounds of general Formula I as described in Example 2.12.

DETAILED DESCRIPTION

[0028] The present disclosure relates to compounds of general Formula I as described below and conjugates comprising such compounds in which the compound is attached, directly or via a linker, to a targeting moiety, such as an antibody.

[0029] In certain embodiments, the compounds exhibit cytotoxic activity. In certain embodiments, the compounds exhibit cytotoxic activity against cancer cells. The cytotoxic activity of the compounds may be exhibited when the free compound is administered, or it may be exhibited when the compound is administered as a conjugate and delivered to a cell by means of the targeting moiety. Accordingly, some embodiments relate to compounds of general Formula I that exhibit cytotoxic activity as free compounds. Some embodiments relate to compounds of general Formula I that exhibit cytotoxic activity in the context of a conjugate targeted to a specific cell type or types, such as an antibody conjugate.

[0030] Certain embodiments of the present disclosure relate to the use of the compounds and conjugates in the treatment of a disease or disorder in a subject, for example, an immunological disease or a cancer. Definitions

[0031] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings. Trade names used herein are intended to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.

[0032] As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0033] The use of the word "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one."

[0034] As used herein, the terms "comprising," "having," "including" and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term "consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term "consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.

[0035] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Chemical Definitions

[0036] The term "acyl," as used herein, refers to the group -C(0)R, where R is hydrogen or alkyl, wherein alkyl is as defined herein.

[0037] The term "acyloxy", as used herein, refers to -OC(0)-alkyl, wherein alkyl is as defined herein. Examples of acyloxy include, but are not limited to: formyloxy, acetoxy, propionyloxy, isobutyryloxy, pivaloyloxy, and the like.

[0038] The term "acylthio", as used herein, refers to -SC(0)-alkyl, wherein alkyl is as defined herein. Examples of acylthio include, but are not limited to: formylthio, acetylthio, propionylthio, isobutyrylthio, pivaloylthio, and the like.

[0039] The term "alkoxycarbonyl", as used herein, refers to -C(0)0-alkyl, wherein alkyl is as defined herein. Examples of alkoxycarbonyl include, but are not limited to: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, sec-butoxycarbonyl, isobutoxycarbonyl, Z-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl, t- pentyloxycarbonyl, neo-pentyloxycarbonyl, 1-methylbutoxycarbonyl, 2-methylbutoxycarbonyl, n- hexyloxycarbonyl, and the like.

[0040] The term "alkenyldiyl", as used herein, refers to a straight or branched unsaturated hydrocarbon di-radical containing the specified number of carbon atoms, and one or more carbon- carbon double bonds, e.g. , d- e alkenyldiyl, C2-C4 alkenyldiyl, or C2 alkenyldiyl. Examples of alkenyldiyl include, but are not limited to: ethenyldiyl, n-propenyldiyl, isopropenyldiyl, n- butenyldiyl, sec-butenyldiyl, isobutenyldiyl, i-butenyldiyl, pentenyldiyl, isopentenyldiyl, t- pentenyldiyl, neo-pentenyldiyl, 1-methylbutenyldiyl, 2-methylbutenyldiyl, n-hexenyldiyl, and the like. [0041] The term "alkyl", as used herein, refers to a straight or branched saturated hydrocarbon radical containing the specified number of carbon atoms, e.g. ,

alkyl, C₁ -C4 alkyl, or C₂ alkyl. Examples of alkyl include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl, /-butyl, pentyl, isopentyl, 7-pentyl, neo-pentyl, 1-methylbutyl, 2-methylbutyl, n- hexyl, and the like.

[0042] The term "alkyldiyl", as used herein, refers to a straight or branched saturated hydrocarbon di-radical containing the specified number of carbon atoms, e.g. , Ci-Ce alkyldiyl, C1-C4 alkyldiyl, or C2 alkyldiyl. Examples of alkyldiyl include, but are not limited to: methyldiyl, ethyldiyl, n- propyldiyl, isopropyldiyl, n-butyldiyl, sec-butyldiyl, isobutyldiyl, 7-butyldiyl, pentyldiyl, isopentyldiyl, 7-pentyldiyl, neo-pentyldiyl, 1-methylbutyldiyl, 2-methylbutyldiyl, n-hexyldiyl, and the like.

[0043] The term "alkylamino", as used herein, refers to -NH-alkyl, wherein alkyl is as defined herein. Examples of alkylamino include, but are not limited to: methylamino, ethylamino, n- propylamino, isopropylamino, n-butylamino, sec-butylamino, isobutylamino, 7-butylamino, pentylamino, isopentylamino, 7-pentylamino, neo-pentylylamino, 1 -methylbutylamino, 2- methylbutylamino, n-hexylylamino, and the like.

[0044] The term "alkyloxy", as used herein, refers to -O-alkyl, wherein alkyl is as defined herein. Examples of alkyl include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, sec-butoxy, isobutoxy, 7-butoxy, pentyloxy, isopentyloxy, 7-pentyloxy, neo-pentyloxy, 1- methylbutoxy, 2-methylbutoxy, n-hexyloxy, and the like.

[0045] The terms "alkylthio" and "thio-alkyl" are used interchangeably herein to refer to -S-alkyl, wherein alkyl is as defined herein. Examples of alkylthio include, but are not limited to: methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, sec-butylthio, isobutylthio, t- butylthio, pentylthio, isopentylthio, 7-pentylthio, neo-pentylthio, 1-methylbutylthio, 2- methylbutylthio, n-hexylthio, and the like.

[0046] The term "amino", as used herein, refers to -NH2.

[0047] The term "amino-cycloalkyl", as used herein, refers to a cycloalkyl group substituted with one amino substituent, as those terms are defined herein. Examples of amino-cycloalkyl include, but are not limited to: aminocyclopropyl, aminocyclobutyl, aminocyclopentyl, aminocyclohexyl, and the like. [0048] The term "amino-alkyl", as used herein, refers to an alkyl group substituted with one amino substituent, as those terms are defined herein. Examples of amino-alkyl include, but are not limited to: aminomethyl, aminoethyl, amino-n -propyl, amino-isopropyl, amino-n-butyl, amino-sec- butyl, amino-isobutyl, amino-7-butyl, amino-pentyl, amino-isopentyl, amino-7-pentyl, amino-neo- pentyl, amino-l-methylbutyl, amino-2-methylbutyl, amino-n-hexyl, and the like.

[0049] The term "amino-aryl", as used herein, refers to an aryl group substituted with one amino substituent, as those terms are defined herein. Examples of amino-aryl include, but are not limited to: amino-phenyl, amino-naphthalenyl, and the like.

[0050] The term "amino-heteroaryl", as used herein, refers to a heteroaryl group substituted with one amino substituent, as those terms are defined herein. Examples of amino-heteroaryl include, but are not limited to, amino-pyridyl, amino-benzofuranyl, amino-pyrazinyl, and the like.

[0051] The term "amino-heterocyclyl", as used herein, refers to an heterocyclyl group substituted with one amino substituent, as those terms are defined herein. Examples of amino-heterocyclyl include, but are not limited to: amino-pyrrolidinyl, amino-piperidinyl, and the like. [0052] The term "aryl", as used herein, refers to a radical derived from a 6- to 12-membered mono- or bicyclic hydrocarbon ring system wherein at least one ring aromatic. Examples of aryl include, but are not limited to: phenyl, naphthalenyl, 1,2,3,4-tetrahydro-naphthalenyl, 5,6,7,8- tetrahydro-naphthalenyl, indanyl, and the like.

[0053] The term "aryl-alkyl", as used herein, refers to an alkyl group substituted with one aryl substituent, as those terms are defined herein. Examples of aryl-alkyl include, but are not limited to: benzyl, phenethyl, phenylpropyl, naphthalenylmethyl, and the like.

[0054] The term "aryldiyl", as used herein, refers to a divalent radical derived from a 6- to 12- membered mono- or bicyclic hydrocarbon ring system wherein at least one ring aromatic. Examples of aryldiyl include, phenyldiyl, naphthalenyldiyl, 1,2,3,4-tetrahydro-naphthalenyldiyl, 5,6,7,8-tetrahydro-naphthalenyldiyl, indanyldiyl, and the like.

[0055] The term "carboxamide", as used herein, refers to -C(0)NH2.

[0056] The term "carboxyl", as used herein, refers to -C(0)OH. [0057] The term "cyano", as used herein, refers to -CN.

[0058] The term "cycloalkyl", as used herein, refers to a cyclic saturated hydrocarbon radical containing the specified number of carbon atoms, e.g., C3-C7 alkyl. Examples of cycloalkyl include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. [0059] The term "cycloalkyl-alkyl", as used herein, refers to an alkyl group substituted with one cycloalkyl substituent, as those terms are defined herein. Examples of cycloalkyl-alkyl include, but are not limited to: cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl, cyclohexylethyl, and the like.

[0060] The term "cycloalkyldiyl", as used herein, refers to a divalent cyclic saturated hydrocarbon radical containing the specified number of carbon atoms, e.g., C3-C7 cycloalkyldiyl, or C4-C7 alkyldiyl. Examples of cycloalkyldiyl include, but are not limited to: cyclopropyldiyl, cyclobutyldiyl, cyclopentyldiyl, cyclohexyldiyl, and the like.

[0061] The term "guanidino", as used herein, refers to -NH-C(=NH)-NH₂.

[0062] The term "halo", as used herein, refers to -F, -CI, -Br, and -I. [0063] The term "haloacyl", as used herein, refers to -C(0)-haloalkyl, wherein haloalkyl is as defined herein. Examples of haloacyl include, but are not limited to: difluoroacetyl, trifluoroacetyl, 3,3,3-trifluoropropanoyl, pentafluoroproponyl, and the like.

[0064] The term "haloalkoxy", as used herein, refers to -O-haloalkyl, wherein haloalkyl is as defined herein. Examples of haloalkoxy include, but are not limited to: difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, and the like.

[0065] The term "haloalkyl", as used herein, refers to an alkyl group as defined herein substituted with from one or more halogens. When more than one halogen is present then they may be the same or different and selected from the group consisting of F, CI, Br, and I. Examples of haloalkyl groups include, but are not limited to: fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, and the like. [0066] The term "heteroaryl", as used herein, refers to a radical derived from a 6- to 12-membered mono- or bicyclic ring system wherein at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of a heteroatom include, but are not limited to: O, S, N, and the like. Examples of heteroaryl include, but are not limited to: pyridyl, benzofuranyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolinyl, benzoxazolyl, benzothiazolyl, lH-benzimidazolyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrrolyl, indolyl, lH-benzoimidazol-2-yl, benzo[l,3]dioxol-5-yl, 3,4- dihydro-2H-benzo[l,4]oxazin-7-yl, 2,3-dihydro-benzofurn-7-yl, 2,3-dihydro-indol-l-yl, and the like.

[0067] The term "heteroaryldiyl", as used herein, refers to a divalent radical derived from a 6- to 12-membered mono- or bicyclic ring system wherein at least one ring atom is a heteroatom and at least one ring is aromatic. Examples of a heteroatom include, but are not limited to: O, S, N, and the like. Examples of heteroaryldiyl include, but are not limited to: thiazolyldiyl, 2,4-thiazolyldiyl, triazolyldiyl, l,2,3-triazolyl-l,4-diyl, pyridyldiyl, benzofuranyldiyl, pyrazinyldiyl, pyridazinyldiyl, pyrimidinyldiyl, triazinyldiyl, quinolinyldiyl, benzoxazolyldiyl, benzothiazolyldiyl, 1H- benzimidazolyldiyl, isoquinolinyldiyl, quinazolinyldiyl, quinoxalinyldiyl, pyrrolyldiyl, indolyldiyl, lH-benzoimidazol-2-yldiyl, benzo[l,3]dioxol-5-yldiyl, 3,4-dihydro-2H-benzo[l,4]oxazin-7-yldiyl, 2,3-dihydro-benzofurn-7-yldiyl, 2,3-dihydro-indol-l-yldiyl, and the like.

[0068] The term "heterocyclyl", as used herein, refers to a radical derived from a 3- to 12- membered mono- or bicyclic non-aromatic ring system wherein at least one ring atom is a heteroatom. Examples of a heteroatom include, but are not limited to: O, S, N, and the like. A heterocyclyl substituent can be attached via any of its available ring atoms, for example, a ring carbon, or a ring nitrogen. In some embodiments, the heterocyclyl group is a 3-, 4-, 5-, 6- or 7- membered containing ring. Examples of a heterocyclyl group include, but are not limited to: aziridin-l-yl, aziridin-2-yl, azetidin-l-yl, azetidin-2-yl, azetidin-3-yl, piperidin-l-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, morpholin-4-yl, piperazin-l-yl, piperazin-2-yl, piperazin-3-yl, piperazin-4-yl, pyrrolidin-l-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, [l,3]-dioxolan-2-yl, thiomorpholin-4-yl, [l,4]oxazepan-4-yl, l,l-dioxo- ⁶-thiomorpholin-4-yl, azepan-l-yl, azepan-2-yl, azepan-3-yl, azepan-4-yl, octahydro-quinolin-l-yl, octahydro- isoquinolin-2-yl, and the like. [0069] The term "heterocyclyl-alkyl", as used herein, refers to an alkyl group substituted with one heterocyclyl substituent, as those terms are defined herein. Examples of heterocyclyl-alkyl include, but are not limited to: azetidin-3 -ylmethyl, piperidin-1 -ylmethyl, piperidin-2 -ylmethyl, piperidin-3- ylmethyl, piperidin-4-ylmethyl, morpholin-2-ylmethyl, morpholin-3 -ylmethyl, morpholin-4- ylmethyl, piperazin-l-ylmethyl, piperazin-2 -ylmethyl, piperazin-3 -ylmethyl, piperazin-4-ylmethyl, pyrrolidin-1 -ylmethyl, pyrrolidin-2 -ylmethyl, pyrrolidin-3 -ylmethyl, [1, 3] -dioxolan-2 -ylmethyl, thiomoφholin-4-ylmethyl, [l,4]oxazepan-4-ylmethyl, l,l-dioxo-lλ⁶-thiomoφholin-4-ylmethyl, azepan-1 -ylmethyl, azepan-2 -ylmethyl, azepan-3 -ylmethyl, azepan-4-ylmethyl, octahydro- quinolin-1 -ylmethyl, octahydro-isoquinolin-2-yl, and the like. [0070] The term "heterocyclyldiyl", as used herein, refers to a divalent radical derived from a 3- to 12-membered mono- or bicyclic non-aromatic ring system wherein at least one ring atom is a heteroatom. Examples of a heteroatom include, but are not limited to: O, S, N, and the like. A heterocyclyldiyl substituent can be attached via any two of its available ring atoms, for example, ring carbons, or ring nitrogens. In some embodiments, the heterocyclyldiyl is a 3-, 4-, 5-, 6- or 7- membered containing ring. Examples of a heterocyclyldiyl group include, but are not limited to: aziridin-l-yldiyl, aziridin-2-yldiyl, azetidin-l-yldiyl, azetidin-2-yldiyl, azetidin-3 -yldiyl, piperidin- 1-yldiyl, piperidin-2-yldiyl, piperidin-3 -yldiyl, piperidin-4-yldiyl, morpholin-2-yldiyl, morpholin- 3-yldiyl, morpholin-4-yldiyl, piperazin-1 -yldiyl, piperazin-2 -yldiyl, piperazin-3 -yldiyl, piperazin-4- yldiyl, pyrrolidin-1 -yldiyl, pyrrolidin-2 -yldiyl, pyrrolidin-3 -yldiyl, [1,3] -dioxolan-2 -yldiyl, thiomorpholin-4-yldiyl, [l,4]oxazepan-4-yldiyl, l, l-dioxo- 6-thiomorpholin-4-yldiyl, azepan-1- yldiyl, azepan-2-yldiyl, azepan-3-yldiyl, azepan-4-yldiyl, octahydro-quinolin-1 -yldiyl, octahydro- isoquinolin-2 -yldiyl, and the like.

[0071] The term "hydroxyl", as used herein, refers to -OH.

[0072] The term "hydroxy-alkyl", as used herein, refers to an alkyl group substituted with one hydroxy substituent, as those terms are defined herein. Examples of hydroxy-alkyl include, but are not limited to: hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, hydroxy-isopropyl, hydroxy-n- butyl, hydroxy-sec-butyl, hydroxy-isobutyl, hydroxy-7-butyl, hydroxy-pentyl, hydroxy-isopentyl, hydroxy-7-pentyl, hydroxy-neo-pentyl, hydroxy- 1 -methylbutyl, hydroxy-2-methylbutyl, hydroxy-n- hexyl, and the like. [0073] The term "nitro", as used herein, refers to -NO2. [0074] The term "oxo", as used herein, refers to =0. [0075] The term "thio", as used herein, refers to -SH.

[0076] As used herein, "substituted" indicates that at least one hydrogen atom of the chemical group is replaced by a non-hydrogen substituent or group, the non-hydrogen substituent or group can be monovalent or divalent. When the substituent or group is divalent, then it is understood that this group is further substituted with another substituent or group. When a chemical group herein is "substituted" it may have up to the full valance of substitution; for example, a methyl group can be substituted by 1, 2, or 3 substituents, a methylene (methyldiyl) group can be substituted by 1 or 2 substituents, a phenyl group can be substituted by 1, 2, 3, 4, or 5 substituents, a naphthyl group can be substituted by 1, 2, 3, 4, 5, 6, or 7 substituents, and the like, unless otherwise specified. Likewise, "substituted with one or more substituents" refers to the substitution of a group with one substituent up to the total number of substituents physically allowed by the group. Further, when a group is substituted with more than one group they can be identical or they can be different. [0077] The term "protecting group," as used herein, refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl and amino groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Groups can be selectively incorporated into compounds of the present disclosure as precursors. For example, an amino group can be placed into a compound described herein as an azido group that can be chemically converted to the amino group at a desired point in the synthesis. Generally, groups are protected or present as a precursor that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time. Further representative protecting or precursor groups are discussed in Agrawal, et al, Protocols for Oligonucleotide Conjugates, Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1 72. Examples of hydroxyl protecting groups include, but are not limited to, /-butyl, 7-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1- ethoxyethyl, l-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, 7-butyldimethylsilyl, 7-butyldiphenylsilyl (TBDPS), triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9- fluorenylmethyl carbonate, mesylate and tosylate. Examples of amino protecting groups include, but are not limited to, carbamate -protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1 -methyl -1 -(4 -biphenylyl)ethoxycarbonyl (Bpoc), 7-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide- protecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl.

[0078] "Stable compound" and "stable structure", as used herein, refer to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. [0079] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0080] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10 -sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethane sulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene- 1, 5 -disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2 -naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0081] "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Ammonium, sodium, potassium, calcium, and magnesium salts are particularly useful in this context. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

[0082] As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, compounds may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the like, as well as the corresponding solvated forms. Compounds may be true solvates, or may merely retain adventitious water or be a mixture of water plus some adventitious solvent. Some embodiments of the present disclosure relate to solvates of the compounds described herein. Other Definitions

[0083] The term "antibody" refers to a full-length immunoglobulin molecule or a functionally active portion of a full-length immunoglobulin molecule, i.e. , a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof. The immunoglobulin may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The antibodies may be derived from any species. Certain embodimens relate to antibodies of human, murine, camelid or rabbit origin. The antibodies may be polyclonal, monoclonal, multi-specific (e.g., bispecific), human, humanized, chimeric, linear or single chain antibodies. The term "antibody" also includes diabodies, maxibodies, minibodies, scFv fragments, Fab fragments, F(ab') fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope -binding fragments of any of the above which immunospecifically bind to a target antigen.

[0084] An antibody "which binds" an antigen of interest is one capable of binding that antigen with sufficient affinity that the antibody is useful in targeting a cell expressing the antigen.

[0085] The term "intracellular metabolite" refers to a compound resulting from a metabolic process or reaction inside a cell on a composition described herein (e.g. , an antibody drug conjugate (ADC)). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker of the subject composition, or hydrolysis of a functional group such as a hydrazone, ester, or amide within the subject composition. In the context of conjugates, including ADCs, intracellular metabolites include, but are not limited to, antibodies and free drug which have been separated intracellularly, i.e. , after entry, diffusion, uptake or transport into a cell (e.g. , by enzymatic cleavage of an ADC by an intracellular enzyme).

[0086] In the context of conjugates, including ADCs, the terms "intracellularly cleaved" and "intracellular cleavage" refer to metabolic processes or reactions inside a cell on a composition described herein whereby the covalent attachment, e.g., the linker (L), between the drug moiety (D) and the targeting moiety (T) (e.g. , an antibody) is broken, resulting in the free drug dissociated from (T) inside the cell. In certain embodiments, the cleaved moieties of the subject compositions are thus intracellular metabolites (e.g., T, T-L fragment, D-L fragment, and D). Accordingly, some embodiments of the present disclosure relate to compositions that are cleavage products of a composition of Formula II as described herein, which cleavage products include compositions comprising compounds of Formula I as described herein.

[0087] The term "extracellular cleavage" refers a metabolic process or reaction outside a cell on a composition described herein whereby the covalent attachment, e.g. , the linker (L), between the drug moiety (D) and the targeting moiety (T) (e.g. , an antibody) is broken, resulting in the free drug dissociated from (T) outside the cell. In certain embodiments, the cleaved moieties of the subject compositions are thus initially extracellular metabolites (e.g., T, T-L fragment, D-L fragment, and D), which may move intracellularly by diffusion and cell permeability or transport.

[0088] The terms "subject" and "patient" as used herein refer to an animal, typically a mammal, in need of treatment. [0089] The term "mammal" includes humans and both domestic animals, such as laboratory animals, farm animals and household pets (e.g. , cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife, and the like.

[0090] "Optional" or "optionally" means that the subsequently described event of circumstances may or may not occur, and that some embodiments of the present disclosure relate to instances where said event or circumstance occurs and some embodiments relate to instances in which it does not. For example, "optionally substituted aryl" means that the aryl substituent may or may not be substituted and that the description includes both substituted aryl substituents and aryl substituents having no substitution.

[0091] "Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration (or other similar regulatory agency of another jurisdiction) as being acceptable for use in humans or domestic animals. [0092] The term "therapeutically effective amount" refers to that amount of a compound or composition which, when administered to a subject is sufficient to effect treatment of the particular indication to be treated (e.g., cancer or tumor cells in the subject). The amount of a compound or composition described herein which constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0093] "Treating" or "treatment" as used herein covers the treatment of the disease or condition of interest in a subject having the disease or condition of interest, and may include one or more of preventing the disease or condition from occurring for example in a subject predisposed to the condition who has not yet been diagnosed as having it; inhibiting the disease or condition, for example, arresting its development; relieving the disease or condition, for example, causing regression of the disease or condition, and/or relieving one or more symptoms resulting from the disease or condition.

[0094] An "effective amount" in respect of a particular result to be achieved is an amount sufficient to achieve the desired result. For example, an "effective amount" of compound when referred to in the context of the killing of cancer cells, refers to an amount of drug sufficient to produce a killing effect. COMPOUNDS

[0095] In one aspect, the present disclosure relates to compounds of general Formula I:

and pharmaceutically acceptable salts thereof, wherein: R is selected from: amino-Ci-C6 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino- heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from aryl, aryl-G-C6 alkyl, C1-C6 alkyl, G-C6 alkylthio, carboxyl, carboxamide, C3-C7 cycloalkyl, C3-C7 cycloalkyl -G-G alkyl, guanidino, halo, G-G haloalkyl, heterocyclyl, heterocyclyl-G-G alkyl, hydroxyl, and thio; or R is R^NCHCR³)-;

R¹ is selected from: H and G-G alkyl;

R² is G-G alkyl; and

R³ is selected from: H, R¹⁵ and R⁷-CH(CH₃)₂-; or R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl;

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁷ is selected from: H, aryl, G-G cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: G-G acylthio, G-G alkenyl, G-G alkyl, G-G alkylamino, G-G alkyloxy, amino, amino-G-G alkyl, halo, G-G haloalkyl, hydroxyl, hydroxy-G-G alkyl, and thio, wherein G-G alkenyl, G-G alkylamino and G-G alkyloxy are further optionally substituted with one substituent selected from G-G alkylaryl, hydroxyl, and thio;

R⁹ and R¹⁰ are each independently selected from: H, G-G alkyl, aryl, aryl-G-G alkyl, C3-C7 cycloalkyl-G-G alkyl, and hydroxy-G-G alkyl;

R¹⁵ is G-G alkyl;

X is selected from G-G alkenyldiyl and G alkyldiyl, wherein G alkyldiyl is substituted with one substituent selected from: G-G acyloxy, G-G alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹;

Y is heteroaryldiyl, or Y is absent; and Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; with the proviso that when R is R^NCHiR³)-; R¹ is H; R² is methyl; R³ is PhCH(CH₃)₂-; R⁵ is methyl; X is -CH=CH(CH₃)-; Y is absent and Z is absent, then R⁴ is other than fert-butyl. [0096] Certain embodiments relate to compounds having general Formula I as defined above, with the proviso that when R is piperidin-2-yl; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; X is C₂ alkyldiyl substituted with acetoxy; Y is 2,4,-thiazolyl; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl.

[0097] Certain embodiments relate to compounds having general Formula I as defined above, with the proviso that when R is l-methylpiperidin-2-yl; R⁴ is fert-butyl or sec-butyl and R⁵ is methyl, then X is C₂ alkyldiyl as defined above.

[0098] In certain embodiments, in compounds of general Formula I:

X is C₂ alkyldiyl substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹, and

Y is heteroaryldiyl.

The Variable R

[0099] In some embodiments, R is selected from: amino-G-C6 alkyl, amino-aryl, amino-C₃-C7 cycloalkyl, amino-heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from G-C6 alkyl and halo.

[00100] In some embodiments, R is selected from: l-(dimethylamino)-2-methylpropyl, 2-methyl- l-(methylamino)propyl, 1-aminocyclopentyl, 1-aminocyclopropyl, 4-aminophenyl, 2- aminopropan-2-yl, 1-aminocyclohexyl, 3-aminooxetan-3-yl, 2-(methylamino)propan-2-yl, 1- amino-2-methylpropan-2-yl, 2-methylpyrrolidin-2-yl, 2-amino-3-methylbutan-2-yl, 2-aminobutan- 2-yl, 2-methyl-l-(methylamino)propan-2-yl, 2-methylpiperidin-2-yl, 3-fluoropyrrolidin-3-yl, 1,2- dimethylpyrrolidin-2-yl, 2-(dimethylamino)propan-2-yl. [00101] In some embodiments, R is R^NCHCR³)-.

The Variable R¹

[00102] In some embodiments, R¹ is selected from: H and C1-C4 alkyl.

[00103] In some embodiments, R¹ is selected from: H, methyl, and propyl.

[00104] In some embodiments, R¹ is selected from: H, methyl, and isopropyl.

The Variable R²

[00105] In some embodiments, R² is C1-C4 alkyl. In some embodiments, R² is methyl.

The Variable R³

[00106] In some embodiments, R³ is R⁷-CH(CI¾)2-. In some embodiments, R³ is H. In some embodiments, R³ is R¹⁵.

The Variable R⁴

[00107] In some embodiments, R⁴ is C1-C4 alkyl or cyclopropyl.

[00108] In some embodiments, R⁴ is selected from: propyl, butyl, and cyclopropyl

[00109] In some embodiments, R⁴ is selected from: sec-propyl, sec-butyl, fert-butyl, cyclohexyl, cyclopentyl and cyclopropyl.

The Variable R⁵

[00110] In some embodiments, R⁵ is selected from: H and C1-C4 alkyl optionally substituted with C1-C4 acyloxy.

[00111] In some embodiments, R⁵ is C1-C4 alkyl optionally substituted with C1-C4 acyloxy.

[00112] In some embodiments, R⁵ is methyl optionally substituted with C1-C4 acyloxy. The Variable R⁶

[00113] In some embodiments, R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: 1-aminocyclopropyl, 4-aminophenyl, amino, aminomethyl, bromo, fert-butyl, carboxamide, carboxyl, chloro, cyano, cyclopentyl, ethyl, fluoro, hydroxy, isopropyl, methoxy, methyl, nitro, phenyl, pyridin-3-yl, thio, thiomethyl, trifluoromethoxy, and trifluoromethyl.

[00114] In some embodiments, R⁶ is selected from: C1-C4 alkyl, aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1-C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-C3-C7 cycloalkyl, amino -heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-G-C6 alkyl.

[00115] In some embodiments, R⁶ is selected from: 5,6,7,8-tetrahydronaphthalen-l-yl, benzyl, cyclohexyl, ethyl, hexan-2-yl, methyl, naphthalen-2-yl, piperidin-l-yl, phenyl, propyl, pyridin-3-yl, and thien-2-yl, each optionally substituted with one or more substituents selected from: 1- aminocyclopropyl, 4-aminophenyl, amino, aminomethyl, bromo, fert-butyl, carboxamide, carboxyl, chloro, cyano, cyclopentyl, ethyl, fluoro, hydroxy, isopropyl, methoxy, methyl, nitro, phenyl, pyridin-3-yl, thio, thiomethyl, trifluoromethoxy, and trifluoromethyl.

[00116] In some embodiments, R⁶ is selected from: 4-aminobenzyl, 4-(aminomethyl)benzyl, 4- (aminomethyl)phenyl, 4-aminophenyl, 3-aminobenzyl, 3-(aminomethyl)benzyl, 3- (aminomethyl)phenyl, 3-aminophenyl, benzyl, phenyl, 3-mercaptopropyl, 2-mercaptoethyl, 4- (mercaptomethyl)phenyl, 7-tolyl, methyl, 2,4,6-trimethylphenyl, 4-(trifluoromethoxy)phenyl, 2,4,6-triisopropylphenyl, 4-fert-butylphenyl, 4-chlorophenyl, 3-cyanophenyl, 2-nitrophenyl, 4- methoxy-2-nitrophenyl, 4-aminocarbonyl-2-nitrophenyl, 4-methoxyphenyl, phenyl, 2-fluorobenzyl, piperidin-l-yl, o-tolyl, 4-bromophenyl, naphthalen-2-yl, 4-methoxycarbonyphenyl, 2- (trifluoromethyl)benzyl, hexan-2-yl, 2-methoxyethyl, cyclopentylmethyl, cyclohexyl, pyridin-3- ylmethyl, 4-carboxyphenyl, 3-aminophenyl, pyridin-3-yl, thien-2-yl, 4-hydroxyphenyl, 4-(l- aminocyclopropyl)benzyl, 4-(l-aminocyclopropyl)phenyl, 2-methylbenzyl, 4-nitrobenzyl, 4- chlorobenzyl, phenethyl, 4-bromobenzyl, 4-cyanobenzyl, 3-nitrobenzyl, 4-fert-butylbenzyl, 2- nitrobenzyl, 4-nitrophenethyl, 2-chloro-3-methoxycarbonylphenyl, 2-aminophenyl, [1,1 - biphenyl]-4-yl, 4'-amino-[l,r-biphenyl]-4-yl, 4-fluorobenzyl, 3-(trifluoromethyl)benzyl, 3- (trifluoromethoxy)benzyl, 3,4-dichlorobenzyl, 2-cyanobenzyl, 3-chlorobenzyl, 4-amino-2- ethylphenyl, 4-amino-3-(trifluoromethoxy)phenyl, 4-amino-2,3-dimethylphenyl, 4-amino-5, 6,7,8- tetrahydronaphthalen-l-yl, 4-amino-3-methylphenyl, 4-amino-3 -fluorophenyl, 4-amino-3- ethylphenyl, and 4-amino-3-(trifluoromethyl)phenyl.

[00117] R⁶ is aryl or aryl-G-C4 alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-G-C6 alkyl.

[00118] In some embodiments, R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00119] In some embodiments, R⁶ is selected from: 4-aminobenzyl, 4-(aminomethyl)benzyl, 4- (aminomethyl)phenyl, 4-aminophenyl, and benzyl. The Variable R⁷

[00120] In some embodiments, R⁷ is selected from: H, aryl, C3-C7 cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: G-G acylthio, G- G alkenyl, G-G alkyl, G-G alkylamino, G-G alkyloxy, amino, amino-G-G alkyl, halo, G-G haloalkyl, hydroxyl, hydroxy-G-G alkyl, and thio, wherein G-G alkenyl, G-G alkylamino and G-G alkyloxy are further optionally substituted with one substituent selected from /j>-tolyl, hydroxyl, and thio.

[00121] In some embodiments, R⁷ is selected from: H, aryl, G-G cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: (2- hydroxyethyl)amino, (2-mercaptoethyl)amino, 2-(acetylthio)ethoxy, 2-aminoethoxy, 2- hydroxyethoxy, 2-mercaptoethoxy, 3-methoxy, 4-methylstyryl, amino, aminomethyl, chloro, fluoro, hydroxyl, hydroxymethyl, methyl, thio, trifluoromethyl.

[00122] In some embodiments, R⁷ is selected from: H, lH-indol-3-yl, 1 -methyl- lH-indol-3-yl, 2- methoxyphenyl, 3-((2-hydroxyethyl)amino)phenyl, 3-((2-mercaptoethyl)amino)phenyl, 3-(2- hydroxyethoxy)phenyl, 3-(2-mercaptoethoxy)phenyl, 3,5-difluorophenyl, 3,5-dimethylphenyl, 3- chlorophenyl, 3-mercaptophenyl, 3-methoxyphenyl, 3-trifluoromethylphenyl, 4-((2- hydroxyethyl)amino)phenyl, 4-((2-mercaptoethyl)amino)phenyl, 4- 4-(2-hydroxyethoxy)phenyl, 4- (2-mercaptoethoxy)phenyl, 4-mercaptophenyl, 4-methoxyphenyl, cyclohexyl, thien-2-yl, ra-tolyl, and phenyl.

The Variable R⁸

[00123] In some embodiments, R⁸ is selected from aryl, heteroaryl and C5-C7 cycloalkyl, optionally substituted with one substituent selected from amino and hydroxyl.

[00124] In some embodiments, R⁸ is aryl optionally substituted with one substituent selected from amino and hydroxyl.

[00125] In some embodiments, R⁸ is selected from: lH-indol-3-yl, 4-aminophenyl, 4- hydroxyphenyl, 5-hydroxypyridin-2-yl, cyclohexyl, and phenyl.

The Variable R⁹

[00126] In some embodiments, R⁹ is Η. In some embodiments, R⁹ is C1-C4 alkyl. In some embodiments, R⁹ is aryl. In some embodiments, R⁹ is aryl-G-C4 alkyl. In some embodiments, R⁹ is C3-C7 cycloalkyl-Ci-C4 alkyl. In some embodiments, R⁹ is hydroxy-G-C4 alkyl.

The Variable R¹⁰

[00127] In some embodiments, R¹⁰ is Η. In some embodiments, R¹⁰ is C1-C4 alkyl. In some embodiments, R¹⁰ is aryl. In some embodiments, R¹⁰ is aryl-G-C4 alkyl. In some embodiments, R¹⁰ is C3-C7 cycloalkyl-Ci-C4 alkyl. In some embodiments, R¹⁰ is hydroxy-Ci-C4 alkyl.

The Variable R¹¹

[00128] In some embodiments, R¹¹ is selected from: Η, C1-C4 acyl, and C1-C4 alkyl. [00129] In some embodiments, R¹¹ is C1-C6 acyl or G-C6 alkyl.

[00130] In some embodiments, R¹¹ is selected from: Η, acetyl, propyl, ethyl, and methyl. The Variable R

[00131] In some embodiments, R¹² is C1-C4 acyl. In some embodiments, R¹² is C1-C4 alkyl.

The Variable X

[00132] In some embodiments, X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, C1-C6 alkyloxy, hydroxyl, and oxo.

[00133] In some embodiments, X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, and -OC(0)NR⁹R¹⁰. [00134] In some embodiments, X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: C1-C4 acyloxy, C1-C4 alkyloxy, hydroxyl, and -OC(0)NR⁹R¹⁰.

[00135] In some embodiments, X is selected from: -CH=CH- -CH=CH(CH₃)-, -CH₂CH(OH)-, -C(0)CH₂-, -CH₂C(0)-, -CH₂CH(OAc)-, -CH₂CH(OPr)-, -CH₂CH(OEt)-, and -CH₂CH(OMe)-.

The Variable Y

[00136] In some embodiments, Y is 2,4-thiazolyldiyl. In some embodiments, Y is 1,2,3-triazolyl- 1,4-diyl. In some embodiments, Y is absent.

The Variable Z [00137] In some embodiments, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-. In some embodiments, Z is absent.

Certain Combinations

[00138] In some embodiments, R² is G-C₆ alkyl; and R³ is R⁷-CH(CH₃)₂- [00139] In some embodiments, R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl.

[00140] In some embodiments, R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl selected from: pyrrolidinyldiyl, piperidinyldiyl, and azepanyldiyl.

[00141] Certain embodiments of the present disclosure relate to compounds of Formula la:

la and pharmaceutically acceptable salts thereof, wherein:

R¹ is selected from: H and G-C6 alkyl;

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-C6 haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently selected from: H, G-G alkyl, aryl, aryl-G-G alkyl, C3-C7 cycloalkyl-G-G alkyl, and hydroxy-G-G alkyl; X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹;

Y is heteroaryldiyl, or Y is absent; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; and n is 0, 1, or 2.

[00142] Certain embodiments relate to compounds having Formula la as defined above, with the proviso that when R is piperidin-2-yl; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; X is C2 alkyldiyl substituted with acetoxy; Y is 2,4,-thiazolyl; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl.

[00143] Certain embodiments relate to compounds having Formula la as defined above, with the proviso that when R is l-methylpiperidin-2-yl; R⁴ is fert-butyl or sec-butyl and R⁵ is methyl, then X is C2 alkyldiyl as defined above.

[00144] In certain embodiments, in compounds of general Formula la: X is C2 alkyldiyl substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹, and

Y is heteroaryldiyl.

[00145] In certain embodiments in compounds of Formula la, R¹ is selected from: H and C1-C4 alkyl.

[00146] In certain embodiments in compounds of Formula la, R⁴ is C1-C4 alkyl or cyclopropyl.

[00147] In certain embodiments in compounds of Formula la, R⁵ is C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy. In certain embodiments in compounds of Formula la, R⁶ is selected from: C1-C4 alkyl, aryl, aryl- C1-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1-C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-C3-C? cycloalkyl, amino -heteroaryl or amino- heterocyclyl, and the aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C? cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00148] In certain embodiments in compounds of Formula la, X is C₂ alkyldiyl substituted with one substituent selected from: C1-C4 acyloxy, C1-C4 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰.

[00149] In certain embodiments in compounds of Formula la, Y is heteroaryldiyl.

[00150] In certain embodiments in compounds of Formula la, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

[00151] In certain embodiments in compounds of Formula la, Z is absent. [00152] In certain embodiments in compounds of Formula la: is selected from: H and C1-C4 alkyl; is C1-C4 alkyl or cyclopropyl;

R⁵ is C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: C1-C4 alkyl, aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1 -C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-C3-C? cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C? cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl;

R⁸ is selected from: aryl, heteroaryl, and C5-C7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl;

X is C2 alkyldiyl substituted with one substituent selected from: C1-C4 acyloxy, C1-C4 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰;

Y is heteroaryldiyl;

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; and n is 0, 1, or 2. In certain embodiments in compounds of Formula la: R¹ is selected from: H, methyl, and isopropyl;

R⁴ is selected from: sec-propyl, sec-butyl, fert-butyl, cyclohexyl, cyclopentyl and cyclopropyl;

R⁵ is selected from: H and methyl;

R⁶ is selected from: 4-aminobenzyl, 4-(aminomethyl)benzyl, 4-(aminomethyl)phenyl, 4- aminophenyl, 3-aminobenzyl, 3-(aminomethyl)benzyl, 3-(aminomethyl)phenyl, 3- aminophenyl, benzyl, phenyl, 3-mercaptopropyl, 2-mercaptoethyl, 4- (mercaptomethyl)phenyl, 7-tolyl, methyl, 2,4,6-trimethylphenyl, 4- (trifluoromethoxy)phenyl, 2,4,6-triisopropylphenyl, 4-fert-butylphenyl, 4-chlorophenyl,

3- cyanophenyl, 2-nitrophenyl, 4-methoxy-2-nitrophenyl, 4-aminocarbonyl-2-nitrophenyl,

4- methoxyphenyl, phenyl, 2-fluorobenzyl, piperidin-l-yl, o-tolyl, 4-bromophenyl, naphthalen-2-yl, 4-methoxycarbonyphenyl, 2-(trifluoromethyl)benzyl, hexan-2-yl, 2- methoxyethyl, cyclopentylmethyl, cyclohexyl, pyridin-3-ylmethyl, 4-carboxyphenyl, 3- aminophenyl, pyridin-3-yl, thien-2-yl, 4-hydroxyphenyl, 4-(l-aminocyclopropyl)benzyl, 4-(l-aminocyclopropyl)phenyl, 2-methylbenzyl, 4-nitrobenzyl, 4-chlorobenzyl, phenethyl, 4-bromobenzyl, 4-cyanobenzyl, 3-nitrobenzyl, 4-fert-butylbenzyl, 2- nitrobenzyl, 4-nitrophenethyl, 2-chloro-3-methoxycarbonylphenyl, 2-aminophenyl, [1,1 - biphenyl]-4-yl, 4'-amino-[l, r-biphenyl]-4-yl, 4-fluorobenzyl, 3-(trifluoromethyl)benzyl,

3- (trifluoromethoxy)benzyl, 3,4-dichlorobenzyl, 2-cyanobenzyl, 3-chlorobenzyl, 4- amino-2-ethylphenyl, 4-amino-3-(trifluoromethoxy)phenyl, 4-amino-2,3-dimethylphenyl,

4- amino-5,6,7,8-tetrahydronaphthalen-l-yl, 4-amino-3-methylphenyl, 4-amino-3- fluorophenyl, 4-amino-3-ethylphenyl, and 4-amino-3-(trifluoromethyl)phenyl;

R⁸ is selected from: lH-indol-3-yl, 4-aminophenyl, 4-hydroxyphenyl, 5-hydroxypyridin- 2-yl, cyclohexyl, and phenyl;

X is selected from -CH=CH- -CH=CH(CH₃)-, -CH₂CH(OH)-, -C(0)CH₂-, -CH₂C(0)-, -CH₂CH(OAc)-, -CH₂CH(OPr)-, -CH₂CH(OEt)-, and -CH₂CH(OMe)-;

Y is 2,4-thiazolyldiyl, or Y is absent;

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; and n is 0, 1, or 2. Certain embodiments of the present disclosure relate to compounds of Formula lb:

lb harmaceutically acceptable salts thereof, wherein: is selected from: H and G-C6 alkyl; is G-C6 alkyl; is selected from: C1-C4 alkyl and C3-C6 cycloalkyl; R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

Y is heteroaryldiyl, or Y is absent; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent.

[00155] Certain embodiments relate to compounds having Formula lb with the proviso that when R⁷ is phenyl; R¹ is H; R² is methyl; R⁵ is methyl; X is -CH=CH(CH₃)-; Y is absent and Z is absent, then R⁴ is other than fert-butyl. [00156] In certain embodiments, in compounds of Formula lb:

X is C2 alkyldiyl substituted with one substituent selected from: C1-C4 acyloxy, C1-C4 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰; and

Y is heteroaryldiyl. [00157] In certain embodiments in compounds of Formula lb: R¹ is H or methyl; R² is methyl;

R⁴ is selected from: sec-propyl, sec-butyl, fert-butyl, cyclohexyl, cyclopentyl, and cyclopropyl;

R⁵ is selected from: H and methyl;

R⁶ is selected from: 4-aminobenzyl, 4-(aminomethyl)benzyl, 4-(aminomethyl)phenyl, 4- aminophenyl, 3-aminobenzyl, 3-(aminomethyl)benzyl, 3-(aminomethyl)phenyl, 3- aminophenyl, benzyl, phenyl, 3-mercaptopropyl, 2-mercaptoethyl, 4- (mercaptomethyl)phenyl, /j>-tolyl, methyl, 2,4,6-trimethylphenyl, 4- (trifluoromethoxy)phenyl, 2,4,6-triisopropylphenyl, 4-fert-butylphenyl, 4-chlorophenyl,

4- methoxyphenyl, phenyl, 2-fluorobenzyl, piperidin-l-yl, o-tolyl, 4-bromophenyl, naphthalen-2-yl, 4-methoxycarbonyphenyl, 2-(trifluoromethyl)benzyl, hexan-2-yl, 2- methoxyethyl, cyclopentylmethyl, cyclohexyl, pyridin-3-ylmethyl, 4-carboxyphenyl, 3- aminophenyl, pyridin-3-yl, thien-2-yl, 4-hydroxyphenyl, 4-(l-aminocyclopropyl)benzyl, 4-(l-aminocyclopropyl)phenyl, 2-methylbenzyl, 4-nitrobenzyl, 4-chlorobenzyl, phenethyl, 4-bromobenzyl, 4-cyanobenzyl, 3-nitrobenzyl, 4-fert-butylbenzyl, 2- nitrobenzyl, 4-nitrophenethyl, 2-chloro-3-methoxycarbonylphenyl, 2-aminophenyl, [Ι, Γ- biphenyl]-4-yl, 4'-amino-[l, l '-biphenyl]-4-yl, 4-fluorobenzyl, 3-(trifluoromethyl)benzyl, 3-(trifluoromethoxy)benzyl, 3,4-dichlorobenzyl, 2-cyanobenzyl, 3-chlorobenzyl, 4- amino-2-ethylphenyl, 4-amino-3-(trifluoromethoxy)phenyl, 4-amino-2,3-dimethylphenyl, 4-amino-5,6,7,8-tetrahydronaphthalen-l-yl, 4-amino-3-methylphenyl, 4-amino-3- fluorophenyl, 4-amino-3-ethylphenyl, and 4-amino-3-(trifluoromethyl)phenyl;

R⁷ is selected from: H, lH-indol-3-yl, l-methyl-lH-indol-3-yl, 2-methoxyphenyl, 3-((2- hydroxyethyl)amino)phenyl, 3 -((2-mercaptoethyl)amino)phenyl, 3 -(2-

(acetylthio)ethoxy)phenyl, 3-(2-hydroxyethoxy)phenyl, 3-(2-mercaptoethoxy)phenyl, 3- (4-methylstyryl)phenyl, 3-(aminomethyl)phenyl, 3-(hydroxymethyl)phenyl, 3- hydroxyphenyl, 3,5-difluorophenyl, 3,5-dimethylphenyl, 3-aminophenyl, 3-chlorophenyl, 3-mercaptophenyl, 3-methoxyphenyl, 3-trifluoromethylphenyl, 4-((2- hydroxyethyl)amino)phenyl, 4-((2-mercaptoethyl)amino)phenyl, 4-(2-

(acetylthio)ethoxy)phenyl, 4-(2-aminoethoxy)phenyl, 4-(2-hydroxyethoxy)phenyl, 4-(2- mercaptoethoxy)phenyl, 4-(aminomethyl)phenyl, 4-(hydroxymethyl)phenyl, 4- aminophenyl, 4-hydroxyphenyl, 4-mercaptophenyl, 4-methoxyphenyl, cyclohexyl, thien- 2-yl, ra-tolyl, and phenyl;

Y is 2,4-thiazolyldiyl, or Y is absent; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. Certain embodiments of the present disclosure relate to compounds of Formula le:

le and pharmaceutically acceptable salts thereof, wherein: R¹ is selected from: H and G-C6 alkyl; R² is C1-C6 alkyl; and

R³ is selected from H, R¹⁵, and R⁷-CH(CH₃)₂-; or

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl;

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰; R¹⁵ is C1-C6 alkyl; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent.

[00159] Certain embodiments relate to compounds having Formula Ie as defined above, with the proviso that when R¹ is H; R² and R³ taken together with the atoms to which they are each bonded form piperidin-2-yl; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; R¹¹ is acetyl; Z is - C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl.

[00160] In certain embodiments in compounds of Formula Ie:

R¹ and R² are each independently C1-C6 alkyl; and

R³ is H or R¹⁵. [00161] In certain embodiments in compounds of Formula Ie, R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl.

[00162] In certain embodiments in compounds of Formula Ie, R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl selected from: pyrrolidinyldiyl, piperidinyldiyl, and azepanyldiyl. [00163] In certain embodiments in compounds of Formula Ie, R¹ is selected from: H and C1-C4 alkyl.

[00164] In certain embodiments in compounds of Formula Ie, R⁴ is C1-C4 alkyl or cyclopropyl.

[00165] In certain embodiments in compounds of Formula Ie, R⁵ is C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy. [00166] In certain embodiments in compounds of Formula Ie, R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1-C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00167] In certain embodiments in compounds of Formula Ie, R⁶ is aryl or aryl-G-C4 alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00168] In certain embodiments in compounds of Formula Ie, R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl. [00169] In certain embodiments in compounds of Formula Ie, R¹¹ is selected from: G-C6 acyl, G- G alkyl, and -C(0)NR⁹R¹⁰.

[00170] In certain embodiments in compounds of Formula Ie, R¹¹ is selected from: G-C4 acyl, G- G alkyl, and -C(0)NR⁹R¹⁰.

[00171] In certain embodiments in compounds of Formula Ie, R¹¹ is G-G acyl or G-G alkyl. [00172] In certain embodiments in compounds of Formula Ie, R¹¹ is G-G acyl or G-G alkyl. [00173] In certain embodiments in compounds of Formula Ie, Z is absent.

[00174] In certain embodiments in compounds of Formula Ie, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

[00175] In certain embodiments in compounds of Formula Ie: Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-; and

R⁸ is aryl optionally substituted with one substituent selected from amino and hydroxyl. [00176] In certain embodiments in compounds of Formula Ie: selected from: H and G-G alkyl; R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl;

R⁴ is C1-C4 alkyl or cyclopropyl;

R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1 -C4 alkyl is substituted with a substituent selected from: amino -aryl, amino-C3-C? cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino -aryl, amino-C3-C? cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl;

R¹¹ is selected from: H, G-C₆ acyl, Ci-C₆ alkyl, and -C(0)NR⁹R¹⁰; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. In certain embodiments in compounds of Formula Ie:

R¹ is selected from: H and C1-C4 alkyl;

R⁴ is C1-C4 alkyl or cyclopropyl; R⁵ is C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1-C4 alkyl is substituted with a substituent selected from: amino -aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino -aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl;

R⁸ is selected from: aryl, heteroaryl, and C3-C7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl;

R¹¹ is selected from: H, G-C₆ acyl, G-C₆ alkyl, and -C(0)NR⁹R¹⁰; and

Z is absent. Certain embodimemts of the present disclosure relate to compounds of Formula Ix:

Ix and pharmaceutically acceptable salts thereof, wherein:

R¹ is selected from: H and G-C6 alkyl;

R⁴ is selected from: G-G alkyl and G-G cycloalkyl; R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R¹⁴ is selected from: G-G acyloxy, G-G alkyloxy, hydroxyl, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; and n is 0, 1, or 2.

[00179] Certain embodiments relate to compounds having Formula Ix as defined above, with the proviso that when n is 1; R¹ is H; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; R¹⁴ is acetoxy; Z is - C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl. [00180] In certain embodiments in compounds of Formula Ix, R¹ is selected from: H and G-G alkyl.

[00181] In certain embodiments in compounds of Formula Ix, R⁴ is G-G alkyl or cyclopropyl.

[00182] In certain embodiments in compounds of Formula Ix, R⁵ is G-G alkyl optionally substituted with G-G acyloxy. [00183] In certain embodiments in compounds of Formula Ix, R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1-C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00184] In certain embodiments in compounds of Formula Ix, R⁶ is aryl or aryl-G-C4 alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00185] In certain embodiments in compounds of Formula Ix, R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00186] In certain embodiments in compounds of Formula Ix, R⁹ and R¹⁰ are each independently H or C1-C4 alkyl.

[00187] In certain embodiments in compounds of Formula Ix, R¹⁴ is selected from: G-C6 acyloxy, Ci-Ce alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰. [00188] In certain embodiments in compounds of Formula Ix, R¹⁴ is selected from: C1-C4 acyloxy, C1-C4 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰.

[00189] In certain embodiments in compounds of Formula Ix, R¹⁴ is G-C acyloxy or G-C6 alkyloxy.

[00190] In certain embodiments in compounds of Formula Ix, R¹⁴ is G-C4 acyloxy or G-C4 alkyloxy.

[00191] In certain embodiments in compounds of Formula Ix, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-. [00192] In certain embodiments in compounds of Formula Ix: Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-; and

R⁸ is aryl optionally substituted with one substituent selected from amino and hydroxyl. [00193] In certain embodiments in compounds of Formula Ix, Z is absent. [00194] In certain embodiments in compounds of Formula Ix:

R¹ is selected from: H and C1-C4 alkyl;

R⁴ is C1-C4 alkyl or cyclopropyl;

R⁵ is C1-C4 alkyl optionally substituted with C1-C4 acyloxy;

R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1 -C4 alkyl is substituted with a substituent selected from: amino -aryl, amino-C3-C? cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino -aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl;

R¹⁴ is selected from: G-C6 acyloxy, C1-C6 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; and n is 0, 1, or 2.

[00195] In certain embodiments in compounds of Formula Ix: R¹ is selected from: H and C1-C4 alkyl; R⁴ is C1-C4 alkyl or cyclopropyl;

R⁵ is C1-C4 alkyl optionally substituted with C1-C4 acyloxy;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl;

R¹⁴ is selected from: C1-C6 acyloxy, C1-C6 alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰; Z is absent; and n is 0, 1, or 2. Certain embodimemts of the present disclosure relate to compounds of Formula Ih:

Ih harmaceutically acceptable salts thereof, wherein: R¹ is selected from: H and G-C6 alkyl; R² is C1-C6 alkyl; and

R³ is selected from: H, R¹⁵, and R⁷-CH(CH₃)₂-; or

R⁶ is selected from: G-C6 alkyl, aryl, aryl-G-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-C6 haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl;

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰;

R¹⁵ is G-G alkyl; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. In certain embodiments in compounds of Formula Πι, R¹ is G-G alkyl. [00198] In certain embodiments in compounds of Formula Πι:

R¹ and R² are each independently C1-C6 alkyl; and R³ is H or R¹⁵.

[00199] In certain embodiments in compounds of Formula Πι:

R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl.

[00200] In certain embodiments in compounds of Formula Πι:

R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the C1 -C4 alkyl is substituted with a substituent selected from: amino -aryl, amino-C3-C? cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino -aryl, amino-C3-C? cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl.

[00201] In certain embodiments in compounds of Formula Πι:

R¹ is H or C1-C4 alkyl;

R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl; and

[00202] In certain embodiments in compounds of Formula Πι, Z is absent.

[00203] In certain embodiments in compounds of Formula Ih, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

[00204] In certain embodiments in compounds of Formula Πι: R¹ is selected from: H, methyl, and isopropyl; R² is methyl; and R³ is R⁷-CH(CH₃)₂-; or

R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl selected from: pyrrolidinyldiyl, piperidinyldiyl, and azepanyldiyl;

4- methoxyphenyl, phenyl, 2-fluorobenzyl, piperidin-l-yl, o-tolyl, 4-bromophenyl, naphthalen-2-yl, 4-methoxycarbonyphenyl, 2-(trifluoromethyl)benzyl, hexan-2-yl, 2- methoxyethyl, cyclopentylmethyl, cyclohexyl, pyridin-3-ylmethyl, 4-carboxyphenyl, 3- aminophenyl, pyridin-3-yl, thien-2-yl, 4-hydroxyphenyl, 4-(l -aminocyclopropyl)benzyl, 4-(l -aminocyclopropyl)phenyl, 2-methylbenzyl, 4-nitrobenzyl, 4-chlorobenzyl, phenethyl, 4-bromobenzyl, 4-cyanobenzyl, 3-nitrobenzyl, 4-fert-butylbenzyl, 2- nitrobenzyl, 4-nitrophenethyl, 2-chloro-3-methoxycarbonylphenyl, 2-aminophenyl, [Ι, Γ- biphenyl]-4-yl, 4'-amino-[l, r-biphenyl]-4-yl, 4-fluorobenzyl, 3-(trifluoromethyl)benzyl, 3-(trifluoromethoxy)benzyl, 3,4-dichlorobenzyl, 2-cyanobenzyl, 3-chlorobenzyl, 4- amino-2-ethylphenyl, 4-amino-3-(trifluoromethoxy)phenyl, 4-amino-2,3-dimethylphenyl, 4-amino-5,6,7,8-tetrahydronaphthalen-l-yl, 4-amino-3-methylphenyl, 4-amino-3- fluorophenyl, 4-amino-3-ethylphenyl, and 4-amino-3-(trifluoromethyl)phenyl;

R¹¹ is selected from: Η, acetyl, propyl, ethyl, and methyl; and Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. Certain embodiments of the present disclosure relate to compounds of Formula Ik':

and pharmaceutically acceptable salts thereof, wherein: R¹ is H or C1 -C4 alkyl; R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl; and

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰.

[00206] In certain embodiments, in compounds of Formula Ik', R¹ is H, methyl or propyl.

[00207] In certain embodiments, in compounds of Formula Ik', R¹ is H or methyl.

[00208] In certain embodiments, in compounds of Formula Ik', R⁵ is G-G alkyl optionally substituted with one substituent selected from: G-G alkyloxy and G-G acyloxy. [00209] In certain embodiments, in compounds of Formula Ik', R⁵ is G-G alkyl optionally substituted with G-G acyloxy.

[00210] In certain embodiments, in compounds of Formula Ik', R⁶ is selected from: G-G alkyl, aryl, aryl -G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl, wherein the G-G alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio- G-G alkyl. [00211] In certain embodiments in compounds of Formula Ik', R⁶ is aryl or aryl-G-G alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00212] In certain embodiments in compounds of Formula Ik', R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00213] In certain embodiments in compounds of Formula Ik', R¹¹ is C1-C4 acyl or C1-C4 alkyl.

[00214] In certain embodiments in compounds of Formula Ik':

R¹ is methyl; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00215] In certain embodiments in compounds of Formula Ik': R¹ is H; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00216] In certain embodiments in compounds of Formula Ik': R¹ is H; and R⁵ is H or methyl; and

R¹¹ is selected from: H, acetyl, propyl, ethyl, and methyl. [00217] Certain embodiments of the present disclosure relate to compounds of Formula ΙΓ:

ΙΓ and pharmaceutically acceptable salts thereof, wherein: R¹ is H or C1-C4 alkyl;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-C6 haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl; and

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰.

[00218] In certain embodiments, in compounds of Formula ΙΓ, R¹ is H, methyl or propyl.

[00219] In certain embodiments, in compounds of Formula ΙΓ, R¹ is H or methyl.

[00220] In certain embodiments, in compounds of Formula ΙΓ, R⁵ is G-G alkyl optionally substituted with one substituent selected from: G-G alkyloxy and G-G acyloxy.

[00221] In certain embodiments, in compounds of Formula ΙΓ, R⁵ is G-G alkyl optionally substituted with G-G acyloxy.

[00222] In certain embodiments, in compounds of Formula ΙΓ, R⁶ is selected from: G-G alkyl, aryl, aryl -G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl, wherein the G-G alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- C1-C6 alkyl.

[00223] In certain embodiments in compounds of Formula ΙΓ, R⁶ is aryl or aryl-Ci-C4 alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio- Ci-Ce alkyl.

[00224] In certain embodiments in compounds of Formula ΙΓ, R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00225] In certain embodiments in compounds of Formula ΙΓ, R¹¹ is C1-C4 acyl or C1-C4 alkyl.

[00226] In certain embodiments in compounds of Formula ΙΓ: R¹ is methyl; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00227] In certain embodiments in compounds of Formula ΙΓ:

R¹ is H; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00228] Certain embodiments of the present disclosure relate to compounds of Formula Io:

Ιο and pharmaceutically acceptable salts thereof, wherein: R¹ is selected from: H and G-C6 alkyl; R² is C1-C6 alkyl; and R³ is R⁷-CH(CH₃)₂-; or

R⁶ is selected from: G-C6 alkyl, aryl, aryl-G-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R¹¹ is selected from: H, G-G acyl, and G-G alkyl; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. In certain embodiments in compounds of Formula lo, Z is absent. [00230] In certain embodiments in compounds of Formula Io, Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

[00231] In certain embodiments in compounds of Formula Io: R¹ is selected from: H, methyl, and isopropyl; R² is methyl; and R³ is R⁷-CH(CH₃)₂-; or

4- methoxyphenyl, phenyl, 2-fluorobenzyl, piperidin-l-yl, o-tolyl, 4-bromophenyl, naphthalen-2-yl, 4-methoxycarbonyphenyl, 2-(trifluoromethyl)benzyl, hexan-2-yl, 2- methoxyethyl, cyclopentylmethyl, cyclohexyl, pyridin-3-ylmethyl, 4-carboxyphenyl, 3- aminophenyl, pyridin-3-yl, thien-2-yl, 4-hydroxyphenyl, 4-(l-aminocyclopropyl)benzyl, 4-(l-aminocyclopropyl)phenyl, 2-methylbenzyl, 4-nitrobenzyl, 4-chlorobenzyl, phenethyl, 4-bromobenzyl, 4-cyanobenzyl, 3-nitrobenzyl, 4-fert-butylbenzyl, 2- nitrobenzyl, 4-nitrophenethyl, 2-chloro-3-methoxycarbonylphenyl, 2-aminophenyl, [Ι, Γ- biphenyl]-4-yl, 4'-amino-[l,r-biphenyl]-4-yl, 4-fluorobenzyl, 3-(trifluoromethyl)benzyl,

R⁷ is selected from: H, lH-indol-3-yl, l-methyl-lH-indol-3-yl, 2-methoxyphenyl, 3-((2- hydroxyethyl)amino)phenyl, 3 -((2-mercaptoethyl)amino)phenyl, 3 -(2- (acetylthio)ethoxy)phenyl, 3-(2-hydroxyethoxy)phenyl, 3-(2-mercaptoethoxy)phenyl, 3- (4-methylstyryl)phenyl, 3-(aminomethyl)phenyl, 3-(hydroxymethyl)phenyl, 3- hydroxyphenyl, 3,5-difluorophenyl, 3,5-dimethylphenyl, 3-aminophenyl, 3-chlorophenyl, 3-mercaptophenyl, 3-methoxyphenyl, 3-trifluoromethylphenyl, 4-((2- hydroxyethyl)amino)phenyl, 4-((2-mercaptoethyl)amino)phenyl, 4-(2-

R¹¹ is selected from: Η, acetyl, propyl, ethyl, and methyl; and Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent. Certain embodiments of the present disclosure relate to compounds of Formula Ir':

Ir' and pharmaceutically acceptable salts thereof, wherein: R¹ is H or C1-C4 alkyl;

R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy; R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl; and

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰.

[00233] In certain embodiments in compounds of Formula Ir', R¹ is H, methyl or propyl. [00234] In certain embodiments in compounds of Formula Ir', R¹ is H or methyl.

[00235] In certain embodiments, in compounds of Formula Ir', R⁵ is G-G alkyl optionally substituted with one substituent selected from: G-G alkyloxy and G-G acyloxy.

[00236] In certain embodiments, in compounds of Formula Ir', R⁵ is G-G alkyl optionally substituted with G-G acyloxy. [00237] In certain embodiments, in compounds of Formula Ir', R⁶ is selected from: G-G alkyl, aryl, aryl -G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl, wherein the G-G alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio- G-G alkyl.

[00238] In certain embodiments in compounds of Formula Ir', R⁶ is aryl or aryl-G-G alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio- G-G alkyl. [00239] In certain embodiments in compounds of Formula Ir', R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00240] In certain embodiments in compounds of Formula Ir', R¹¹ is C1-C4 acyl or C1-C4 alkyl.

[00241] In certain embodiments in compounds of Formula Ir':

R¹ is methyl; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00242] In certain embodiments in compounds of Formula Ir': R¹ is H; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl.

[00243] Certain embodiments of the present disclosure relate to compounds of general Formula Is':

Is' and pharmaceutically acceptable salts thereof, wherein: R¹ is H or C1-C4 alkyl;

R⁹ and R¹⁰ are each independently H or G-G alkyl; and

R¹¹ is selected from: H, G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰.

[00244] In certain embodiments, in compounds of Formula Is', R¹ is H, methyl or propyl.

[00245] In certain embodiments, in compounds of Formula Is', R¹ is H or methyl.

[00246] In certain embodiments, in compounds of Formula Is', R⁵ is G-G alkyl optionally substituted with one substituent selected from: G-G alkyloxy and G-G acyloxy. [00247] In certain embodiments, in compounds of Formula Is', R⁵ is G-G alkyl optionally substituted with G-G acyloxy.

[00248] In certain embodiments, in compounds of Formula Is', R⁶ is selected from: G-G alkyl, aryl, aryl -G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl, wherein the G-G alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-G alkyl, G-G cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio- G-G alkyl. [00249] In certain embodiments in compounds of Formula Is', R⁶ is aryl or aryl-G-G alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-

C1-C6 alkyl.

[00250] In certain embodiments in compounds of Formula Is', R⁶ is benzyl or phenyl, each optionally substituted with one or more substituent selected from amino and aminomethyl.

[00251] In certain embodiments in compounds of Formula Is', R¹¹ is C1-C4 acyl or C1-C4 alkyl.

[00252] In certain embodiments in compounds of Formula Is':

R¹ is methyl; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00253] In certain embodiments in compounds of Formula Is': R¹ is H; and

R⁵ is -CH2OR¹², where R¹² is selected from: C1-C4 acyl and C1-C4 alkyl. [00254] Certain embodiments relate to compounds of Formula Iw:

Iw harmaceutically acceptable salts thereof, wherein:

R is selected from: amino-G-C6 alkyl, amino-aryl, amino-G-G cycloalkyl , amino- heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from aryl, aryl-G-C6 alkyl, G-C6 alkyl, G-C6 alkylthio, carboxyl, carboxamide, C3-C7 cycloalkyl, C3-C7 cycloalkyl -C1-C6 alkyl, guanidino, halo, G-C6 haloalkyl, heterocyclyl, heterocyclyl-G-G alkyl, hydroxyl, and thio; or R is R^NCHCR³)-;

R¹ is selected from: H and G-C6 alkyl; R² is C1-C6 alkyl; and R³ is R⁷-CH(CH₃)₂-; or

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-Ci-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G- G alkyloxy, G-G alkoxycarbonyl, G-G alkyl, G-G alkylamino, amino, amino-G-G alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, G-G cycloalkyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-G alkyl;

R⁹ and R¹⁰ are each independently selected from: H, G-G alkyl, aryl, aryl-G-G alkyl, C3-C7 cycloalkyl-G-G alkyl, and hydroxy-G-G alkyl; X is selected from C2-C3 alkenyldiyl and G alkyldiyl, wherein G alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹;

R¹³ is selected from: C3-C6 alkyl and C3-C6-cycloalkyl;

Y is heteroaryldiyl, or Y is absent; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; with the proviso that when R is R^NCHCR³)-; R¹ is H; R² is methyl; R³ PhCH(CH₃)₂-; R⁵ is methyl; R¹³ is isopropyl; X is -CH=CH(CH₃)-; Y is absent and Z absent, then R⁴ is other than fert-butyl. [00255] One skilled in the art will appreciate that compounds of general Formula I and sub- formulae described above may be generated from a precursor molecule in which one or more functional groups in the compound are protected with an appropriate protecting group. For example, compounds of general Formula I or sub-formulae having a free amino, hydroxyl or carboxyl group may be generated from a precursor molecule in which the amino, hydroxyl or carboxyl group is protected with a standard protecting group, such as those defined above. Such precursor molecules comprising one or more protecting groups may themselves have cyctotoxic activity. Accordingly, certain embodiments of the present disclosure relate to precursors of compounds of general Formula I and sub-formulae that comprise one or more protecting groups. Some embodiments relate to precursors of compounds of general Formula I and sub-formulae in which one or more functional groups are present in protected form. Some embodiments relate to precursors of compounds of general Formula I and sub-formulae in which one or more amino, hydroxyl or carboxyl functional groups are present in protected form.

[00256] Certain embodiments of the present disclosure relate to compounds shown in Table A.

Table A

Compound 65

[00258] One skilled in the art will appreciate that certain compounds of general Formula I and sub-formulae as described above may possess a sufficiently acidic group, a sufficiently basic group, or both functional groups, to react with a number of organic and inorganic bases, or organic and inorganic acids, to form pharmaceutically acceptable salts. Accordingly, certain embodiments of the present disclosure encompass pharmaceutically acceptable salts of the compounds of general Formula I and sub-formulae as described above.

[00259] The compounds of general Formula I and sub-formulae may be prepared according to relevant published literature procedures that are used by one skilled in the art. Exemplary reagents and procedures for these reactions are provided in the Examples section herein. In certain embodiments, the present disclosure relates to methods of making a compound described herein.

[00260] Although specific embodiments are set out above defining certain combinations of substituents, it is to be understood that all combinations of the embodiments pertaining to the chemical groups represented by variables such as various R groups, X, Y, and Z contained within general Formula I and sub-formulae described herein are specifically encompassed just as if each and every combination was individually explicitly recited, to the extent that such combinations define compounds that result in stable compounds (i.e., compounds that can be isolated, characterized and tested for biological activity), and each forms a separate embodiment for the purposes of the present disclosure even if not specifically identified as such. In addition, all subcombinations of the chemical groups listed in the embodiments describing such variables, as well as all subcombinations of uses and medical indications described herein, are also specifically embraced by the present invention just as if each and every subcombination of chemical groups and subcombination of uses and medical indications was individually and explicitly recited herein. In addition, in the event that a list of substituents is listed for any particular R group, and X, Y, or Z in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the particular embodiment and/or claim and that the remaining list of substituents will be considered to be within the scope of the present disclosure.

[00261] One skilled in the art will appreciate that certain compounds disclosed herein may exhibit tautomerism. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. It is to be understood that the structural formulae herein are intended to represent any tautomeric form of the depicted compound that has the requisite activity and are thus not limited to the specific compound form depicted by the structural formulae, unless stated or shown otherwise. [00262] One skilled in the art will appreciate that compounds of general Formula I and sub- formulae may have one or more chiral centers and therefore can exist as enantiomers and/or diastereoisomers. Thus, compounds of general Formula I and sub-formulae used throughout this disclosure are intended to represent all individual enantiomers and mixtures thereof, unless stated or shown otherwise. Certain embodiments of the present disclosure encompass all such enantiomers, diastereoisomers and mixtures thereof, including but not limited to racemates. Certain embodiments relate to compounds of general Formula I and sub-formulae in a substantially pure enantiomeric, diastereomeric or isomeric form. In this context, "substantially pure" is intended to mean that the compound is in a form that is at least 80% optically pure, that is, a form that comprises at least 80% of a single isomer. In certain embodiments, chiral compounds may be in a form that is at least 85% optically pure, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% optically pure.

[00263] Certain embodiments of the present disclosure encompass compounds of general Formula I and sub-formulae that are isotopically-labelled (i.e. have one or more atoms replaced by an atom having a different atomic mass or mass number). Examples of isotopes that can be incorporated into the compounds described herein in some embodiments include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ⁿC, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶C1, ¹²³I, and ¹²⁵I. Such radiolabeled compounds may be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Isotopically-labelled compounds, for example, those incorporating a radioactive isotope, may also be useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium (³H) and carbon-14 (¹⁴C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium (²H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Substitution with positron emitting isotopes, such as ⁿC, ¹⁸F, ¹⁵0 and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples provided herein using an appropriate isotopically-labeled reagent in place of the noted non-labeled reagent.

[00264] In certain embodiments, the present disclosure encompasses any in vivo metabolic products of the compounds described herein. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, or the like, of the administered compound, primarily due to enzymatic processes. Accordingly, in some embodiments, the present disclosure includes compounds produced by a process comprising administering a compound of general Formula I or a sub-formula described herein to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled version of the compound in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.

[00265] It is to be understood that reference to compounds of general Formula I throughout the remainder of this disclosure, includes in various embodiments, compounds of each of the sub- formulae described above to the same extent as if embodiments reciting each of these sub-formulae individually were specifically recited. Preparation of Compounds of General Formula I

[00266] Compounds of general Formula I may be prepared from known starting materials by standard synthetic chemistry methods. Representative examples of suitable synthetic routes are described in detail in the Examples provided herein. One skilled in the art will recognize that alternative methods may be employed to synthesize compounds of general Formula I, and that the approaches described herein are therefore not intended to be exhaustive, but rather to provide the skilled person with examples of some broadly applicable and practical routes to representative compounds.

CONJUGATES COMPRISING COMPOUNDS [00267] In certain embodiments, the compounds of general Formula I and sub-formulae described herein may be used to form conjugates, in which the compound is conjugated, either directly or via a linker, to a targeting moiety.

[00268] Accordingly, in certain embodiments, the conjugates may be represented by general Formula II:

(T)-(L)-(D)

II

[00269] wherein (T) is a targeting moiety, (L) is an optional linker, and (D) is a monovalent radical of a compound of Formula I.

[00270] In certain embodiments, conjugates of the present disclosure may comprise multiple compounds of general Formula I conjugated to the targeting moiety, for example, by attaching the compounds at different sites on the targeting moiety and/or by employing a linker that allows for attachment of multiple compounds to a single site on the targeting moiety.

[00271] Accordingly, certain embodiments relate to conjugates of general Formula VI:

T-(L-(D)_m),

VI [00272] wherein T is the targeting moiety, L is an optional linker; D is a monovalent radical of a compound of general Formula I; m is an integer between 1 and about 10, and n is an integer between 1 and about 20. In certain embodiments, m is between about 1 and about 5, or between 1 and 2. In some embodiments, m is 1. In some embodiments, n is between 1 and 10, for example, between 1 and 8, between 2 and 8, between 2 and 6, or between 2 and 4. In some embodiments, L may be absent. In some embodiments, L is present.

[00273] One skilled in the art will appreciate that compounds of general Formula I are peptide- based and thus may be considered to have a C-terminus and an N-terminus. In certain embodiments, a compound of general Formula I may be conjugated to the targeting moiety or linker via a suitably reactive group at either the C- or N-terminus of the molecule. Such a reactive group may already be present in the compound, or the compound may be modified by standard techniques to introduce a suitable reactive group. In some embodiments, the compound of general Formula I is conjugated via the C-terminus of the molecule. In some embodiments, the compound of general Formula I is conjugated via the N-terminus of the molecule. [00274] In some embodiments, (T) is an antibody. Accordingly, certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising compounds of Formula I (D).

[00275] As will be appreciated by the skilled artisan, a wide variety of means are available to covalently link (T)-(L)-(D). For example, when (L) is present, a wide variety of linker technologies known in the art may be used to link (T) to (D). Further, (T), (L), and (D) may be modified in any suitable manner, as recognized by the person of skill in the art, in order to facilitate conjugate formation.

Targeting Moiety (Ύ)

[00276] The Targeting moiety (T) of the conjugates described herein includes within its scope any unit of a (T) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. In one aspect, (T) acts to deliver the compound of general Formula I (D) to the particular target cell population with which (T) reacts. Such (T) moities include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments, smaller molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), and other molecules or substances capable of binding to a target cell.

[00277] A (T) may form a bond to a linker unit (L) or the compound (D). A (T) can form a bond to a (L) unit via a heteroatom of the (T). Heteroatoms that may be present on a (T) include sulfur (for example, from a sulfhydryl group of a (T)), oxygen (for example, from a carbonyl, carboxyl or hydroxyl group of a (T)) and nitrogen (for example, from a primary or secondary amino group of a (T)). These heteroatoms may be naturally present on (T), or may be introduced into (T) via chemical modification using techniques known in the art. [00278] In certain embodiments, the conjugate includes a linker (L) and (T) is conjugated to the linker via a sulfhydryl group on (T). For example, a (T) may be selected that has a naturally occurring sulfhydryl group such that (T) bonds to (L) via the sulfur atom of the sulfhydryl group. Alternatively, a (T) may be selected that has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SAT A) and 2-iminothiolane hydrochloride (Traut's Reagent). A (T) may also be selected that has one or more carbohydrate groups that can be chemically modified to include one or more sulfhydryl groups.

[00279] In certain embodiments, a (T) may be selected that has one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, for example, Laguzza et al, 1989, J. Med. Chem. 32(3):548-55). The corresponding aldehyde can form a bond with a reactive site on a portion of a (L). Reactive sites that can react with a carbonyl group on a (T) include, but are not limited to, hydrazine and hydroxylamine.

[00280] In some embodiments, a surface lysine residue on (T) may be used to link (T) to (L).

[00281] Alternatively, attachment of (L) to (T) may be achieved by modification of (T) to include additional cysteine residues (see, for example, U.S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p- acetylphenylalanine, formylglycine or /j>-azidomethyl-L-phenylalanine (see, for example, Hofer et al, (2009) Biochemistry 48: 12047-12057; Axup et al, (2012) PNAS 109: 16101-16106; Wu et al, (2009) PNAS 106:3000-3005; Zimmerman et al, (2014) Bioconj. Chem. 25:351-361), to allow for site-specific conjugation.

[00282] Other protocols for the modification of proteins for the attachment or association of (D) are known in the art and include those described in Coligan et al, Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002). [00283] Various targeting moieties suitable for inclusion in drug conjugates are known in the art. For example, the (T) included in the conjugate may be a transferrin, an epidermal growth factor ("EGF"), bombesin, gastrin, a gastrin-releasing peptide, a platelet-derived growth factor, IL-2, IL- 6, a transforming growth factor ("TGF," such as TGF-a or TGF-β), a vaccinia growth factor ("VGF"), insulin, insulin-like growth factor I or II, a lectin or apoprotein from low density lipoprotein. Other examples include engineered protein scaffolds such as those described in International Patent Publication Nos. WO 2012/116453 and WO 2014/012082. In addition, included within (T) in some embodiments are ligands for cell surface receptors derived from various sources, including those derived from human cells, ligands derived from bacteria, and pathogen derived ligands, such as those described in International Patent Publication No. WO 2013/117705.

[00284] In certain embodiments, the (T) for inclusion in the conjugate compositions may be an antibody, such as a polyclonal antibody or monoclonal antibody. The antibody may be directed to a particular antigenic determinant, such as for example, a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof. Methods of producing polyclonal antibodies are known in the art. A monoclonal antibody (mAb) to an antigen of interest may also be prepared using various techniques well known in the art. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV -hybridoma technique (Cole et al. , 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). The Selected Lymphocyte Antibody Method (SLAM) (Babcook, et al , 1996, Proc Natl Acad Sci USA, 93(15):7843-8; McLean et al., 2005, J Immunol. 174(8):4768-78) may also be used to generate monoclonal antibodies. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. [00285] In certain embodiments, (T) may be a monoclonal antibody. The monoclonal antibody may be, for example, a human monoclonal antibody, a humanized monoclonal antibody, an antibody fragment, or a chimeric antibody (for example, a human-mouse antibody). Human monoclonal antibodies may be made by any of numerous techniques known in the art (see, for example, Teng et al, 1983, Proc. Natl. Acad. Sci. USA 80:7308-7312; Kozbor et al, 1983, Immunology Today 4:72-79; Olsson et al, 1982, Meth. Enzymol. 92:3-16; Huse et al, 1989, Science 246: 1275-1281 and McLean et al J Immunol. 2005 Apr 15; 174(8):4768-78).

[00286] In certain embodiments, the antibody included in the conjugate may be a bispecific antibody. Methods for making bispecific antibodies are known in the art. For example, traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Milstein et al, 1983, Nature 305:537-539; International Patent Publication No. WO 93/08829, Traunecker et al, 1991, EMBO J. 10:3655-3659). Alternatively, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences as described in International Patent Publication No. WO 94/04690. For example, the bispecific antibodies can have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.

[00287] Other useful methods for preparing bispecific antibodies include those described in International Patent Publication Nos. WO 2012/032080; WO 2012/058768 and WO 2013/063702, and in European Patent Publication No. EP 2 560 993.

[00288] Additional methods for generating bispecific antibodies are described in Suresh et al, 1986, Methods in Enzymology 121 :210; Rodrigues et al, 1993, J. Immunology 151 :6954-6961; Carter et al, 1992, Bio/Technology 10: 163-167; Carter et al, 1995, J. Hematotherapy 4:463-470; Merchant et al, 1998, Nature Biotechnology 16:677-681.

[00289] Bifunctional antibodies are also described in European Patent Publication No. EP 0 105 360. As described in this reference, hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof. Methods for obtaining such hybrid antibodies are described for example, in International Patent Publication No. WO 83/03679, and European Patent Publication No. EP 0 217 577.

[00290] In certain embodiments, (T) may be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to a target antigen (for example, a cancer antigen, a viral antigen, a microbial antigen, or other antibodies bound to cells or matrix). In this regard, "functionally active" means that the fragment, derivative or analog is able to recognize the same antigen as that recognized by the antibody from which the fragment, derivative or analog is derived. For example, the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (for example, the BIAcore assay) (see, for example, Kabat et al, 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat et al, 1980, J. Immunology 125(3):961-969).

[00291] Other useful antibody fragments include, but not limited to, F(ab')2 fragments, Fab fragments, Fab', Fv fragments and heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (for example, as described in U.S. Patent No. 4,946,778; Bird, (1988) Science 242:423-42; Huston et al , (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883; and Ward et al, (1989) Nature 334:544-54). Antibodies suitable for inclusion in the conjugate compositions in some embodiments may be heavy chain only antibodies (such as those generated using the Humabody® VH platform from Crescendo Biologies, Cambridge, UK) or camelid-derived antibodies (see, for example, International Patent Publication No. WO 2010/001251). [00292] In certain embodiments, recombinant antibodies, such as chimeric or humanized monoclonal antibodies, comprising both human and non-human portions, may be used. Such recombinant antibodies can be made using standard recombinant DNA techniques (see, for example, U.S. Patent Nos. 4,816,567 and 4,816,397). Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, for example, U.S. Patent No. 5,585,089). Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Patent Publication Nos. WO 87/02671 and WO 86/01533; European Patent Publication Nos. 012 023; 0 184 187; 0 171 496 and 0 173 494; U.S. Patent Nos. 4,816,567 and 5,225,539; Berter et al, (1988) Science 240: 1041-1043; Liu et al, (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al , (1987) J. Immunol. 139:3521-3526; Sun et al, (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura ei /., (1987) Cancer. Res. 47:999-1005; Wood et al, (1985) Nature 314:446-449; Shaw et al, (1988) J. Natl. Cancer Inst. 80: 1553-1559; Morrison, (1985) Science 229: 1202-1207; Oi et al, (1986) BioTechniques 4:214; Jones et al, (1986) Nature 321 :552-525; Verhoeyan et al , (1988) Science 239: 1534; and Beidler et al, (1988) J. Immunol. 141 :4053-4060.

[00293] In some embodiments, completely human antibodies may be used. Human antibodies can be prepared, for example, using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen and monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93. Other methods are described in, for example, U.S. Patent Nos. 5,625, 126; 5,633,425; 5,569,825; 5,661,016 and 5,545,806, and in International Patent Publication No. WO 2009/127771.

[00294] Human antibodies that recognize a selected epitope also can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, for example a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (see, for example, Jespers et al , (1994) Biotechnology 12:899-903). Other methods of generating human antibodies include the use of phage display libraries (see, for example, Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381; Marks et al, (1991) J. Mol. Biol. 222:581; Quan and Carter, (2002) "The rise of monoclonal antibodies as therapeutics," in Anti-lgE and Allergic Disease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp. 427-469) and technologies such as mAbLogix™ (Intrexon, Blacksburg, VA), the Adimab discovery platform (Adimab, Lebanon, NH) and the iTab platform (Igenica, Burlingame, CA).

[00295] In certain embodiments, (T) may be a fusion protein of an antibody or a functionally active antibody fragment thereof. For example, an antibody or antibody fragment can be fused via a covalent bond (for example, a peptide bond) at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, such as at least a 10, 20 or 50 amino acid portion of the protein) that is not an antibody.

[00296] Analogs and derivatives of antibodies that may be useful in the conjugate compositions in some embodiments include antibodies modified by additional of further binding motifs (for example, Zybodies™, Zygenia, Gaithersburg, MD), and those that have been modified by, for example, one or more of formylation, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or linkage to a cellular antibody unit or other protein. Additionally, an analog or derivative may contain one or more unnatural amino acids.

[00297] Antibodies for use in the conjugate compositions include in some embodiments antibodies having amino acid modifications (for example, substitutions, deletions or additions) in a region that interacts with an Fc receptor (see, for example, International Patent Publication Nos. WO 97/34631 and WO 2013/004842, and European Patent Publication No. EP 2 552 957). [00298] Antibodies immunospecific for a target antigen can be obtained commercially or other source or produced by various methods known to one of skill in the art such as, for example, chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank database or a similar database, scientific publications, or by routine cloning and sequencing.

[00299] In certain embodiments, the targeting moiety comprised by the conjugate is an antibody or fragment thereof that binds to a cancer antigen. Examples of antibodies that target cancer antigens and which are useful in the treatment of cancer include, but are not limited to, humanized anti-HER2 monoclonal antibody, HERCEPTIN® (trastuzumab; Genentech); RITUXAN® (rituximab; Genentech), a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin' s lymphoma; OvaRex (AltaRex Corporation, MA), a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY), an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; Vitaxin (Medlmmune, Inc., MD), is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite, MA), a humanized IgGl antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA), a humanized anti- CD33 IgG antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ), a humanized anti-CD22 IgG antibody for the treatment of non- Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA), a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolym (Techniclone, Inc., CA), a radiolabeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA), a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma; Avastin (Genentech, Inc., CA), an anti-VEGF humanized antibody for the treatment of lung and colorectal cancers; Epratuzamab (Immunomedics, Inc., NJ and Amgen, CA), an anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ), a humanized anti-CEA antibody for the treatment of colorectal cancer. [00300] Other examples include BR96 mAb (Trail et al , (1993) Science 261 :212-215); BR64 (Trail et al, (1997) Cancer Research 57: 100-105); mAbs against the CD40 antigen, such as S2C6 mAb (Francisco et al, (2000) Cancer Res. 60:3225-3231) and chimeric and humanized variants thereof; mAbs against the CD33 antigen; mAbs against the EphA2 antigen; mAbs against the CD70 antigen, such as 1F6 mAb and 2F2 mAb and chimeric and humanized variants thereof, and mAbs against the CD30 antigen, such as AC10 (Bowen et al, (1993) J. Immunol. 151 :5896-5906; Wahl et al, (2002) Cancer Res. 62(13):3736-42) and chimeric and humanized variants thereof. Many other internalizing antibodies that bind to tumor associated antigens can be used and have been reviewed (see, for example, Franke et al, (2000) Cancer Biother. Radiopharm. 15:459 76; Murray, (2000) Semin. Oncol. 27:64 70; Breitling et al, Recombinant Antibodies, John Wiley & Sons, New York, 1998).

[00301] Other cancer antigens (or tumor-associated antigens (TAAs)) that may be targeted by antibodies include, but are not limited to, CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific membrane antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gplOO (melanoma), MARTI (melanoma), prostate specific antigen (PSA) (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product (carcinomas).

[00302] In certain embodiments, the targeting moiety comprised by the conjugate is an antibody or fragment thereof that binds to an antigen associated with an immunological disease. Examples of antibodies that target antigens associated with immunological diseases and which are useful in the treatment of autoimmune and rheumatologic diseases include those that target cytokines, B cells or co-stimulation molecules. Anti-cytokine antibodies include, for example, anti-tumor necrosis factor (TNF)-a, anti-interleukin (IL)-l, and anti-IL-6 antibodies. Examples of antibodies which target B cells include, for example, anti-CD20 antibodies, which are useful for B-cell depletion, and anti-B lymphocyte stimulator (BLyS) antibodies, which are useful for B cell receptor (BCR) modulation.

[00303] Examples of antibodies that target such antigens and are used in the treatment of autoimmune and rheumatologic diseases include, but are not limited to, abatacept (Orencia), a CTLA-4 IgGl fusion that binds to CD80/86; adalimumab (Humira), a human anti-TNF-a antibody; anakinra (Kineret), an anti-IL-1 antibody; belimumab (Benlysta), a human monoclonal anti -BLyS antibody; canakinumab (Ilaris), an anti-IL-1 antibody; certolizumab pegol (Cimzia), a pegylated Fab fragment of a humanized anti-TNF-a monoclonal antibody; efalizumab (Raptiva), a humanized anti-CDl la antibody; epratuzumab, an IgGl monoclonal anti-CD22 antibody; golimumab (Simponi), a fully human monoclonal IgGl anti-TNF-a antibody; infliximab (Remicade), a chimeric anti-TNF-a antibody; natalizumab (Tysabri), a humanized anti-alpha-4 integrin antibody; ofatumumab (Arzerra), a fully human monoclonal anti-CD20 antibody; rilonacept (Arcalyst), an anti-IL-1 antibody; rituximab (Rituxan, Mabthera), a chimeric human monoclonal anti-CD20 antibody; sifalimumab, an anti-IFN-a monoclonal antibody; tocilizumab (Actemra, Roactemra), a recombinant monoclonal IgGl anti -human IL-6 receptor (IL-6R) antibody; ustekinumab (Stelara), an anti-IL-12/IL-23 antibody, and vedolizumab (Entyvio), a humanized anti-integrin α4,β7 antibody. Linker Moiety (L)

[00304] The conjugate compositions optionally include a linker moiety (L), which is a bifunctional or multifunctional moiety capable of linking one or more compounds of general Formula I to targeting moiety (T). In some embodiments, the linker may be bifunctional (or monovalent) such that it links a single compound to a single site on (T). In some embodiments, the linker may be multifunctional (or polyvalent) such that it links more than one compound to a single site on (T). Multifunctional linkers may also be used to link one compound to more than one site on (T) in some embodiments.

[00305] The linkers include a functional group capable of reacting with the target group or groups on (T) and one or more functional groups capable of reacting with a target group on the compound of general Formula I (D). Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Non-limiting examples of functional groups for reacting with free cysteines or thiols include maleimide, haloacetamide, haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Also useful in this context are "self-stabilizing" maleimides as described in Lyon et al, (2014) Nat. Biotechnol. 32: 1059-1062. Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters such as N-hydroxysuccinamide (NHS) esters or sulfo-NHS esters, imido esters such as Traut's reagent, isothiocyanates, aldehydes and acid anhydrides such as diethylenetriaminepentaacetic anhydride (DTP A). Other examples include succinimido-l, l,3,3-tetra-methyluronium tetrafluoroborate (TSTU) and benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Non-limiting examples of functional groups capable of reacting with an electrophilic group on (T) or (D) (such as an aldehyde or ketone carbonyl group) include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide. [00306] In certain embodiments in which (T) is an antibody, a linker that includes a functional group that allows for bridging of two interchain cysteines on the antibody binding construct may be used, such as a ThioBridge™ linker (Badescu et al, (2014) Bioconjug. Chem. 25 : 1124-1136), a dithiomaleimide (DTM) linker (Behrens et al, 2015, Mol. Pharm. 12:3986-3998), a dithioaryl(TCEP)pyridazinedione based linker (Lee et al, (2016) Chem. Sci. 7:799-802) or a dibromopyridazinedione based linker (Maruani et al, (2015) Nat. Commun. 6:6645).

[00307] A variety of linkers for linking drugs to antibodies and other targeting moieties are known in the art, including hydrazone-, disulfide- and peptide-based linkers.

[00308] Suitable linkers typically are more chemically stable to conditions outside the cell than to conditions inside the cell, although less stable linkers may be contemplated in certain situations, such as when (D) has a low toxicity to normal cells. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitive or reduction-sensitive. Non-cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-cytotoxin moiety.

[00309] Suitable cleavable linkers include, for example, peptide-containing linkers cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In certain embodiments, the linker may be a dipeptide-containing linker, such as a valine-citrulline (Val-Cit) or a phenylalanine-lysine (Phe-Lys) linker. Other examples of suitable dipeptides for inclusion in cleavable linkers include Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, lie-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me₃Lys-Pro, PhenylGly-(D)Lys, Met- (D)Lys, Asn-(D)Lys, Pro-(D)Lys and Met-(D)Lys. Linkers may also include longer peptide sequences in some embodiments, such as the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe- Lys or (D)Ala-Phe-Lys, or the tetrapeptides Gly-Phe-Leu-Gly or Ala-Leu-Ala-Leu.

[00310] Additional suitable cleavable linkers include disulfide-containing linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Other suitable linkers include linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.

[00311] A further example of a cleavable linker is a linker comprising a β-glucuronide, which is cleavable by β-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf et al, (2002) Curr. Pharm. Des. 8: 1391-1403).

[00312] Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative and self-elimination groups, stretchers or hydrophilic moieties.

[00313] Self-immolative and self-elimination groups that find use in linkers include, for example, /j>-aminobenzyloxycarbonyl (PABC) and /j>-aminobenzyl ether (PABE) groups, and methylated ethylene diamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5 -methanol derivatives as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al. (1990) J. Org. Chem. 55:5867-5877). Self-immolative/self-elimination groups, alone or in combination are often included in peptide-based linkers, but may also be included in other types of linkers. In some embodiments, the linker may include one or more self-immolative and self- elimination groups, for example, a PABC group, a PABE group, or a combination of a PABC or PABE group and an MED.

[00314] Other examples include the linkers described in International Patent Publication No. WO 2016/04084.

[00315] Stretchers that find use in linkers for drug conjugates include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties and may be particularly useful with hydrophobic drugs

[00316] In certain embodiments, the linker included in the conjugate compositions of the present disclosure is a peptide-based linker of general Formula VII:

VII wherein:

Z is a functional group capable of reacting with the target group on (T);

Str is a stretcher;

AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]_m forms a protease cleavage site;

X is a self-immolative group;

D is a monovalent radical of a compound of general Formula I; n is 0 or 1; m is 1, 2 or 3, and o is 0, 1 or 2.

[00317] In some embodiments, in general Formula VII:

[00318] In some embodiments, in general Formula VII:

O O

-(CH2)p— C— . — (CH₂CH₂0)_q— C— . — (CH2)_p— (CH₂CH₂0)_q

O

I I

(CH₂CH₂0)_q— (CH₂)_p— C— (CH2)_p -C-N-(CH2)p

or O R

(CH2)_p— C-N-(CH₂CH₂0)_q wherein

R is H or C1-C6 alkyl; p is an integer between 2 and 10, and q is an integer between 1 and 10.

[00319] In some embodiments of general Formula VII, in which:

O O O

1 1 I I I I

— (CH^p— C— . — (CH₂CH₂0)_q— C— . — (CH2)_p— (CH₂CH₂0)_q— C—

Str is

O

1 1

— (CH₂CH₂0)_q— (CH₂)p— C— (CHz)_p -C-N-(CH₂)_p

or

O R O

— (CH₂)p— C-N-(CH₂CH₂0)_q— C-

R is H or optionally substituted G-C6 alkyl.

[00320] Examples of substituents when R is substituted Ci -Ce alkyl include various hydrophilic groups, such as hydroxyl or a PEG group.

[00321] In some embodiments, in general Formula VII:

O o

I I

Str is — (CH2)_p -C- — (CH₂CH₂0)_q— C—

or O

— (CH₂CH₂0)_q— (CH₂)_p— C— wherein p and q are as defined above.

[00322] In some embodiments, in general Formula VII:

O O

s_{tr is}— (CH₂)_p -C- _or— (CH₂CH₂0)_q— (CH2)_p-C- ₅ wherein p is an integer between 2 and 6, and q is an integer between 2 and 8.

[00323] In some embodiments, in general Formula VII:

AAi-[AA2]m is selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile- Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, lie-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me₃Lys-

Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit- Val, Gly-Cit-Val, (D)Phe-Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly and Ala-Leu-Ala- Leu.

[00324] In some embodiments, in general Formula VII: m is 1 (i.e. AAi-[AA₂]_m is a dipeptide). [00325] In some embodiments, in general Formula VII:

AAi-[AA2]m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

[00326] In some embodiments, in general Formula VII: each X is independently selected from /j>-aminobenzyloxycarbonyl (PABC), p- aminobenzyl ether (PABE) and methylated ethylene diamine (MED).

[00327] In some embodiments, in general Formula VII: n is 1. [00328] In some embodiments, in general Formula VII: o is 1 or 2.

[00329] In some embodiments, in general Formula VII: o is 0 (i.e. X is absent).

[00330] In some embodiments, in general Formula VII:

O O

_C b_†tr i -s— (CH2)_D P— C— or— (CH₂CH₂0) ^/ _a ^ci— (CH₂)_D— C— , w ,herein p is an integer between 2 and 6, and q is an integer between 2 and 8; m is 1 and AAi-[AA2]_m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit; each X is independently selected from /j>-aminobenzyloxycarbonyl (PABC), p- aminobenzyl ether (PABE) and methylated ethylene diamine (MED); n is 1, and o is 1 or 2.

In some embodiments, in general Formula VII:

O O

I I I I

Str _is— (CH2)p— C— _or— (CH₂CH₂0)_q— (CH₂)_p— C— _{^ wherem p 1S m mteger} between 2 and 6, and q is an integer between 2 and 8; m is 1 and AAi-[AA2]_m is a dipeptide selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Τφ-Cit; n is 1, and o is 0.

[00332] In some embodiments, the linker is a disulfide-containing linker and the conjugate has general Formula VIII:

VIII wherein:

T is the targeting moiey;

D is the compound of general Formula I;

Y is -(0¼)_Ρ- or -(Ο¼Ο¼0)_¾-, wherein p and q are each independently an integer between 1 and 10; each R is independently H or G-C6 alkyl; n is 1, 2 or 3, and

_H i?_

wherein N C represents an amide bond formed between the linker and the ε-amino group of a surface lysine on (T).

[00333] Examples of commonly used cleavable linkers that may find use in the conjugate compositions of the present disclosure in some embodiments include, but are not limited to, linkers comprising SPDB, sulfo-SPDB, hydrazone, Val-Cit, maleidocaproyl (MC or mc), mc -Val-Cit, mc- Val-Cit-PABC, Phe-Lys, mc-Phe-Lys or mc-Phe-Lys-PABC. [00334] Various non-cleavable linkers are known in the art for linking drugs to targeting moieties and may be useful in the conjugate compsitions of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N- sulfosuccinimidyl ester moiety for reaction with the cell binding agent, as well as a maleimido- or haloacetyl-based moiety for reaction with the drug, or vice versa. An example of such a non- cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols,— SH) on the drug moiety, while the sulfo-NHS ester is reactive toward primary amines (as found in lysine and the protein or peptide N-terminus). Other non-limiting examples of such linkers include those based on N-succinimidyl 4- (maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)- cyclohexane-l-carboxy-(6-amidocaproate) ("long chain" SMCC or LC-SMCC), κ- maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N- succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m- maleimidobenzoyl -N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(P-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4- (p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those comprising a haloacetyl-based functional group such as N-succinimidyl-4- (iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[00335] Other examples of non-cleavable linkers include maleimidocarboxylic acids, such as maleimidocaproyl (MC).

[00336] In certain embodiments, (T) is conjugated to a compound of general Formula I (D) via a linker (L) that comprises a linker moiety as described in International Patent Publication No. WO 2015/095953. Accordingly, certain embodiments of the present disclosure relate to conjugate compositions of general Formula III:

III wherein: (T) is a targeting moiety; n is an integer between 1 and 10;

(D ^•l) has general Formula (IV):

IV wherein R, R⁴, R⁵, X, Y, and Z are as described herein supra for general Formula I or any sub -formula thereof, and

R^6a is selected from: d-Ce alkyldiyl, aryldiyl, C3-C7 cycloalkyldiyl, heteroaryldiyl, and heterocyclyldiyl, each optionally substituted with one or more substituents selected from: G-C6 alkyloxy, G-C6 alkoxycarbonyl, G-C6 alkyl, G-C6 alkylamino, amino, amino-G-C6 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, aryl, carboxamide, carboxyl, C3-C7 cycloalkyl, cyano, G-C6 haloalkyl, G-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-C6 alkyl;

(Ι^)-(Τ) has general Formula (V):

V wherein each AA is independently an amino acid; p is an integer from 0 to 25; (L²) is optionally the remaining portion of linker (L¹), and wherein the -NH- group bonded to R^6a in Formula IV forms a peptide bond (JPB) with (AA)i in Formula V, wherein the JPB is enzymatically cleavable, and wherein (AA)i-(AA)n, taken together comprises an amino acid sequence capable of facilitating enyzmatic cleavage of the JPB. [00337] It will be appreciated that various linker moieties known in the art, and particularly those known to be useful in the context of drug delivery, may be used in the conjugates described herein. Such linkers include, but are not limited to those described in International Patent Publication Nos. WO 2012/171020; WO 2012/113847; WO 2010/138719; WO 2007/103288; WO 2008/083312; WO 2003/068144; WO 2004/016801; WO 2009/134976; WO 2009/134952; WO 2009/134977; WO 2002/08180; WO 2004/043493; WO 2007/018431; WO 2003/026577; WO 2005/077090; WO 2005/082023; WO 2007/011968; WO 2007/038658; WO 2007/059404; WO 2006/110476; WO 2005/112919, and WO 2008/103693; U.S. Patent Nos. 6,756,037; 7,087,229; 7,122, 189; 7,332,164; 5,556,623; 5,643,573; 5,665,358; 8,288,352; 5,028,697; 5,006,652; 5,094,849; 5,053,394; 5, 122,368; 5,387,578; 5,547,667; 622,929; 5,708, 146; 6,468,522; 6,103,236; 6,638,509; 6,214,345; and 6,759,509; European Patent Application No. 2 326 349; Kim et al, (2010) Bioconjugate Chemistry, 21(8): 1513-1519; Dommerholt et al, (2010) Angew. Chem. Int. Ed., 49:9422-9425; van Hest and van Delft, (2011) ChemBioChem, 12: 1309-1312; and the range of linkers available from Concords Biotherapeutics (San Diego, CA) and Synaffix BV (Oss, NL). [00338] Selection of an appropriate linker for a given conjugate may be readily made by the skilled person having knowledge of the art and taking into account relevant factors, such as the site of attachment to the targeting moiety, any structural constraints of the drug and the hydrophobicity of the drug (see, for example, review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). Drug Moiety (D)

[00339] (D) is a compound of Formula I as described herein. It will be recognized by a worker skilled in the art that compounds described herein may be appropriately modified to facilitate a conjugation reaction with (L), or if (L) is not present, with (T). The skilled person will also appreciate that, depending on the exact structure of (D), various points of attachment on (D) may be used, for example, the C-terminus, the N-terminus or a side chain of (D) may form the point of attachment.

[00340] In certain embodiments, compounds of general Formula I for inclusion in the conjugate compositions have, or can be modified to include, a suitable functional group at the N- or C- terminus for conjugation to the linker or targeting moiety. In some embodiments, compounds of general Formula I for inclusion in the conjugate compositions have, or can be modified to include, a suitable functional group at the C-terminus for conjugation to the linker or targeting moiety. Examples of suitable functional groups include, but are not limited to, hydroxyls, thiols, amino groups, carboxylic acid groups, acyl halides, halide groups, amides, esters, cyano groups, azide groups, and the like. Preparation of the Conjugates

[00341] The conjugate compositions may be prepared by one of several routes known in the art, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). For example, conjugation may be achieved by reaction of the targeting moiety (T) with a bivalent linker reagent to form a T-L intermediate via a covalent bond, followed by reaction with an activated (D) moiety; or reaction of (D) with a linker reagent to form a D-L intermediate via a covalent bond, followed by reaction with (T). Such conjugation methods may be employed with a variety of compounds, targeting moieties and linkers to prepare the conjugate compositions described herein. Various prepared linkers and linker components are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al, (2002) J. Org. Chem. 67: 1866-1872; Frisch et al, (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)).

[00342] Several specific examples of methods of preparing antibody drug conjugates are known in the art, for example, those described in U.S. Patent Nos 8,624,003 (pot method), 8,163,888 (one- step), and 5,208,020 (two-step method). Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer. In addition, various antibody drug conjugation services are available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramel Pharma Solutions (Grangemouth, UK).

[00343] The average number of drugs conjugated to the targeting moiety (referred to as the "drug- to-antibody ratio" or DAR) may be determined by standard techniques such as UV7VIS spectroscopic analysis, ELISA -based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS. In addition, distribution of drug-linked forms (for example, the fraction of targeting moieties containing zero, one, two, three, etc. drugs) may also optionally be analyzed. Various techniques are known in the art to measure such distribution, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al, (2011) mAbs 3: 161-172).

TESTING

[00344] The compounds of general Formula I and/or the conjugate compositions may be tested for activity using standard techniques. Non-limiting examples of suitable testing methods are provided in the Examples section.

[00345] For example, the cytotoxicity of the compounds or conjugates can be assayed in vitro using a suitable cell line, typically a cancer cell line. In general, cells of the selected test cell line are grown to an appropriate density and the candidate compound is added. After an appropriate incubation time (for example, about 48 to 72 hours), cell survival is assessed. Methods of determining cell survival are well known in the art and include, but are not limited to, the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11 :48-50; O'Brien et al, (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No. 5,501,959), the sulforhodamine assay (Rubinstein et al, (1990) J. Natl. Cancer Inst. 82: 113-118) or the neutral red dye test (Kitano et al, (1991) Euro. J. Clin. Investg. 21 :53-58; West et al, (1992) J. Investigative Derm. 99:95-100). Other methods include those described in Current Protocols in Toxicology (Costa et al. (Ed.), John Wiley & Sons, New York, NY). Cytotoxicity is determined by comparison of cell survival in the treated culture with cell survival in one or more control cultures, for example, untreated cultures and/or cultures pre-treated with a control compound (typically a known therapeutic). A variety of cancer cell-lines suitable for testing candidate compounds are known in the art and many are commercially available (for example, from the American Type Culture Collection, Manassas, VA).

[00346] The ability of the compounds or conjugates to inhibit tumour growth or proliferation in vivo can be determined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, Inc., New York, NY). In general, current animal models for screening anti -tumour compounds are xenograft models, in which a human tumour has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumour xenografts, implanted by sub-cutaneous injection or implantation and used in tumour growth assays; human solid tumour isografts, implanted by fat pad injection and used in tumour growth assays; human solid tumour orthotopic xenografts, implanted directly into the relevant tissue and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.

[00347] For example, the compounds or conjugates can be tested in vivo on solid tumors using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumor fragment, or implanted with an appropriate number of cancer cells, on day 0. The animals bearing tumors are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumors, tumors are allowed to develop to the desired size, animals having insufficiently developed tumors being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Animals not bearing tumors may also be subjected to the same treatments as the tumor- bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumor. Chemotherapy generally begins from 3 to 22 days after grafting, depending on the type of tumor, and the animals are observed every day. Test articles can be administered to the animals, for example, by i.p. injection or bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial. Tumors can be measured after a pre-determined time period, or monitored continuously by measuring about 2 or 3 times a week until the tumor reaches a predetermined size and/or weight, or until the animal dies if this occurs before the tumor reaches the pre-determined size/weight. The animals are then sacrificed and the tissue histology, size and/or proliferation of the tumor assessed. [00348] For the study of the effect of the compounds or conjugates on leukaemias, the animals are grafted with a particular number of cells, and the anti-tumor activity is determined by the increase in the survival time of the treated mice relative to the controls. To study the effect of the compounds or conjugates on tumor metastasis, tumor cells are typically treated with the compound/conjugate ex vivo and then injected into a suitable test animal. The spread of the tumor cells from the site of injection is then monitored over a suitable period of time. [00349] In vivo toxic effects of the compounds/conjugates can be evaluated by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed.

PHARMACEUTICAL COMPOSITIONS [00350] For the purposes of administration, the compounds or conjugates of the present disclosure are typically formulated as pharmaceutical compositions. Certain embodiments of the present disclosure thus relate to pharmaceutical compositions comprising a compound or conjugate described herein and a pharmaceutically acceptable carrier, diluent or excipient. The compound or conjugate is typically present in the composition in an amount which is effective to treat the particular disease or condition of interest with acceptable toxicity to the patient. The activity of compounds described herein can be determined by one skilled in the art using standard techniques, for example, as described in the Examples below.

[00351] The compounds or conjugates described herein in an appropriate pharmaceutical composition may be administered via various accepted modes of administration for the disease to be ttreated. The pharmaceutical compositions may, for example, be formulated in solid, semi solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical compositions are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container comprising the pharmaceutical composition in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy (22nd ed.) eds. Loyd V. Allen, Jr., et al, Pharmaceutical Press, 2012. The composition to be administered will, in any event, contain a therapeutically effective amount of a compound or conjugate described herein, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest. [00352] A pharmaceutical composition described herein may be in the form of a solid or liquid. Accordingly, the carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form, or the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. [00353] When intended for oral administration, the pharmaceutical compositions typically are either solid or liquid form, including semi solid, semi liquid, suspension and gel forms.

[00354] As a solid composition for oral administration, the pharmaceutical compositions may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

[00355] When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[00356] The pharmaceutical compositions may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be, for example, for oral administration or for delivery by injection. When intended for oral administration, the pharmaceutical compositions typically contain, in addition to the active compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[00357] Liquid pharmaceutical compositions, whether solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution (such as physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[00358] Pharmaceutical compositions may be formulated for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[00359] Pharmaceutical compositions may be formulated for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[00360] Pharmaceutical compositions may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.

[00361] Pharmaceutical compositions may be prepared in dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds described herein may be delivered in single phase, bi phasic, or tri phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. [00362] The pharmaceutical compositions described herein may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a compound described herein with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non covalently interact with the compound described herein so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

[00363] The compounds and conjugates described herein are administered in a therapeutically effective amount, which one skilled in the art will appreciate will vary depending upon a variety of factors understood such as the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; any drug combination and/or the severity of the particular disorder or condition. Appropriate dosages and concentrations of the compounds or conjugates can be readily determined by one skilled into the art taking into account factors such as those outlined above.

[00364] In certain embodiments, compounds and conjugates as described herein may be administered as part of a combination therapy. Accordingly, a pharmaceutical composition comprising a compound or conjugate may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation which contains the compound or conjugate and one or more additional active agents, as well as administration of the compound or conjugate and each active agent in its own separate pharmaceutical dosage formulation. Where separate dosage formulations are used, the formulation comprising the compound or conjugate and the formulation comprising the additional active agent(s) may be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially.

METHODS OF USE

[00365] Certain embodiments of the present disclosure relate to the use of the compounds and conjugates described herein as cytotoxic agents. Accordingly, in certain embodiments, the compounds and conjugates may be used to inhibit proliferation of a target cell population, such as a cancer cell population, either in vitro or in vivo. [00366] Certain embodiments of the present disclosure relate to the use of the compounds and conjugates described herein in therapy. Some embodiments relate to methods of using a compound or conjugate described herein in the treatment of a disease or disorder.

[00367] Given the characteristics of the compounds and conjugates described herein, one skilled in the art will appreciate that the compounds and conjugates may be indicated for use to treat various diseases where exertion of a cytotoxic or cytostatic effect on a target cell is desirable. Non- limiting examples of diseases that may be treated in some embodiments include benign and malignant tumors; leukemia and lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; autoimmune disease; inflammatory disease; fibrosis, and infectious disease.

[00368] Certain embodiments of the present disclosure relate to methods of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate described herein.

[00369] A therapeutically effective amount of compound or conjugate in respect of cancer treatment may exert one or more of the following effects: reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and potentially stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and potentially stop) tumor metastasis; inhibit, to some extent, tumor growth; increase survival time; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the compound may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

[00370] Certain embodiments relate to the use of a compound or conjugate as described herein in a method of inhibiting tumor growth in a subject. Some embodiments relate to the use of a compound or conjugate as described herein in a method of killing cancer cells in vitro. Some embodiments relate to the use of a compound or conjugate as described herein in a method of killing cancer cells in vivo in a subject having a cancer. Some embodiments relate to the use of a compound or conjugate as described herein in a method of increasing the survival time of a subject having cancer, comprising administering to the subject a therapeutically effective amount of the compound or conjugate. [00371] Examples of cancers which may be may be treated or stabilized in certain embodiments include hematologic neoplasms, including leukemias, myelomas and lymphomas; carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as "solid tumors," examples of commonly occurring solid tumors include, but are not limited to, cancer of the bladder, brain, breast, cervix, colon, endometrium, head and neck, kidney, lung, ovary, pancreas, prostate, salivary gland, stomach or uterus, non-small cell lung cancer and colorectal cancer. Various forms of lymphoma also may result in the formation of a solid tumor and, therefore, are also often considered to be solid tumors.

[00372] Certain embodiments relate to the use of a compound or conjugate described herein in the treatment of a solid tumor. Solid tumors include, but are not limited to, sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer (for example, gastrointestinal cancer), oral cancer, nasal cancer, throat cancer, squamous cell carcinoma (for example, of the lung), basal cell carcinoma, adenocarcinoma (e.g. , of the lung), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, non-small cell lung cancer, epithelial carcinoma, glioma, glioblastoma, multiforme astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma. [00373] Certain embodiments relate to the use of a compound or conjugate described herein in the treatment of a hematological (or blood-borne) cancer. Blood-borne cancers include, but are not limited to, acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia "AML", acute promyelocyte leukemia "APL", acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, and multiple myeloma.

[00374] Certain embodiments relate to the use of a compound or conjugate described herein in the treatment of a lymphoma. Lymphomas include, but are not limited to, Hodgkin disease, non- Hodgkin lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera.

[00375] Certain embodiments relate to the use of a compound or conjugate as described herein in methods for treating or preventing cancer in combination with an additional method of treatment. The additional method of treatment may be, for example, treatment with chemotherapeutic or other anti -cancer agent(s), radiation or surgery.

[00376] Examples of chemotherapeutic agents commonly used alone or in combination in the treatment of cancers include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, treosulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1 -TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; triazines such as decarbazine; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; epipodophyllins, such as etoposide, teniposide, topotecan, 9-aminocamptothecin, camptothecin orcrisnatol; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g. , Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins (for example, A2 and B2), cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino- doxorubicin, cyanomoφholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; photodynamic therapies, such as vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy- hypocrellin A (2BA-2-DMHA); folic acid analogues such as denopterin, pteropterin, and trimetrexate; dpurine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as ALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate and trimetrexate; antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, floxuridine, doxifluridine and ratitrexed; and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX®, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g. , TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); vinca alkaloid; vinorelbine (NAVELBINE®); velcade; revlimid; thalidomide; IMiD3; lovastatin; verapamil; thapsigargin; 1 -methyl -4-phenylpyridinium; cell cycle inhibitors such as staurosporine; novantrone; edatrexate; daunomycin; mtoxantrone; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); vitamin D3 analogs, such as EB 1089, CB 1093 and KH 1060; retinoids such as retinoic acid; L-asparaginase; nitrosoureas; procarbizine; BCNU and plicamycin.

[00377] Other examples of anti-cancer agents include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti -estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, megastrol, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON® toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RTVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti- androgens such as flutamide, bicalutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC -alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor, and ABARELFX® rmRH.

[00378] Certain embodiments relate to the use of a compound or conjugate described herein in a method of treating an immunological disease. Immunological diseases are characterized by inappropriate activation of immune cells and can be classified, for example, by the type(s) of hypersensitivity reaction(s) that underlie the disorder. These reactions are typically classified into four types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immunity (CMI) reactions (also referred to as delayed-type hypersensitivity (DTH) reactions). (See, for example, Fundamental Immunology (William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993)).

[00379] Specific examples of such immunological diseases include the following: rheumatoid arthritis, autoimmune demyelinative diseases (for example, multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g. , Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier' s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti -phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch- Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton- Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure. [00380] In certain embodiments, the immunological disease may be a disorder of B lymphocytes (for example, systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Thl -lymphocytes (for example, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or acute graft versus host disease), or Th2 -lymphocytes (for example, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease).

[00381] In certain embodiments, the immunological disease may be T cell-mediated, which may include activated T cells.

[00382] Certain embodiments relate to the use of a compound or conjugate described herein in a method of treating fibrosis. Fibrosis can occur in many tissues within the body, typically as a result of inflammation or damage, examples include but are not limited to; lungs (for example, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis), liver (for example, cirrhosis), and heart (for example, endomyocardial fibrosis, old myocardial infarction, atrial fibrosis). Other examples of fibrosis disorders include mediastinal fibrosis (soft tissue of the mediastinum), myelofibrosis (bone marrow), retroperitoneal fibrosis (soft tissue of the retroperitoneum), progressive massive fibrosis (lungs), nephrogenic systemic fibrosis (skin), Crohn's disease (intestine), keloid (skin), scleroderma/systemic sclerosis (skin, lungs), arthrofibrosis (knee, shoulder, other joints), Peyronie's disease (penis), Dupuytren's contracture (hands, fingers) and some forms of adhesive capsulitis (shoulder). [00383] Certain embodiments relate to the use of a compound or conjugate described herein in a method of treating an infectious disease. In this context, the compound or conjugate may be used directly on certain infectious agents or pathogens, or may be used to exert a cytostatic or cytotoxic effect on a host cell that harbors or otherwise provides for the infectious agent or pathogen. [00384] The following Examples illustrate various methods of making compounds described herein, i.e., compounds of Formula I and related formulae. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of Formula I not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described herein.

[00385] The following examples are provided for purposes of illustration, not limitation.

EXAMPLES

EXAMPLE 1: SYNTHESIS OF COMPOUNDS OF GENERAL FORMULA I [00386] An example of a synthetic protocol that may be used to prepare certain compounds of general Formula I is provided as Scheme 1 in FIG. 1 and described in the following Example.

General Procedure 2 - DCC/DMAP Mediated N- Acyl Sulfonamide Formation.

[00387] To a stirred solution of the acid in dichloromethane was added a solution of the sulfonamide (1.3 equivalents, in dichloromethane, N,N-dimethylformamide, or a mixture thereof, as necessary). Dicyclohexylcarbodiimide (1.2 equivalents) was added and subsequently N,N- dimethylaminopyridine (1.2 equivalents). Reaction course was monitored by HPLC-MS (typically 16 h) and excess by-products could be precipitated by the addition of diethyl ether. Solids were removed by filtration and washed with 1 : 1 diethyl ether/dichloromethane. The combined organic layers were concentrated, and the residue was purified by silica gel chromatography or optionally prep-HPLC to give the desired N-acyl sulfonamide. General Procedure 6 - HATU Mediated Peptide Bond Formation.

[00388] To a stirred solution of the carboxylic acid in a minimal amount of dichloromethane or NyV-dimethylformamide or mixture thereof, at 0°C was added HATU ( 1.05-1.2 equivalents) and either N,N-diisopropylamine (2-4 equivalents) or 2,4,6-collidine (2-4 equivalents). Stirring was continued for a brief induction period (5-20 minutes) at which time the reaction was charged with a solution of the amine in dichloromethane. The reaction was allowed to warm to room temperature and monitored for progress by HPLC-MS. Upon completion, volatiles were removed under reduced pressure and the residual material was purified by silica gel chromatography or reverse phase HPLC to furnish amide in adequate purity. General Procedure 9 - Boc Group Removal

[00389] To a solution of the Boc-protected construct in dichloromethane was added 10% v/v trifluoroacetic acid. Reaction course was monitored by HPLC-MS. Upon completion, all volatiles were removed under reduced pressure. The residual material was purified either by reverse phase HPLC, silica gel chromatography or precipitation from a mixture of cold methanol/dichloromethane/diethyl ether.

General Procedure 10 - Ester Saponification.

[00390] To a solution of the ester containing compound in 1,4-dioxane or methanol was added lithium hydroxide ( 10 equivalents) and water (10% v/v). The reaction was allowed to stir at room temperature or optionally heated to 50 °C. Reaction course was monitored by HPLC-MS. Upon completion, volatiles were removed under reduced pressure, the aqueous layer was pH adjusted if necessary and washed successively with dichloromethane or ethyl acetate. The organic phases were pooled, dried over MgS04, filtered and concentrated. The reaction product was either used "as is" or purified by silica gel chromatography as necessary.

Example 1.1 : Methyl 2-formylthiazole-4-carboxylate (Compound 1).

[00391] Compound 1 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. ¾ NMR (400 MHz, DMSO) δ 9.99 (s, 1H), 8.99 (s, 1H), 3.90 (s, 3H).

Example 1.2: (S^)-2-Methyl-_JV-(3-methylbutan-2-ylidene)propane-2-sulfinamide (Compound 2).

[00392] Compound 2 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. ¾ NMR (400 MHz, DMSO-t/₆) δ 2.58 (sept, J = 6.9 Hz, 1H), 2.27 (s, 3H), 1.14 (s, 9H), 1.07 (d, J= 6.8 Hz, 2H). Example 1.3: Methyl 2-((i?,£)-3-((S)-teri-Butylsulfinylimino)-l-hydroxy-4- methylpentyl)thiazole-4-carboxylate (Compound 3).

[00393] Compound 3 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. Ή NMR (400 MHz, Chloroform- ) δ 8.13 (s, 1H), 6.62 (d, J= 8.4 Hz, 1H), 5.30 (s, 2H), 5.13 (m, 1H), 3.94 (s, 3H), 3.44 - 3.21 (m, 2H), 2.86 (p, J= 6.7 Hz, 1H), 1.30 (s, 9H), 1.21 (dd, J= 6.8, 5.6 Hz, 6H).

Example 1.4: Methyl 2-((LR,3i?)-3-((S)-l,l-Dimethylethylsulfinamido)-l-hydroxy-4- methylpentyl)thiazole-4-carboxylate (Compound 4).

[00394] Compound 4 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. To a solution of compound 3 (1.60 g, 4.44 mmol) in 12 mL THF cooled to -75 °C under N₂, was added Ti(OEt)₄ (1.86 mL, 2.02 g, 8.88 mmol) then solid NaBFL (672 mg, 17.76 mmol). The reaction was allowed to warm to -45 °C, then maintained at that temperature for one hour. To quench, aliquots of MeOH were added, keeping the temperature below -30 °C, until gas evolution ceased. The mixture was then allowed to warm to rt, 30 mL of dlrbO was added and the precipitates were filtered over Celite® while washing with 300 mL EtOAc. The filtrate was then washed three times with brine, dried over Na2SC>4, filtered, and concentrated to a yellow oil. Flash chromatography (20-100% EtO Ac/Hex) yielded the title compound (1.24 g, 77%) as a clear, viscous oil. Ή NMR (400 MHz, Chloroform- ) δ 8.15 (s, lH), 5.54 (d, J = 6.6 Hz, 1H), 5.20 (m, 1H), 3.96 (s, 3H), 3.46 (m, J = 12.0, 8.1, 4.0 Hz, 1H), 3.35 (d, J = 8.5 Hz, 1H), 2.30 (ddd, J = 14.7, 11.7, 3.0 Hz, 1H), 2.07 (s, 1H), 1.93 (ddd, J = 14.8, 11.5, 3.5 Hz, 1H), 1.69 (s, 3H), 1.31 (s, 9H), 0.94 (dd, J = 14.1, 6.7 Hz, 6H). m/z calcd. for G5H26N2O4S2 = 362.13 Found [M+H]⁺ = 363.6. Example 1.5: Methyl 2-((4i?,6i?)-3-((S)-teri-Butylsulfinyl)-4-isopropyl-l,3-oxazinan-6- yl)thiazole-4-carboxylate (Compound 5).

[00395] Compound 5 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. To a solution of compound 4 (2.88 g, 7.94 mmol) was added solid paraformaldehyde (4.8 g, 159 mmol) and the reaction was heated to 70 °C in a sealed vessel for three days. When no starting material remained, the reaction was cooled, filtered through Celite and concentrated to a yellow oil. This was purified by flash chromatography (17-70% EtOAc/CH₂Cl₂) to yield the title compound (2.25 g, 76%) as a sticky foam. ¾ NMR (400 MHz, Chloroform-t δ 8.19 (s, 1H), 5.32 (s, 1H), 5.29 - 5.17 (m, 2H), 4.78 (d, J = 11.6 Hz, 1H), 3.97 (s, 3H), 3.09 (dd, J = 10.2, 5.4 Hz, 1H), 2.49 - 2.32 (m, 2H), 2.19 (m, 1H), 1.24 (s, 9H), 1.08 (t, J= 6.7 Hz, 6H). Example 1.6: Methyl 2-((li?,3i?)-l-Hydroxy-4-methyl-3-(methylamino)pentyl)thiazole-4- carboxylate (Compound 6).

[00396] Compound 6 was synthesized according to the procedures described for the ethyl ester analog in U.S. Patent No. 8,476,451 in comparable yield. To a stirred solution of compound 5 (2.25 g, 6.01 mmol) in 30 mL of 9: 1 MeCN/EtOH was added solid NaBH₃CN (377 mg, 6.01 mmol). Next 4 M HCl in dioxane (6.01 mL, 24.0 mmol) was added dropwise over 30 min to evolve a small amount of gas and form a turbid solution. After 90 min, no starting material was detectable by HPLC. The solvent was removed in vacuo, and the residue was purified by flash chromatography (1-20% (5% NH40H/MeOH)/CH₂Ci2) to give the title compound (1.66 g, quant.) as a clear oil. Ή NMR (400 MHz, Methanol-d4) δ 8.40 (s, 1H), 5.19 (dd, J = 6.5, 5.1 Hz, 1H), 3.94 (s, 3H), 3.01 - 2.93 (m, 1H), 2.68 (s, 3H), 2.22 - 2.07 (m, 3H), 1.00 (d, J = 6.9 Hz, 6H). m/z calcd. for C₁₂H₂₀N₂O₃S = 272.12 Found [M+H]⁺ = 273.6.

Example 1.7: Methyl 2-((LR,3i?)-l-(teri-Butyldimethylsilyloxy)-4-methyl-3- (methylamino)pentyl)thiazole-4-carboxylate (Compound 7).

[00397] To a solution of compound 6 (1.07 g, 3.93 mmol) in dichloromethane (20 mL) was added i-butyldimethylsilyl chloride (1.18 g, 7.86 mmol) and imidazole (535 mg, 7.86 mmol). The reaction was complete after one hour by TLC (10% (5% OH/MeOHyCT^Ck), and white precipitate had formed. The precipitate was filtered off, washed with dichloromethane, concentrated, and the residue purified by flash chromatography (2-20% (5% NH40H/MeOH)/CH₂Cl₂) to give the title compound (1.24 g, 82%) as a clear, viscous oil. ¾ NMR (400 MHz, Chloroform- ) δ 8.14 (s, lH), 5.36 (dd, J= 7.3, 4.3 Hz, 1H), 3.96 (s, 3H), 2.65 - 2.56 (m, 1H), 2.47 (s, 3H), 2.09 - 1.89 (m, 2H), 1.82 (ddd, J = 14.1, 9.0, 4.2 Hz, 1H), 0.96 (s, 9H), 0.89 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H), 0.17 (s, 3H), 0.01 (s, 3H). m/z calcd. for C18H34N2O3SS- = 386.21 Found [M+H]⁺ = 387.7. Example 1.8: Methyl 2-((5i?,7i?,10S)-10-sec-Butyl-7-isopropyl-2,2,3,3,8,14,14-heptamethyl- 9,12-dioxo-4,13-dioxa-8,ll-diaza-3-silapentadecan-5-yl)thiazole-4-carboxylate (Compound 8).

[00398] To a cooled (0 °C) solution of Boc-isoleucine-OH (1.06 g, 4.60 mmol) in dichloromethane (5 mL) were added HATU (1.49 g, 3.91 mmol) and 2,4,6-collidine (1.21 mL, 9.20 mmol). The suspension was stirred for 15 minutes, at which time compound 7 was added as a solution in dichloromethane and the reaction was allowed to warm to room temperature with stirring continued overnight at which time HPLC-MS indicated complete consumption of compound 7. The reaction mixture was concentrated to dryness, then taken up in a combination of saturated NaHCC and ethyl acetate (50 mL), and extracted. The aqueous was extracted twice more with 50 mL EtOAc, then the combined organics were washed with saturated ammonium chloride, brine, dried over sodium sulfate, filtered and concentrated to afford a clear, yellow oil. The product was purified by flash chromatography (5-50% EtO Ac/Hex) to afford the title compound (390 mg, 28%) as a clear oil. Ή NMR (400 MHz, Chloroform-d) δ 8.12 (s, 1H), 5.22 (d, J = 9.7 Hz, 1H), 4.90 (dd, J= 7.3, 3.1 Hz, 1H), 4.48 (dd, J= 9.7, 6.6 Hz, 1H), 3.95 (s, 3H), 3.04 (s, 3H), 2.14 - 2.02 (m, 1H), 1.88 - 1.75 (m, 1H), 1.77 - 1.69 (m, 1H), 1.63 - 1.58 (m, 2H), 1.43 (s, 9H), 1.20 - 1.07 (m, 1H), 0.99 (d, J = 6.1 Hz, 6H), 0.96 (s, 9H), 0.86 (t, J = 7.4 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.21 (s, 3H), -0.04 (s, 3H).

Example 1.9: 2-((5i?,7i?,10S)-10-sec-Butyl-7-isopropyl-2,2,3,3,8,14,14-heptamethyl-9,12- dioxo-4,13-dioxa-8,ll-diaza-3-silapentadecan-5-yl)thiazole-4-carboxylic Acid (Compound 9).

[00399] To a solution of compound 8 (390 mg, 0.65 mmol) in 1,4-dioxane (12 mL) was added 0.5 M lithium hydroxide monohydrate (6.5 mL, 3.25 mmol). The resulting suspension stirred and monitored by HPLC-MS and TLC (50% (2% AcOH/EtOAc)/Hex). After 90 min, the reaction was complete, and it was concentrated under reduced pressure to a small volume to remove most of the dioxane. To the residual aqueous suspension, saturated ammonium chloride (5 mL) was added to form a milky precipitate. This was extracted three times with EtOAc (30 mL), washed once with brine, dried over sodium sulfate, filtered and concentrated to afford the title compound (336 mg, 88%) as a sticky white foam. ¾ NMR (400 MHz, Methanol-^) δ 7.98 (s, 1H), 4.60 - 4.48 (m, 1H), 4.43 (t, J = 8.9 Hz, 1H), 3.11 (s, 3H), 2.23 - 2.1 1 (m, 1H), 2.09 - 2.05 (m, 1H), 1.92 - 1.72 (m, 2H), 1.66 (dqd, J = 15.1, 7.4, 3.0 Hz, 1H), 1.44 (s, 9H), 1.24 (s, lH), 1.24 - 1.12 (m, 1H), 1.02 (d, J = 6.7 Hz, 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.97 (s, 9H), 0.93 (t, J= 7.4 Hz, 3H), 0.83 (d, J = 6.7 Hz, 3H), 0.20 (s, 3H), -0.08 (s, 3H). Example 1.10: tert-Butyi (2S,3S)-l-(((li?,3i?)-l-(teri-Butyldimethylsilyloxy)-4-methyl-l-(4-(4- (2,2,2-trifluoroacetamido)phenylsulfonylcarbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)- 3-methyl-l-oxopentan-2-ylcarbamate (Compound 10).

[00400] The title compound was prepared from compound 9 by following general procedure 2. ¾ NMR (400 MHz, Methanol-^) δ 8.21 (s, 1H), 8.07 (d, J = 7.1 Hz, 2H), 7.98 - 7.90 (m, 1H), 7.85 (d, J = 7.1 Hz, 2H), 4.50 (t, J = 4.7 Hz, OH), 4.36 (d, J = 8.0 Hz, 1H), 3.48 (ddt, J = 10.5, 6.6, 3.0 Hz, 1H), 3.07 (s, 3H), 2.16 - 1.99 (m, 3H), 1.95 - 1.82 (m, 3H), 1.75 (ddt, J = 13.4, 8.2, 5.1 Hz, 4H), 1.61 (tt, J = 11.1, 3.4 Hz, 2H), 1.52 - 1.28 (m, 13H), 1.22 - 1.08 (m, 3H), 1.03 (d, J = 6.6 Hz, 4H), 1.00 - 0.76 (m, 21H), 0.20 (s, 3H), -0.07 (s, 3H). Example 1.11: 2-((lR,3R)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)-_JV-(4-(2,2,2- trifluoroacetamido)phenylsulfonyl)thiazole-4-carboxamide (Compound 12).

[00401] The title compound was prepared from compound 10 by following General Procedures 9 and 6. ¾ NMR (400 MHz, Methanol-^) δ 8.07 - 8.00 (m, 3H), 7.76 (d, J = 8.7 Hz, 2H), 4.74 (d, J = 8.8 Hz, 1H), 4.65 (d, J = 10.6 Hz, 1H), 3.17 (s, 3H), 2.92 (d, J = 1 1.3 Hz, 1H), 2.58 (d, J = 10.9 Hz, 1H), 2.29 - 2.24 (m, 1H), 2.17 (s, 3H), 2.13 - 2.00 (m, 1H), 1.97 - 1.86 (m, 2H), 1.77 (d, J = 12.2 Hz, 2H), 1.66 - 1.51 (m, 5H), 1.31 (s, 3H), 1.29 - 1.16 (m, 1H), 1.00 (d, J = 7.2 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H). m/z calcd. for C32H45F3N6O7S2 = 746.27 Found [M+H]⁺ = 747.9.

Example 1.12: (S,£)-Ethyl 4-(teri-Butoxycarbonyl(methyl)amino)-2,5-dimethylhex-2-enoate, Boc-ICD-OEt (Compound 13).

[00402] The title compound was synthesized from {S,E)-ehy\ -{tert- butoxycarbonyl(methyl)amino)-2,5-dimethylhex-2-enoate (synthesized according to US 7,579,323 B l) using General Procedure 9 and subsequently with Boc-Isoleucine-OH and using General Procedure 6. NMR provided for a sample treated with TFA to remove the Boc group and resolve rotamers in the spectrum. Ή NMR (400 MHz, Chloroform- ) δ 6.68 (dd, J = 9.5, 1.8 Hz, 1H), 5.33 (s, OH), 4.97 (t, J = 9.9 Hz, 1H), 4.36 (d, J = 4.1 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.56 (s, 1H), 2.96 (s, 3H), 2.07 - 1.83 (m, 5H), 1.53 (s, 1H), 1.34 (t, J = 7.1 Hz, 3H), 1.12 (d, J = 7.0 Hz, 3H), 1.00 - 0.83 (m, 9H). Example 1.13: (S,£)-4-((2S,3S)-2-((i?)-l-Isopropylpiperidine-2-carboxamido)-_JV,3- dimethylpentanamido)-2,5-dimethylhex-2-enoic Acid (Compound 16).

[00403] The title compound was synthesized from Boc-ICD-OEt by following General Procedure 9 and reacting the liberated amine with D-(N-isopropyl)-pipecolic acid using General Procedure 6. Finally, the C-terminal carboxylate was liberated using General Procedure 10 prior to purification by preparative scale HPLC. Ή NMR (400 MHz, Methanol-^) δ 6.78 (dd, J = 9.5, 1.6 Hz, 1H), 5.04 (t, J = 10.1 Hz, 1H), 4.57 (dd, J = 8.6, 4.8 Hz, 1H), 4.01 - 3.92 (m, 1H), 3.56 - 3.41 (m, 2H), 3.12 (s, 3H), 3.04 - 2.93 (m, 1H), 2.13 (d, J = 13.2 Hz, 1H), 2.09 - 1.99 (m, 2H), 1.96 (d, J = 12.5 Hz, 2H), 1.90 (d, J = 1.5 Hz, 3H), 1.89 - 1.79 (m, 2H), 1.68 - 1.56 (m, 2H), 1.34 (d, J = 6.7 Hz, 3H), 1.29 (d, J = 6.8 Hz, 3H), 1.27 - 1.18 (m, 1H), 0.97 - 0.91 (m, 9H), 0.88 (d, J = 6.6 Hz, 3H). C24H43N3O4 calcd. m/z = 437.33 found [M+H]⁺ = 438.95

Example 1.14: (S,£)-4-((2S,3S)-7V,3-Dimethyl-2-((S)-3-methyl-2-(methylamino)-3- phenylbutanamido)pentanamido)-2,5-dimethylhex-2-enoic Acid (Compound 17).

[00404] The title compound was synthesized from Boc-ICD-OEt by following General Procedure 9 and reacting the liberated amine with (5)-2-(feri-butoxycarbonyl(methyl)amino)-3-methyl-3- phenylbutanoic acid using General Procedure 6. Finally, the C-terminal carboxylate was liberated using General Procedure 10 and the Boc group removed by General Procedure 9 prior to purification by preparative scale HPLC. ¾ NMR (400 MHz, Methanol-^) δ 7.53 (d, J = 7.4 Hz, 2H), 7.46 (t, J= 7.6 Hz, 2H), 7.35 (t, J= 7.2 Hz, 1H), 6.78 (d, J= 9.7 Hz, 1H), 5.03 (t, J= 10.0 Hz, 1H), 4.80 (d, J = 8.7 Hz, 1H), 4.09 (s, 1H), 3.79 - 3.64 (m, 1H), 3.16 (s, 3H), 2.47 (s, 3H), 2.11 -

2.00 (m, 1H), 1.93 (s, 3H), 1.74 - 1.61 (m, 1H), 1.48 (s, 3H), 1.40 (s, 3H), 1.34 - 1.23 (m, 1H),

1.01 - 0.88 (m, 12H). C27H43N3O4 calcd. m/z = 473.33 found [M+H]⁺ = 474.98

Example 1.15: (S^)-4-((2S,3S)-2-Amino-7V,3-dimethylpentanamido)-2,5-dimethyl-7V-(4-(2,2,2- trifluoroacetamido)phenylsulfonyl)hex-2-enamide (Compound 21).

I l l

[00405] The title compound was prepared from Boc-ICD-OEt according to General Procedure 10, followed by N-acyl sulfonamide generation with 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide according to General Procedure 2, followed by General Procedure 9. ^l NMR (400 MHz, Chloroform-t δ 8.00 - 7.85 (m, 2H), 7.76 (d, J = 8.8 Hz, 2H), 6.39 (dd, J = 9.2, 1.8 Hz, 1H), 4.45 - 4.30 (m, 1H), 4.14 (d, J= 4.1 Hz, 1H), 2.82 (s, 3H), 2.08 - 1.91 (m, 1H), 1.67 (s, J= 1.5 Hz, 3H), 1.41-1.35 (m, J= 13.3, 7.6, 3.2 Hz, 1H), 1.10 - 0.88 (m, 4H), 0.77 (ddd, J= 17.2, 9.0, 5.4 Hz, 9H).

Example 1.16: (i?)-N-((2S,3S)-l-(((S^)-2,5-Dimethyl-6-oxo-6-(4-(2,2,2- trifluoroacetamido)phenylsulfonamido)hex-4-en-3-yl)(methyl)amino)-3-methyl-l-oxopentan- 2-yl)-l-isopropylpiperidine-2-carboxamide (Compound 24).

[00406] The title compound was prepared from Compound 21 and N-iso-propyl-D-pipecolic acid according to General Procedure 6. ¾ NMR (400 MHz, Methanol-d4) 5 8.13 - 8.04 (m, 2H), 7.97 - 7.88 (m, 2H), 6.46 (dd, J= 9.1, 1.6 Hz, 1H), 4.94 (t, J= 9.9 Hz, 1H), 4.60 - 4.52 (m, 1H), 3.95 (dd, J = 11.8, 2.9 Hz, 2H), 3.55 - 3.40 (m, 2H), 3.12 (s, 3H), 3.04 - 2.90 (m, 1H), 2.12 (d, J = 12.2 Hz, 1H), 2.08 - 1.98 (m, 2H), 1.95 (d, J= 13.1 Hz, 3H), 1.89 - 1.78 (m, 4H), 1.67 - 1.55 (m, 2H), 1.33 (d, J = 6.7 Hz, 3H), 1.27 (d, J = 6.7 Hz, 3H), 0.95 - 0.84 (m, 12H). C32H4₈F₃N506S calcd. m/z = 687.33 found [M+H]⁺ = 688.99.

EXAMPLE 2: SYNTHESIS OF COMPOUNDS OF GENERAL FORMULA I AND CONJUGATES THEREOF

General Method I - Lithium hydroxide ester hydrolysis

[00407] An aqueous solution of LiOH H₂0 (1.0 M, 4.0 eq) was added to stirring mixture of an appropriate ester (1.0 eq) in 1,4-dioxane (0.1 M). The resulting mixture was stirred for 4 h and then concentrated in vacuo. The residue was dissolved in ¾0 and treated with 1.0 M citric acid. The resulting milky suspension was extracted with Et₂0 (4x) and the combined organics was washed with brine (lx), dried

filtered and concentrated in vacuo to afford the desired acid as a white solid.

General Method II - EDC mediated N-&zy\ sulfonamide formation on tubulophenylalanine

[00408] To a solution of ALM00553-016 (1.0 eq) in CH₂C1₂ (0.1 M) was added an appropriate sulfonamide (1.05 eq) and DMAP (1.05 eq) followed by the addition of DMF dropwise until a clear solution was obtained. EDC HCl (1.05 eq) was then added, and the mixture was stirred at ambient temperature. After 16-24 h, the mixture was concentrated in vacuo, and the residue was partitioned between 1.0 M HC1 and EtOAc. After extracting the aqueous layer with EtOAc (2x), the organics were combined and washed with brine (lx), dried (Na2<SO4), filtered, concentrated in vacuo and purified by silica gel chromatography with 10-100% (2% AcOH/EtOAc) in hexanes to afford the desired N-acyl sulfonamide as a white solid.

General Method III - TFA deprotection o

F,C OH

Η,Ν λ

CH2CI2 [00409] TFA (100 eq) was added to a stirring mixture of an appropriate Boc-protected amine (1.0 eq) in CH2CI2. After stirring for 30 minutes, the solution was concentrated in vacuo and co- evaporated with CH2CI2 (2x) and PhMe (2x) to afford the desired amine as a trifluoroacetic acid salt. General Method IV - HATU coupling of tubulophenylalanine sulfonamide

[00410] To a solution of intermediate 3 (1.0 eq) in DMF (0.015 M) was added HATU (1.3 eq) and DIPEA (4.0 eq), and the resulting solution was stirred at ambient temperature. After 0.5 h, a solution of an appropriate amine (1.1 eq) in DMF (0.015 M) was added and stirred at ambient temperature for an additional 0.5 h. The reaction mixture was concentrated in vacuo and purified by silica gel column chromatography with 1-10% MeOH/CFbC to afford the desired amide as a white solid. General Method V - d-methylpipecolic acid HATU coupling

[00411] DIPEA (4.0 eq) was added to a stirring mixture of (i?)-l-methylpiperidine-2-carboxylic acid (1.5 eq) and HATU (2.0 eq) in DMF. The yellow solution was stirred at ambient temperature for 5-10 minutes. The resulting activated acid was added to a stirring mixture of an appropriate amine (1.0 eq) in DMF. After stirring for 30 minutes at ambient temperature, the mixture was purified by silica gel column chromatography with 100% EtOAc, followed by 2-20% MeOH/GrbC to afford the desired amide as a white solid.

General Method VI - Lithium hydroxide saponification

oxane ₂

[00412] An aqueous solution of LiOH H₂0 (1.0 M, 8.0 eq) was added to stirring mixture of an appropriate trifluoroacetamide (1.0 eq) in 1,4-dioxane (0.02 M). The resulting mixture was stirred for 18 h and then concentrated in vacuo. The residue was dissolved in ¾0, treated with a 0.25 M phosphate buffer (pH 6.2) and then extracted with CH2CI2 (4x). The combined organics was washed with brine (lx), dried

filtered and concentrated in vacuo. The residue was dissolved in minimal MeOH/GrbCh mixtures and precipitated with cold hexanes to afford the desired amine as a white solid. General Method VII - Copper EDC coupling with MT-VC-OH

[00413] To a stirring solution of MT-VC-OH (3.0 eq) in CH₂C1₂/DMF (8: 1, 0.05 M), HOAt (3.0 eq) and EDC HCl (2.9 eq) were added. After 15 min, the appropriate amine (1.0 eq) was added followed by the immediate addition of Q1CI2 (3.0 eq). After 1 h, the resulting suspension was diluted with MeOH, filtered through celite and concentrated in vacuo. The residue was purified by flash chromatography 1-20% MeOH/CH2Cl2 to give the desired compound as a white solid.

General Method VIII - Copper EDC coupling with Boc-VC-OH

[00414] A mixture of (i?)-2-((5)-2-(feri-butoxycarbonylamino)-3-methylbutanamido)-5- ureidopentanoic acid (3.0 eq, prepared according to US2010/0233190 Al), EDC HCl (2.9 eq), and HOAt (3.0 eq) in CH2CI2 DMF (8: 1 v/v) was stirred at ambient temperature for 5-10 minutes. The resulting activated acid was added to a stirring solution of an appropriate amine (1.0 eq) in CH2CI2/DMF (8: 1 v/v) followed by the immediate addition of CuC (3.0 eq). The resulting olive green mixture was stirred at ambient temperature for 0.5 h then treated with N-(2- hydroxyethyl)ethylenediamine (4-8 eq) and diluted with MeOH. The blue mixture was concentrated in vacuo and the residue was purified by silica gel chromatography with 100% EtOAc, followed by 2-20% MeOH/GrhCh to afford the coupled dipeptide as a white solid. General Method IX - Addition of MT-TFP

[00415] To a stirring solution of an appropriate amine (1.0 eq) and MT-TFP in 1,4-dioxane was added an aqueous solution of NaHCC (4.5 eq). The resulting mixture was stirred at ambient temperature for 3 h and concentrated in vacuo. The residue was purified by silica gel chromatography with mixtures MeOH/Ci^C to afford the desired linkable material as a white solid.

General Method X - Borane-mediated reduction of TV-oxides

[00416] Bis(pinacolato)diboron (5.0 eq) was added to a stirring suspension of an appropriate N- oxide (1.0 eq) in MeCN. The mixture was stirred for 0.5 h at ambient temperature and concentrated in vacuo. The residue was purified by silica gel chromatography with mixtures of MeOH/Ci^Ch to afford the reduced product as a white solid.

General Method XI - DCC-mediated TV-acyl sulfonamide formation on Boc-Ile-TubV(Oi?)- OH

[00417] A mixture of an appropriate acid (1.0 eq), an appropriate sulfonamide (1.3 eq), DMAP (1.5 eq) and DCC (1.5 eq) in CH2CI2 (0.02 M) was stirred at ambient temperature for 24-72 h. The mixture was passed through Celite and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography with 1-6% MeOH/CEbC to afford the desired N- acyl sulfonamide as an off-white solid.

General Method XII - Acetylation of secondary alcohol

[00418] Acetic anhydride (35 eq) was added dropwise to a cold (0°C) stirring solution of an appropriate secondary alcohol (1.0 eq) in pyridine (0.1 M). The resulting mixture was stirred at ambient temperature for 17 h, concentrated in vacuo and co-evaporated with PhMe (3x). The oily residue was purified by silica gel column chromatography with 100% EtOAc, follow by 5-20% MeOH/CEbC to afford the desired acetate as an off-white solid.

General Method XIII - Boc-protection of sulfonamides NHBoc

[00419] A solution of di-fert-butyldicarbonate (1.2-1.9 eq) and an appropriate aminosulfonamide (1.0 eq) in 1,4-dioxane (0.1 M) was stirred at reflux for 23-45 h. The mixture was concentrated in vacuo and the pale yellow oily residue was partitioned between EtOAc and H2O. The aqueous layer was extracted with EtOAc (2x). The combined organics was washed with brine (lx), dried (Mg<SO4), filtered, and concentrated in vacuo. The resulting yellow foam was triturated and co- evaporated with Et20 (3x) to afford the desired carbamate as a pale yellow solid.

General Method XIV - Hydrogenolysis of azide

[00420] To a solution of the appropriate azide (1.0 eq) in MeOH (0.2 M) was added 10% Pd/C (15 mol% Pd). The reaction mixture was purged with N2 and then with ¾. The resulting black suspension was stirred at ambient temperature for 18 h under ¾ ( 1 atm) and then passed through Celite. The filtrate was concentrated in vacuo and co-evaporated with CH2CI2 (3x) to afford the desired primary amine as a white solid.

[00421] EXAMPLES 2.1 TO 2.5 describe the preparation of certain compounds of general Formula I and drug-linkers comprising these compounds. The synthetic scheme is provided as Scheme 2 in FIG. 2 and employs the following general intermediates.

Intermediate 1: Ethyl 2-((6S,9i?,lli?)-6-((S)-sec-butyl)-9-isopropyl-2,2,8-trimethyl-4,7-dioxo- 3,12-dioxa-5,8-diazatetradecan-ll-yl)thiazole-4-carboxylate

[00422] To a cold (0°C) stirring solution of ethyl 2-((li?,3i?)-3-((2^3,S)-2-((tert- butoxycarbonyl)amino)-N,3-dimethylpentanamido)-l-hydroxy-4-methylpentyl)thiazole-4- carboxylate (1 eq, available commercially) and TBAI (0.25 eq) in DMF (0.05 M) was added successively iodoethane (7.0 eq) and NaH (60%, 1.8 eq). The resulting mixture was stirred cold under N2 for 0.5 h then carefully quenched with H2O and extracted with EtOAc (4x). The combined organics was successively washed with H2O (2x) and brine (2x) then dried (MgS04), filtered and concentrated in vacuo to afford the desired ethyl ether 2 as a pale yellow film in 98% yield. Ή NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 5.26 (d, J = 9.7 Hz, 1H), 4.49 (dd, J = 9.6, 7.0 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 4.38 (t, J = 6.5 Hz, 1H), 3.55 (q, J = 7.6, 6.8 Hz, 2H), 3.05 (s, 3H), 1.86 - 1.47 (m, 6H), 1.46 - 1.38 (m, 12H), 1.26 (t, J = 7.0 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H). Mass calculated for ^eH^NsOeS+H^ 528.3, found 528.3.

Intermediate 2: 2-((6S,9i?,lli?)-6-((S)-sec-Butyl)-9-isopropyl-2,2,8-trimethyl-4,7-dioxo-3,12- dioxa-5,8-diazatetradecan-ll-yl)thiazole-4-carboxylic acid

[00423] Prepared according to general method I from Intermediate 1 (93 mg, quant.). ¾ NMR (400 MHz, Chloroform-t δ 8.24 (s, 1H), 5.29 (d, J = 9.7 Hz, 1H), 4.48 (dd, J = 9.7, 7.4 Hz, 1H), 4.43 - 4.33 (m, 1H), 3.60 (tdd, J = 8.6, 5.0, 1.6 Hz, 2H), 3.07 (s, 3H), 2.04 (d, J = 8.2 Hz, 2H), 1.79 (ddd, J = 9.9, 6.7, 2.8 Hz, 2H), 1.73 - 1.55 (m, 2H), 1.43 (s, 9H), 1.28 (t, J = 7.0 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). Mass calculated for (C₂₄H₄iN₃0₆S+H)⁺ 500.3, found 500.6.

Intermediate 3: (2S,4i?)-4-Amino-2-methyl-5-phenylpentanoic acid

[00424] (2S,4i?)-((2S,5i?)-2-isopropyl-5-methylcyclohexyl) 4-(fert-butoxycarbonylamino)-2- methyl-5-phenylpentanoate (584 mg, 1.31 mmol, prepared according to Siankar et al , Org. Biomol. Chem. 2013, 1 1 , 2273-2287), was pulverized with a mortar and pestle and suspended in 6 M HC1 (6 mL). The suspension was heated to reflux for 2 h. Upon cooling to rt, the solution was extracted once with 10 mL EtOAc, and this organic layer was back extracted with 5 mL dftO. The combined aqueous layers were concentrated by coevaporation several times with MeCN until a white solid was left (306 mg, 96%). Ή NMR (400 MHz, DMSO-t/₆) δ 12.31 (s, 1H), 8.12 (s, 3H), 7.35 (t, J = 7.5 Hz, 2H), 7.31 - 7.23 (m, 3H), 3.00 (dd, J = 13.8, 5.5 Hz, 1H), 2.78 (dd, J = 13.8, 8.0 Hz, 1H), 2.60 (td, J = 13.4, 12.9, 6.2 Hz, 1H), 1.81 (ddd, J = 14.2, 8.8, 5.4 Hz, 1H), 1.53 (ddd, J = 14.1, 7.8, 5.5 Hz, 1H), 1.04 (d, J = 7.0 Hz, 3H). Mass calculated for (Ci₂Hi₇N0₂+H)⁺ 208.14, found 208.6.

Intermediate 4: (2S,4i?)-4-(teri-Butoxycarbonylamino)-2-methyl-5-phenylpentanoic acid

[00425] Intermediate 3 (291 mg, 1.20 mmol) was suspended in MeCN (20 mL) and di-fert-butyl- dicarbonate (330 mg, 1.43 mmol, 1.2 eq) was added, then 1 M NaOH (3.6 mL, 3.6 mmol, 3 eq) was added and the reaction was stirred overnight at rt. When HPLC indicated no starting material remained, the solution was acidified to pH < 4 with cone. HCl, then solvents were removed in vacuo. The residue was diluted with dH₂0 (30 mL) and extracted twice with 100 mL ether. The combined organics were dried over NaCl(_sat), dried over Na2<S04(_S), filtered, and concentrated in vacuo to yield a white solid (340 mg, 93%). ¾ NM# (400 MHz, Chloroform-d) δ 7.43 - 7.05 (m, 5H), 6.32 (s, OH, minor rotamer NH), 4.57 (d, J = 9.0 Hz, 1H, major rotamer NH), 4.10 - 3.79 (m, 1H), 2.94 - 2.44 (m, 3H), 2.02 - 1.76 (m, 1H), 1.55 (s, 3H, minor rotamer ¾u), 1.43 (s, 6H, major rotamer ¾u), 1.38 - 1.25 (m, 1H), 1.20 (d, J = 6.9 Hz, 3H). Mass calculated form (Ci₇H₂₅N0₄-H)⁺ 306.16, found 306.3.

Intermediate 5: 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)ethoxy)ethoxy)ethoxy)propanoate (MT-TFP)

[00426] To a stirred solution of MT-OH (0.520 g, 1.73 mmol, 1 eq) in dichloromethane (10 mL) was added 2,3,5,6-tetrafluorophenol (0.301 g, 1.81 mmol, 1.05 eq), followed by N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.348 g, 1.81 mmol, 1.05 eq). The reaction was stirred at room temperature. After 18hr, the reaction solvent was evaporated under reduced pressure and the resulting residue was purified by flash chromatography to yield the title compound (0.323 g, 42% yield). ¾ NMR (400 MHz, Chloroform- ) δ 7.02 (tt, J = 9.9, 7.1 Hz, 1H), 6.71 (s, 2H), 3.90 (t, J= 6.3 Hz, 2H), 3.75 (td, J = 5.6, 0.9 Hz, 2H), 3.70 - 3.64 (m, 6H), 3.63 (s, 4H), 2.98 (t, J = 6.3 Hz, 2H). m/z calc'd for G9H19F4NO7 449.11. Found [M+H]⁺ = 450.48, [M+Na]⁺ = 472.48 Intermediate 6: (14S,17S)-l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo- 17-(3-ureidopropyl)-3,6,9-trioxa-13,16-diazaoctadecan-18-oic acid (MT-VC-OH)

[00427] Prepared according to International Patent Publication No. WO 2015/095953. Intermediate 7: 2,2,2-Trifluoro-/V-(3-(sulfamoylmethyl)phenyl)acetamide

[00428] Trifluoroacetic anhydride (0.45 mL, 3.24 mmol) was added to a stirring suspension of (3- aminophenyl)methanesulfonamide (0.5 g, 2.56 mmol) in 1,4-dioxane (5 mL). The resulting tan- coloured suspension was stirred at ambient temperature for 22 h. The mixture filtered and washed with CH2CI2 to afford the desired product as a white solid in 98% (0.7 g) yield. Ή NMR (400 MHz, DMSO-t/e) δ 11.35 (s, 1H), 7.73 - 7.64 (m, 2H), 7.42 (t, J = 7.8 Hz, lH), 7.23 (dt, J = 7.7, 1.3 Hz, 1H), 6.88 (s, 2H), 4.28 (s, 2H). Mass calculated for (C₉H₉F₃N₂O₃S-H)- 281.0, found 281.6.

EXAMPLE 2.1

Intermediate 1-i: teri-Butyl (2i?,4S)-4-methyl-5-oxo-l-phenyl-5-(4-(2,2,2- trifluoroacetamido)phenylsulfonamido)pentan-2-ylcarbamate

[00429] Prepared according to general method II from Intermediate 4 and 2,2,2-trifluoro-N-(4- sulfamoylphenyl)acetamide (75 mg, 75%) 'ΗΝΜΛ (400 MHz, Chloroform- ) δ 1 1.30 (s, 1H), 8.36 (s, 1H), 8.13 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.35 - 7.22 (m, 3H), 7.06 - 6.99 (m, 2H), 4.54 (d, J = 9.2 Hz, 1H), 3.88 - 3.64 (m, 1H), 2.70 (dd, J = 14.1, 6.7 Hz, 1H), 2.65 (dd, J = 14.2, 6.6 Hz, 1H), 2.42 - 2.32 (m, 1H), 1.70 (t, J= 13.1 Hz, 1H), 1.48 (s, 9H), 1.35 - 1.25 (m, 1H), 1.07 (d, J= 6.7 Hz, 3H). Mass calculated for (CzsHsoFsNsOeS+H^ 558.19, found 558.5.

Intermediate 1-ii: (2S,4i?)-4-Amino-2-methyl-5-phenyWV-(4-(2,2,2- trifluoroacetamido)phenylsulfonyl)pentanamide

[00430] Prepared according to general method III from intermediate 1-i (82.7 mg, quant.) ¾ NMR (400 MHz, Methanol-^) δ 8.03 (d, J = 8.8 Hz, 2H), 7.86 (d, J= 8.8 Hz, 2H), 7.41 - 7.24 (m, 3H), 7.23 - 7.06 (m, 2H), 3.38 - 3.34 (m, 1H), 2.71 (dd, J= 14.2, 6.0 Hz, 1H), 2.66 - 2.48 (m, 2H), 1.97 (dt, J = 15.2, 7.8 Hz, lH), 1.63 (dt, J = 14.4, 5.7 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H). Mass calculated for (C₂oH₂₂F₃N30₄S+H)⁺ 458.14, found 458.4. Intermediate 1-iii: teri-Butyl (2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-((2i?,4S)-4-methyl-5- oxo-l-phenyl-5-(4-(2,2,2-trifluoroacetamido)phenylsulfonamido)pentan-2- ylcarbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3-methyl-l-oxopentan-2-ylcarbamate

[00431] Prepared from according to general method IV from intermediates 2 and 1-ii (35.9 mg, 64%). ¾ NMR (400 MHz, Chloroform- ) δ 8.28 (s, 1H), 8.20 (d, J = 8.7 Hz, 2H), 8.18 (s, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 9.1 Hz, 1H), 7.31 - 7.15 (m, 3H), 7.07 (d, J = 6.4 Hz, 2H), 5.36 (d, J = 9.7 Hz, 1H), 4.50 (dd, J = 9.7, 7.4 Hz, 1H), 4.30 (d, J = 10.2 Hz, 1H), 4.26 - 4.13 (m, 1H), 3.57 (q, J = 7.3 Hz, 2H), 3.09 (s, 3H), 2.83 (d, J = 6.6 Hz, 2H), 2.42 (td, J = 12.8, 12.3, 7.6 Hz, 1H), 2.10 - 1.60 (m, 9H), 1.43 (s, 9H), 1.28 (t, J = 7.0 Hz, 3H), 1.05 (d, J= 6.7 Hz, 3H), 1.04 - 0.98 (m, 6H), 0.95 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H). Mass calculated for

939.40, found 939.7. Compound 25: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((2i?,4S)-4-methyl-5-oxo-l-phenyl-5- (4-(2,2,2-trifluoroacetamido)phenylsulfonamido)pentan-2-yl)thiazole-4-carboxamide

[00432] Prepared according to general methods III and V from intermediate 1-iii (32.6 mg, 89%). 'Η ΝΜΛ (400 MHz, Methanol-^) δ 8.12 (s, 1H), 8.01 (d, J= 8.5 Hz, 2H), 7.83 (d, J= 8.5 Hz, 2H), 7.25 - 7.08 (m, 5H), 4.76 (d, J= 7.8 Hz, 1H), 4.46 - 4.36 (m, 1H), 4.21 - 4.09 (m, 1H), 3.85 - 3.65 (m, 1H), 3.56 (ddd, J = 14.0, 7.0, 2.8 Hz, 2H), 3.30 - 3.20 (m, 1H), 3.17 (s, 3H), 3.11 - 3.02 (m, 1H), 2.84 - 2.74 (m, 2H), 2.53 (s, 3H), 2.50 - 2.43 (m, 1H), 2.15 - 1.44 (m, 15H), 1.25 (t, J = 7.0 Hz, 3H), 1.08 (d, J = 7.0 Hz, 3H), 1.04 (d, J= 6.8 Hz, 3H), 1.01 (d, J= 6.5 Hz, 3H), 0.94 (t, J= 7.4 Hz, 3H), 0.86 (d, J= 6.6 Hz, 3H). Mass calculated for (C₄6H64F₃N₇0₈S2+H)⁺ 964.43, found 964.8.

Compound 26: A'-((2i?,4S)-5-(4-Aminophenylsulfonamido)-4-methyl-5-oxo-l-phenylpentan-2- yl)-2-((LR,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4-carboxamide

[00433] Prepared according to general method VI from compound 25 (29.5 mg, quant.). ¾ NMR (400 MHz, Methanol-^) δ 8.14 (s, 1H), 7.72 (d, J= 8.8 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 4.76 (d, J = 7.6 Hz, 1H), 4.44 - 4.33 (m, 2H), 4.09 - 4.02 (m, 1H), 3.62 - 3.54 (m, 1H), 3.51 (q, J = 7.1 Hz, 2H), 3.24 (d, J = 61.8 Hz, 3H), 2.80 (qd, J = 13.8, 7.3 Hz, 2H), 2.61 (s, 3H), 2.52 - 2.42 (m, 1H), 2.15 - 1.44 (m, 16H), 1.25 (t, J = 7.0 Hz, 3H), 1.13 - 0.98 (m, 9H), 0.98 - 0.92 (m, 3H), 0.86 (d, J = 6.6 Hz, 3H). Mass calculated for (C₄₄H₆₅N₇0₇S2+H)⁺ 868.45, found 868.9. Compound 27: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((2i?,4S)-5-(4-((14S,17S)-l-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa- 13,16-diazaoctadecanamido)phenylsulfonamido)-4-methyl-5-oxo-l-phenylpentan-2- yl)thiazole-4-carboxamide

[00434] Prepared according to general method VII from intermediates 6 and compound 26 (3.5 mg, 7%). ¾ NMi? (600 MHz, Methanol-^) δ 8.05 (s, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 7.4 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 7.3 Hz, 2H), 6.71 (s, 2H), 4.77 (s, 1H), 4.46 (dd, J = 9.4, 4.7 Hz, 1H), 4.35 - 4.25 (m, 1H), 4.12 (d, J = 6.8 Hz, 1H), 4.06 - 3.95 (m, 1H), 3.65 (ddt, J = 9.4, 6.2, 3.3 Hz, 4H), 3.58 (t, J = 5.6 Hz, 2H), 3.54 - 3.46 (m, 4H), 3.46 (d, J = 7.0 Hz, 10H), 3.40 - 3.35 (m, 2H), 3.14 - 3.06 (m, 1H), 3.05 (s, 3H), 3.05 - 2.98 (m, 1H), 2.95 (t, J = 12.8 Hz, 1H), 2.74 - 2.64 (m, 2H), 2.64 (s, 3H), 2.47 (t, J = 5.7 Hz, 2H), 2.40 - 2.33 (m, 1H), 2.08 - 1.99 (m, 2H), 1.97 - 1.92 (m, 2H), 1.89 - 1.77 (m, 4H), 1.80 - 1.62 (m, 3H), 1.56 - 1.41 (m, 5H), 1.13 (t, J = 7.0 Hz, 3H), 0.95 - 0.91 (m, 6H), 0.91 - 0.85 (m, 9H), 0.82 (t, J = 7.4 Hz, 3H), 0.74 (d, J = 6.5 Hz, 3H). Mass calculated for

1407.71, found 1408.3.

EXAMPLE 2.2

Intermediate 2-i: tert-Butyi (2i?,4S)-4-methyl-5-oxo-l-phenyl-5-((4-(2,2,2- trifluoroacetamido)phenyl)methylsulfonamido)pentan-2-ylcarbamate

[00435] Prepared according to general method II from intermediate 4 and 2,2,2-trifluoro-N-(4- sulfamoylphenyl)acetamide (100 mg, 53%). Ή NMR (400 MHz, Chloroform- ) δ 10.95 (s, IH), 7.74 (s, IH), 7.55 (d, J= 8.6 Hz, 2H), 7.47 (d, J= 8.4 Hz, 2H), 7.35 - 7.21 (m, 3H), 7.00 (d, J= 6.6 Hz, 2H), 4.80 (d, J = 14.1 Hz, IH), 4.62 (d, J = 14.2 Hz, IH), 4.38 (d, J = 9.1 Hz, IH), 3.72 - 3.58 (m, IH), 2.75 (dd, J = 14.9, 5.2 Hz, IH), 2.63 (dd, J = 15.0, 8.2 Hz, IH), 2.35 (dt, J = 8.0, 4.6 Hz, IH), 2.07 (s, IH), 1.36 (s, 9H), 1.42 - 1.26 (m, IH), 1.16 (s, 3H). Mass calculated for (C₂₆H₃₂F₃N₃0₆S-H)⁺ = 570.20, found 270.1.

Intermediate 2-ii: (2S,4i?)-4-Amino-2-methyl-5-phenyWV-(4-(2,2,2- trifluoroacetamido)benzylsulfonyl)pentanamide

[00436] Prepared according to general method III from intermediate 2-i (77 mg, quant.). ¾ NMR (400 MHz, DMSO-t/e) δ 11.60 (s, IH), 11.37 (s, IH), 7.80 (s, 3H), 7.68 (d, J = 8.6 Hz, 2H), 7.42 - 7.23 (m, 8H), 4.69 (s, 2H), 2.82 (d, J= 6.6 Hz, 2H), 2.61 - 2.52 (m, IH), 1.85 (dt, J = 14.2, 7.1 Hz, IH), 1.56 (dt, J = 13.7, 6.6 Hz, IH), 1.03 (d, J = 6.9 Hz, 3H). Mass calculated for (C2iH₂4F3N30₄S+H)⁺ 472.15, found 472.0.

Intermediate 2-iii tert-Butyl (2S,3S)-l-(((LR,3i?)-l-ethoxy-4-methyl-l-(4-((2i?,4S)-4-methyl-5- oxo-l-phenyl-5-((4-(2,2,2-trifluoroacetamido)phenyl)methylsulfonamido)pentan-2- ylcarbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3-methyl-l-oxopentan-2-ylcarbamate

[00437] Prepared from according to general method IV from intermediates 2 and 2-ii (93.4 mg, 97%). 'H NMR (400 MHz, Chloroform- ) δ 11.64 (s, IH), 8.06 (s, IH), 7.74 (s, IH), 7.61 - 7.49 (m, 4H), 7.32 - 7.15 (m, 3H), 7.04 (d, J = 7.1 Hz, 2H), 5.17 (d, J = 9.7 Hz, IH), 4.84 (d, J = 14.3 Hz, IH), 4.66 (d, J= 14.2 Hz, IH), 4.47 (dd, J = 9.7, 7.2 Hz, IH), 4.41 - 4.36 (m, IH), 4.22 (d, J =

9.6 Hz, IH), 4.07 - 4.02 (m, IH), 3.52 (q, J = 6.9 Hz, 2H), 3.00 (s, 3H), 2.89 - 2.84 (m, 2H), 2.83 (s, 4H), 2.45 - 2.32 (m, IH), 2.00 - 1.61 (m, 3H), 1.43 (s, 9H), 1.24 (t, J = 6.9 Hz, 4H), 1.15 (d, J =

6.7 Hz, 3H), 1.03 - 0.95 (m, 6H), 0.91 (t, J = 7.4 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H). Mass calculated for

953.41, found 953.5. Compound 28: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((2i?,4S)-4-methyl-5-oxo-l-phenyl-5- ((4-(2,2,2-trifluoroacetamido)phenyl)methylsulfonamido)pentan-2-yl)thiazole-4-carboxamide

[00438] Prepared according to general methods III and V from intermediate 2-iii (68 mg, quant.). Ή NMR (400 MHz, DMSO-t/e) δ 8.18 (s, IH), 7.95 - 7.82 (m, IH), 7.61 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.28 - 7.11 (m, 5H), 4.63 (t, J= 8.4 Hz, IH), 4.53 (s, IH), 4.31 - 4.23 (m, IH), 4.22 - 4.12 (m, 3H), 3.17 (d, J = 4.9 Hz, IH), 3.10 (d, J = 8.4 Hz, 2H), 3.00 (s, 3H), 2.93 - 2.73 (m, 2H), 2.45 - 2.22 (m, 6H), 1.99 - 1.38 (m, 14H), 1.12 (t, J = 6.9 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 5.1 Hz, 3H), 0.88 (d, J = 5.0 Hz, 3H), 0.82 (q, J = 7.5, 6.7 Hz, 3H), 0.70 (d, J = 6.5 Hz, 3H). Mass calculated for (C₄7H₆₆F₃N₇0₈ S2+H)⁺ 978.44, found 978.5. Compound 29: A'-((2i?,4S)-5-((4-Aminophenyl)methylsulfonamido)-4-methyl-5-oxo-l- phenylpentan-2-yl)-2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4-carboxamide

[00439] Prepared according to general method VI from compound 28 (43 mg, 73%). ¾ NMR (400 MHz, Methanol-^) δ 8.12 (d, J = 3.9 Hz, 1H), 7.30 - 7.15 (m, 5H), 7.09 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), 4.75 (d, J = 7.9 Hz, 1H), 4.55 (d, J = 14.1 Hz, 1H), 4.46 (d, J = 14.0 Hz, 1H), 4.39 (d, J = 11.3 Hz, 1H), 4.31 - 4.25 (m, 1H), 3.51 (q, J = 6.9 Hz, 2H), 3.24 - 3.10 (m, 2H), 3.16 (s, 3H), 2.94 (d, J= 6.7 Hz, 2H), 2.55 (d, J= 25.1 Hz, 3H), 2.48 (s, 3H), 2.16 - 1.57 (m, 12H), 1.57 - 1.38 (m, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.14 (d, J = 7.2 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.95 (t, J = 7.5 Hz, 3H), 0.86 (d, J = 6.5 Hz, 2H). Mass calculated for (C₄5H67N70₇S2+H)⁺ 882.46, found 882.8.

Intermediate 2-iv: teri-Butyl (S)-l-((S)-l-(4-((iV-((2S,4i?)-4-(2-((LR,3i?)-3-((2S,3S)-N,3- dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4- methylpentyl)thiazole-4-carboxamido)-2-methyl-5- phenylpentanoyl)sulfamoyl)methyl)phenylamino)-l-oxo-5-ureidopentan-2-ylamino)-3- m ethyl- l-oxobutan-2-ylcarbam ate

[00440] Prepared according to general methods III and VIII from compound 29 (37 mg, 67%). ¾ NMR (400 MHz, Methanol-^) δ 8.16 (s, 1H), 7.62 (d, J= 8.4 Hz, 2H), 7.34 (d, J= 8.4 Hz, 2H), 7.29 - 7.15 (m, 5H), 4.74 (d, J = 7.5 Hz, 1H), 4.57 (s, 2H), 4.52 (dd, J = 8.8, 5.1 Hz, 1H), 4.43 - 4.25 (m, 3H), 3.92 (d, J = 6.8 Hz, 1H), 3.66 (d, J = 11.8 Hz, 1H), 3.52 (q, J = 6.9 Hz, 2H), 3.46 - 3.38 (m, 1H), 3.26 - 3.06 (m, 2H), 3.15 (s, 3H), 3.00 - 2.82 (m, 3H), 2.70 (s, 3H), 2.50 - 2.45 (m, 2H), 2.21 - 1.50 (m, 20H), 1.46 (s, 9H), 1.23 (t, J = 7.0 Hz, 3H), 1.13 (d, J= 6.9 Hz, 3H), 1.05 (d, J = 6.7 Hz, 3H), 1.02 - 0.89 (m, 12H), 0.85 (d, J = 6.5 Hz, 3H). Mass calculated for (C₆iH₉₅NiiOi₂S₂+H)⁺ 1238.67, found 1239.1.

Compound 30: 2-((li?,3i?)-3-((2S,3S)-N,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-^-((2i?,4S)-5-((4-((14S,17S)-l-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-

13,16-diazaoctadecanamido)phenyl)methylsulfonamido)-4-methyl-5-oxo-l-phenylpentan-2- yl)thiazole-4-carboxamide

[00441] Prepared from intermediates 2-iv and 5 according to general procedures III and IX. 11.7 mg (54%)¹H NMR (400 MHz, Methanol-^) δ 8.17 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.34 (d, J =

8.3 Hz, 2H), 7.27 - 7.15 (m, 5H), 6.83 (s, 2H), 4.76 (d, J = 7.9 Hz, 1H), 4.60 - 4.29 (m, 6H), 4.21 (d, J = 6.9 Hz, 1H), 3.75 (t, J = 5.9 Hz, 2H), 3.69 (t, J = 5.1 Hz, 2H), 3.63 (t, J = 5.4 Hz, 2H), 3.58 (s, 12H), 3.29 - 3.20 (m, 1H), 3.16 (s, 3H), 3.15 - 3.07 (m, 2H), 2.91 (d, J = 6.8 Hz, 2H), 2.73 - 2.39 (m, 7H), 2.23 - 1.45 (m, 17H), 1.31 (s, 2H), 1.23 (t, J= 6.9 Hz, 3H), 1.13 (d, J = 7.1 Hz, 3H),

1.04 (d, J = 6.8 Hz, 3H), 1.02 - 0.97 (m, 9H), 0.94 (t, J = 7.4 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H). Mass calculated for (C₆₉Hio₄Ni₂0i₆S₂+H)⁺ 1421.72, found 1422.3. EXAMPLE 2.3

Intermediate 3-i: tert-Butyl (2i?,4S)-4-methyl-5-oxo-l-phenyl-5-(4-((2,2,2- trifluoroacetamido)methyl)phenylsulfonamido)pentan-2-ylcarbamate

[00442] Prepared according to general method II from intermediate 4 and 2,2,2-trifluoro-N-(4- sulfamoylbenzyl)acetamide (55 mg, 50%). ¾ NMR (400 MHz, Chloroform- ) δ 11.16 (s, 1H), 8.12 (d,J= 8.4 Hz, 2H), 7.44 (d, J= 8.4 Hz, 2H), 7.37 - 7.22 (m, 3H), 7.05 - 6.98 (m, 2H), 6.70 (s, 1H), 4.66 (dd, J= 15.6, 6.3 Hz, 1H), 4.60 (dd, J= 14.3, 6.0 Hz, 1H), 4.50 (d, J= 9.3 Hz, 1H), 3.83 - 3.69 (m, 1H), 2.66 (d,J= 6.6 Hz, 2H), 2.41 -2.31 (m, 1H), 1.71 (t,J= 13.5 Hz, 1H), 1.47 (s, 9H), 1.33 - 1.19 (m, 1H), 1.07 (d, J= 6.7 Hz, 3H). Mass calculated for (C₂₆H₃₂F₃N₃0₆S+H)⁺ 572.20, found 572.1.

Intermediate 3-ii: (2S,4i?)-4-Amino-2-methyl-5-phenyl-_JV-(4-((2,2,2- trifluoroacetamido)methyl)phenylsulfonyl)pentanamide

[00443] Prepared according to general method III from intermediate 3-i (51 mg, 99%). ¾ NMR (400 MHz, Methanol-^) δ 8.00 (d, J= 8.5 Hz, 2H), 7.49 (d, J= 8.4 Hz, 2H), 7.38 (t, J= 7.2 Hz, 2H), 7.33 (t, J= 7.2 Hz, 1H), 7.20 (d, J= 8.5 Hz, 2H), 4.52 (s, 2H), 3.40 (p, J= 6.6 Hz, 1H), 2.81 (dd,J= 14.3, 6.1 Hz, 1H), 2.68 (dd,J= 14.2, 8.1 Hz, 1H), 2.55 (dq,J= 14.3, 7.2 Hz, 1H), 1.95 (dt, J= 14.7, 7.5 Hz, 1H), 1.63 (dt,J= 14.4, 5.9 Hz, 1H), 1.11 (d,J= 7.0 Hz, 3H). Mass calculated for (C2iH₂4F3N30₄S+H)⁺ 472.15, found 472.0. Intermediate 3-iii: tert-Butyi (2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-((2 ?,4S)-4-methyl-5- oxo-l-phenyl-5-(4-((2,2,2-trifluoroacetamido)methyl)phenylsulfonamido)pentan-2- ylcarbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3-methyl-l-oxopentan-2-ylcarbamate

[00444] Prepared from according to general method IV from intermediates 2 and 3-ii (67 mg, 99%). ¹H NMR (400 MHz, Chloroform- ) δ 8.17 (s, 1H), 8.16 (d, J= 8.1 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.39 - 7.19 (m, 5H), 7.07 (d, J = 7.9 Hz, 2H), 5.20 (d, J = 9.6 Hz, 1H), 4.64 (d, J = 6.0 Hz, 2H), 4.50 (t, J= 9.9, 8.4 Hz, 1H), 4.44 - 4.34 (m, 1H), 4.30 (d, J= 9.6 Hz, 1H), 4.27 - 4.21 (m, 1H), 3.57 (q, J = 7.0 Hz, 2H), 3.07 (s, 3H), 2.83 (d, J = 6.5 Hz, 2H), 2.48 - 2.34 (m, 1H), 2.18 - 1.62 (m, 8H), 1.43 (s, 9H), 1.28 (t, J = 7.1 Hz, 3H), 1.08 - 0.97 (m, 9H), 0.92 (t, J = 7.5 Hz, 3H), 0.89 (d, J= 6.4 Hz, 3H). Mass calculated for

953.41, found 953.8.

Compound 31 : 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((2i?,4S)-4-methyl-5-oxo-l-phenyl-5- (4-((2,2,2-trifluoroacetamido)methyl)phenylsulfonamido)pentan-2-yl)thiazole-4-carboxamide

[00445] Prepared according to general methods III and V from intermediate 3-iii (53.4 mg, 79%). ¾ NMR (400 MHz, Methanol-^) δ 8.10 (s, 1H), 7.98 (d, J= 8.2 Hz, 2H), 7.45 (d, J= 8.0 Hz, 2H), 7.27 - 7.10 (m, 5H), 4.75 (d, J = 7.8 Hz, 1H), 4.56 - 4.51 (m, 2H), 4.41 (d, J = 10.1 Hz, 1H), 4.12 - 4.07 (m, 1H), 3.57 (dq, J = 7.3, 2.1 Hz, 2H), 3.24 - 3.18 (m, 2H), 3.16 (s, 3H), 2.82 (d, J = 6.8 Hz, 2H), 2.50 - 2.45 (m, 4H), 2.14 - 1.31 (m, 16H), 1.25 (t, J = 6.9 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.03 (d, J= 6.7 Hz, 3H), 1.00 (d, J= 6.6 Hz, 3H), 0.93 (t, J= 7.4 Hz, 3H), 0.86 (d, J= 6.6 Hz, 3H). Mass calculated for: (C47H₆6F₃N70₈S2+H)⁺ 978.44, found 978.9. Compound 32: A'-((2i?,4S)-5-(4-(Aminomethyl)phenylsulfonamido)-4-methyl-5-oxo-l- phenylpentan-2-yl)-2-((li?,3i?)-3-((2S,3S)-N,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4-carboxamide

[00446] Prepared according to general method VI from compound 31 (42.8 mg, 91%). ¾ NMR (400 MHz, Methanol-^) δ 8.12 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.27 - 7.10 (m, 5H), 4.75 (d, J= 7.5 Hz, 1H), 4.49 - 4.38 (m, 2H), 4.23 (s, 2H), 4.17 - 4.06 (m, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.77 - 3.61 (m, 2H), 3.60 - 3.52 (m, 2H), 3.45 (d, J = 10.6 Hz, 2H), 3.16 (s, 3H), 3.04 - 2.93 (m, 1H), 2.82 (d, J = 6.9 Hz, 2H), 2.71 (s, 3H), 2.53 - 2.44 (m, 2H), 2.18 - 1.46 (m, 9H), 1.25 (t, J = 7.0 Hz, 3H), 1.11 - 1.03 (m, 6H), 1.01 (d, J = 6.7 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H), 0.86 (d, J= 6.5 Hz, 3H). Mass calculated for (C₄5H67N70₇S2+H)⁺ 882.46, found 882.8.

Intermediate 3-iv: teri-Butyl (S)-l-((S)-l-(4-(iV-((2S,4i?)-4-(2-((LR,3i?)-3-((2S,3S)-N,3- dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4- methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoyl)sulfamoyl)benzylamino)- l-oxo-5-ureidopentan-2-ylamino)-3-methyl-l-oxobutan-2-ylcarbamate

[00447] Prepared according to general methods VIII and X from compound 32 (16.1 mg, 28%). ¾ NMR (400 MHz, Methanol-^) δ 8.14 (s, 1H), 7.92 (d, J= 8.2 Hz, 2H), 7.42 (d, J= 8.0 Hz, 2H), 7.27 - 7.13 (m, 5H), 4.77 (d, J = 8.0 Hz, 1H), 4.57 (s, 1H), 4.52 - 4.36 (m, 4H), 4.26 (s, 1H), 3.88 (d, J = 6.7 Hz, 1H), 3.56 (dd, J = 7.1, 2.9 Hz, 2H), 3.16 (s, 3H), 3.18 - 3.02 (m, 2H), 2.85 (d, J = 6.9 Hz, 2H), 2.52 (s, 2H), 2.45 (s, 3H), 2.11 - 1.46 (m, 21H), 1.44 (s, 9H), 1.25 (t, J= 6.9 Hz, 3H), 1.06 (d, J = 7.0 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H), 0.98 - 0.88 (m, 9H), 0.85 (d, J= 6.7 Hz, 3H). Mass calculated for (CeiHwNiiOizSz+H 1238.67, found 1239.2.

Compound 33: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-A'-((2i?,4S)-5-(4-((4S,7S)-20-(2,5-dioxo- 2,5-dihydro-lH-pyrrol-l-yl)-7-isopropyl-3,6,9-trioxo-4-(3-ureidopropyl)-12,15,18-trioxa- 2,5,8-triazaicosyl)phenylsulfonamido)-4-methyl-5-oxo-l-phenylpentan-2-yl)thiazole-4- carboxamide

[00448] Prepared according to general method IX from intermediate 3-iv (13.5 mg, 98%). ¾ NMR (400 MHz, Methanol-^) δ 8.15 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 7.27 - 7.13 (m, 5H), 6.83 (s, 2H), 4.77 (d, J = 8.0 Hz, 1H), 4.53 - 4.34 (m, 5H), 4.19 (d, J = 7.0 Hz, 1H), 3.73 (t, J = 6.2 Hz, 2H), 3.70 (t, J = 5.5 Hz, 2H), 3.66 - 3.51 (m, 11H), 3.16 (s, 3H), 3.12 - 3.07 (m, 4H), 2.86 (d, J = 7.1 Hz, 2H), 2.54 (q, J = 6.1 Hz, 2H), 2.42 (s, 4H), 2.18 - 1.89 (m, 6H), 1.89 - 1.56 (m, 10H), 1.56 - 1.36 (m, 5H), 1.35 - 1.23 (m, 1H), 1.25 (t, J = 7.0 Hz, 3H), 1.07 (d, J = 6.9 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 1.01 (d, J = 6.5 Hz, 3H), 0.96 (d, J = 6.7 Hz, 6H), 0.93 (t, J = 7.5 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). Mass calculated for

1421.72, found 1422.4.

EXAMPLE 2.4

Intermediate 4-i: tert-butyi ((2i?,4S)-4-Methyl-5-oxo-l-phenyl-5-(((4-((2,2,2- trifluoroacetamido)methyl)phenyl)methyl)sulfonamido)pentan-2-yl)carbamate

[00449] Prepared according to general method II from 2,2,2-trifluoro-N-(4- (sulfamoylmethyl)benzyl)acetamide (71 mg, 63%). 'H NMR (400 MHz, CDC1₃) δ 10.77 (s, 1H), 7.47 (d, J = 7.9 Hz, 2H), 7.31 (dt, J = 8.4, 4.1 Hz, 3H), 7.28 - 7.22 (m, 3H), 7.02 (d, J = 7.2 Hz, 2H), 4.79 (d, J= 14.0 Hz, 1H), 4.65 (d, J= 14.0 Hz, 1H), 4.44 - 4.26 (m, 3H), 3.80 - 3.60 (m, 1H), 2.70 (ddd, J = 22.6, 14.7, 6.6 Hz, 2H), 2.43 - 2.27 (m, 1H), 1.83 (t, J= 12.0 Hz, 1H), 1.38 (s, 10H), 1.17 (d, J= 6.8 Hz, 3H). Mass calculated for (C₂₇H₃4F₃N306S+H)⁺ 586.21, found 586.1.

Intermediate 4-ii: (2S,4i?)-4-Amino-2-methyl-5-phenyl-yV-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)sulfonyl)pentanamide

[00450] Prepared according to general method III from 4-i (68 mg, quant.). Ή NMR (400 MHz, MeOD) δ 7.49 - 7.27 (m, 9H), 4.73 (q, J = 14.2 Hz, 2H), 4.48 (s, 2H), 3.00 (dd, J = 14.3, 6.3 Hz, 1H), 2.88 (dd, J = 14.3, 7.8 Hz, 1H), 2.56 (dd, J = 13.7, 7.2 Hz, 1H), 2.06 (dt, J = 14.4, 7.1 Hz, 1H), 1.75 (dt, J = 14.4, 6.3 Hz, 1H), 1.15 (d, J = 7.1 Hz, 3H). Mass calculated for (C₂₂H₂₆F₃N30₄S+H)⁺ 486.16, found 486.1. Intermediate 4-iii: tert-B tyl ((2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-(((2i?,4S)-4-methyl- 5-oxo-l-phenyl-5-(((4-((2,2,2-trifluoroacetamido)methyl)phenyl)methyl)sulfonamido)pentan- 2-yl)carbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3-methyl-l-oxopentan-2- yl) carbamate

[00451] Prepared according to general method IV from intermediates 4-ii and 2 (79 mg, quant.). ¾ NMR (400 MHz, CDC1₃) δ 11.39 (d, J= 31.2 Hz, 1H), 8.03 (s, lH), 7.47 (d, J= 8.0 Hz, 2H), 7.32 - 7.17 (m, 7H), 7.06 (d,J= 6.8 Hz, 2H), 6.55 (s, 1H), 5.15 (d,J= 9.7 Hz, 1H), 4.78 (d,J = 13.9 Hz, 1H), 4.66 (d, J= 14.2 Hz, 1H), 4.44 (d, J= 9.5 Hz, 1H), 4.38 (t, J= 6.0 Hz, 2H), 4.22 (d, J= 11.8 Hz, 1H), 4.17-4.04 (m, 1H), 3.51 (q,J= 6.9 Hz, 2H), 3.00 (s, 2H), 2.85 -2.74 (m, 4H), 2.43-2.31 (m, 1H), 2.09 - 1.59 (m, 7H), 1.41 (s, 9H), 1.23 (t,J=7.0Hz, 3H), 1.13 (d,J=6.7Hz, 3H), 0.99 - 0.94 (m, 6H), 0.89 (t, J= 7.3 Hz, 3H), 0.85 (d, J= 6.6 Hz, 3H). Mass calculated for (C₄₆H₆₅F₃N609S2+H)⁺ 967.42, found 967.8.

Compound 34: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentananiido)-l-ethoxy-4-methylpentyl)-7V-((2i?,4S)-4-methyl-5-oxo-l-phenyl-5- (((4-((2,2,2-trifluoroacetamido)methyl)phenyl)methyl)sulfonamido)pentan-2-yl)thiazole-4- carboxamide

[00452] Prepared according to general methods III and V from intermediate 4-iii (58 mg, 72% over 2 steps). ¾ NMR (400 MHz, MeOD) δ 8.14 (s, 1H), 7.37 (d,J= 7.9 Hz, 2H), 7.30 - 7.22 (m, 6H), 7.21 - 7.16 (m, 1H), 4.75 (d, J= 7.6 Hz, 1H), 4.65 - 4.53 (m, 3H), 4.45 - 4.30 (m, 5H), 3.75 (dt,J= 13.2, 6.5 Hz, 2H), 3.53 - 3.46 (m, 1H), 3.25 (dd, J= 14.8, 7.4 Hz, 2H), 3.17 (s, 3H), 2.94 (d,J=6.4Hz, 2H), 2.51 (s, 3H), 2.14 - 1.58 (m, 14H), 1.21 (t,J=6.8Hz, 3H), 1.13 (d,J=7.0Hz, 3H), 1.04 (d, J= 6.7 Hz, 3H), 1.00 - 0.92 (m, 6H), 0.86 (d, J= 6.6 Hz, 3H). Mass calculated for (C₄₈H₆₈F3N₇0₈S2+H)⁺ 992.45, found 992.6.

Compound 35: 7V-((2i?,4S)-5-(((4-(Aminomethyl)phenyl)methyl)sulfonamido)-4-methyl-5-oxo- l^henylpentan-2-yl)-2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4-carboxamide

[00453] Prepared according to general method VI from compound 34 (34.5 mg, 72%). Mass calculated for (C₄₆H₆9N70₇S2+H)⁺ 896.47, found 896.6.

Compound 36: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-7V-((2i?,4S)-5-(((4-((4S,7S)-20-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-7-isopropyl-3,6,9-trioxo-4-(3-ureidopropyl)-12,15,18- trioxa-2,5,8-triazaicosyl)phenyl)methyl)sulfonamido)-4-methyl-5-oxo-l-phenylpentan-2- yl)thiazole-4-carboxamide

[00454] Prepared according to general methods VIII, III and IX from compound 35 (4.8 mg, 28%). Mass calculated for (C₇oHio6Ni20i₆S2)⁺ 1435.73, found 1436.2.

EXAMPLE 2.5

Intermediate 5-i: tert-butyl ((2i?,4S)-4-methyl-5-oxo-l-phenyl-5-(((3-(2,2,2- trifluoroacetamido)phenyl)methyl)sulfonamido)pentan-2-yl)carbamate

[00455] Prepared according to general method II from 2,2,2 rifluoro-N-(3- (sulfamoylmethyl)phenyl)acetamide (72 mg, 66%). ¾ NMR (400 MHz, CDC1₃) δ 10.41 (s, IH), 8.21 (s, IH), 7.76 (d, J = 8.0 Hz, IH), 7.51 (s, IH), 7.40 (t, J = 7.9 Hz, IH), 7.33 - 7.17 (m, 5H), 7.03 (d, J = 7.0 Hz, 2H), 4.76 - 4.61 (m, 2H), 4.41 (d, J = 9.3 Hz, IH), 3.76 - 3.59 (m, IH), 2.67 (qd, J = 14.4, 6.4 Hz, 2H), 2.43 - 2.22 (m, IH), 1.79 (t, J = 13.6 Hz, IH), 1.36 (s, 9H), 1.14 (d, J = 6.8 Hz, 3H). Mass calculated for (C₂₆H₃₂F₃N30₆S+H)⁺ 572.20, found 572.1.

Intermediate 5-ii: ^S^H-amino^-methyl-S-phenyl-A^P-^^- trifluoroacetamido)benzyl)sulfonyl)pentanamide

[00456] Prepared according to general method III from 5-i (71 mg, quant.). Ή NMR (400 MHz, MeOD) δ 7.83 (s, IH), 7.59 (dd, J = 8.2, 1.1 Hz, IH), 7.47 - 7.36 (m, 3H), 7.36 - 7.23 (m, 4H), 4.73 (s, 2H), 3.62 - 3.48 (m, IH), 2.93 (ddd, J= 21.9, 14.2, 7.1 Hz, 2H), 2.59 (dd, J = 13.5, 7.0 Hz, IH), 2.08 (dt, J = 14.1, 6.9 Hz, IH), 1.75 (dt, J= 14.5, 6.3 Hz, IH), 1.13 (d, J = 7.1 Hz, 3H). Mass calculated for (Cz^FsNsC S+H^ 472.14, found 472.0. Intermediate 5-iii: teri-butyl ((2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-(((2i?,4S)-4-methyl- 5-oxo-l-phenyl-5-(((3-(2,2,2-trifluoroacetamido)phenyl)methyl)sulfonamido)pentan-2- yl)carbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3-methyl-l-oxopentan-2-yl)carbamate

[00457] Prepared according to general method IV from intermediates 2 and 5-ii (82 mg, quant.). ¾ NMR (400 MHz, CDCI3) δ 10.55 (s, IH), 8.75 (s, IH), 7.99 (s, IH), 7.84 (d, J = 8.8 Hz, IH), 7.54 (s, IH), 7.42 (t, J= 7.9 Hz, IH), 7.34 - 7.16 (m, 6H), 7.08 (d, J= 6.6 Hz, 2H), 5.15 (d, J= 9.3 Hz, 1H), 4.83 (d, J = 14.2 Hz, 1H), 4.65 (d, J = 14.3 Hz, 1H), 4.53 - 4.41 (m, 1H), 4.24 (d, J = 8.8 Hz, 1H), 4.20 - 4.06 (m, 1H), 3.58 - 3.46 (m, 2H), 3.02 (s, 2H), 2.87 - 2.81 (m, 1H), 2.80 (s, 3H), 2.46 - 2.29 (m, 1H), 2.09 - 1.57 (m, 7H), 1.41 (s, 9H), 1.25 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 6.9 Hz, 3H), 0.98 (dd, J = 8.8, 6.9 Hz, 6H), 0.88 (dd, J = 15.6, 7.4 Hz, 6H). Mass calculated for (C₄₅H₆₃F₃N609S2+H)⁺ 953.41, found 953.8.

Compound 37: 2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((2i?,4S)-4-methyl-5-oxo-l-phenyl-5- (((3-(2,2,2-trifluoroacetamido)phenyl)methyl)sulfonamido)pentan-2-yl)thiazole-4- carboxamide

[00458] Prepared according to general methods III and V from 5-iii (42 mg, 54% over 2 steps). Ή NMR (400 MHz, MeOD) δ 8.13 (s, 1H), 7.77 - 7.66 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.33 - 7.13 (m, 6H), 4.80 - 4.56 (m, 3H), 4.42 (d, J= 9.0 Hz, 1H), 4.38 - 4.28 (m, 1H), 3.77 (dt, J= 13.2, 6.5 Hz, 2H), 3.54 (dd, J= 14.7, 7.4 Hz, 2H), 3.28 (dd, J= 14.8, 7.4 Hz, 3H), 3.19 (s, 3H), 2.95 (d, J = 6.7 Hz, 2H), 2.59 (s, 3H), 2.17 - 1.53 (m, 14H), 1.25 (t, J= 7.0 Hz, 3H), 1.16 (d, J= 7.0 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 1.04 - 0.95 (m, 6H), 0.89 (d, J = 6.4 Hz, 3H). Mass calculated for (C47H₆6F₃N70₈S2+H)⁺ 978.44, found 978.6.

Compound 38: yY-((2i?,4S)-5-(((3-aminophenyl)methyl)sulfonamido)-4-methyl-5-oxo-l- phenylpentan-2-yl)-2-((li?,3i?)-3-((2S,3S)-iV,3-dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4-carboxamide

[00459] Prepared according to general method VI from compound 37 (36 mg, quant.). ¾ NMR (400 MHz, Methanol-^) δ 8.10 (s, 1H), 7.71 - 7.66 (m, 2H), 7.37 (t, J = 8.0 Hz, 1H), 7.29 - 7.15 (m, 6H), 4.79 - 4.54 (m, 4H), 4.40 (d, J = 10.2 Hz, 1H), 4.33 - 4.28 (m, 1H), 3.51 (q, J = 6.1 Hz, 2H), 3.30 - 3.20 (m, 2H), 3.16 (s, 3H), 2.92 (d, J = 6.8 Hz, 2H), 2.80 - 2.68 (m, 1H), 2.70 - 2.41 (m, 2H), 2.57 (s, 3H), 2.26 - 1.46 (m, 13H), 1.22 (t, J= 6.9 Hz, 3H), 1.13 (d, J= 7.1 Hz, 3H), 1.04 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H). Mass calculated for (C₄5H67N70₇S2+H)⁺ 882.45, found 882.6.

Compound 39: 2-((li?,3i?)-3-((2S,3S)-iV,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-yV-((2i?,4S)-5-(((3-((14S,17S)-l-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa- 13,16-diazaoctadecan-18-amido)phenyl)methyl)sulfonamido)-4-methyl-5-oxo-l- phenylpentan-2-yl)thiazole-4-carboxamide

[00460] Prepared according to general methods VIII, III and IX from compound 38 (1.6 mg, 6%). Ή NMR (400 MHz, MeOD) δ 8.15 (s, lH), 7.68 (d, J = 8.4 Hz, 2H), 7.32 - 7.14 (m, 7H), 6.86 (s, 2H), 4.79 (d, J = 7.8 Hz, 1H), 4.70 (d, J = 13.6 Hz, 1H), 4.63 - 4.53 (m, 2H), 4.42 (d, J = 11.6 Hz, 1H), 4.26 (d, J = 6.9 Hz, 2H), 3.79 (t, J = 6.7 Hz, 3H), 3.72 (t, J = 5.4 Hz, 3H), 3.69 - 3.56 (m, 14H), 3.54 (d, J = 6.8 Hz, 2H), 3.25 - 3.12 (m, 7H), 2.96 (d, J = 6.4 Hz, 2H), 2.66 - 2.41 (m, 7H), 2.27 - 1.49 (m, 14H), 1.25 (t, J = 7.0 Hz, 3H), 1.15 (d, J = 7.1 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.02 (t, J = 6.2 Hz, 9H), 0.96 (d, J = 7.6 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H). Mass calculated for

1421.71, found 1422.1.

[00461] EXAMPLES 2.6 TO 2.9 describe the preparation of certain compounds of general Formula I and drug-linkers comprising these compounds. The synthetic scheme is provided as Scheme 3 in FIG. 3. EXAMPLE 2.6

Intermediate 6-i: tert-Butyi ((2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-(((4-(2,2,2- trifluoroacetamido)phenyl)sulfonyl)carbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3- methyl-l-oxopentan-2-yl)carbamate

[00462] Prepared according to general method XI from intermediate 2 and 2,2,2-trifluoro-N-(4- sulfamoylphenyl)acetamide (46 mg, 82%). 'HNMR (400 MHz, MeOD) δ 8.32 (s, 1H), 8.17-8.11 (m, 2H), 7.94 - 7.90 (m, 2H), 4.42 (t,J= 9.9 Hz, 2H), 3.56 (q,J= 7.0 Hz, 2H), 3.15 (s, 3H), 2.17 - 1.94 (m, 3H), 1.85 (s, 3H), 1.66 (ddt, J= 15.1, 7.6, 3.8 Hz, 1H), 1.44 (s, 9H), 1.24 (t, J= 7.0 Hz, 3H), 1.00 (dd, J = 6.7, 4.9 Hz, 6H), 0.95 (t, J = 7.4 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H). Mass calculated for

750.3, found 750.6.

Compound 40: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-(2,2,2- trifluoroacetamido)phenyl)sulfonyl)thiazole-4-carboxamide

[00463] Prepared according to general methods III and V from intermediate 6-i (42 mg, 89%). ¾ NMR(400 MHz, MeOD) δ 8.13 (s, 1H), 8.05 - 7.98 (m, 2H), 7.79 (d,J= 8.8 Hz, 2H), 4.69 (d,J = 7.7 Hz, 1H), 4.48 (s, 2H), 3.75 (p,J= 6.6 Hz, 1H), 3.48 (s, 4H), 3.23 (s, 3H), 2.98 (s, 2H), 2.13 (d, J= 13.7 Hz, 2H), 1.94 (t,J= 15.2 Hz, 5H), 1.81 (d,J= 13.5 Hz, 2H), 1.63 (d,J= 12.6 Hz, 2H), 1.44- 1.34 (m, 3H), 1.21 (q,J=7.1Hz, 3H), 1.06 (d,J=6.8Hz, 3H), 1.00- 0.86 (m, 8H). Mass calculated for

775.3, found 775.7. Compound 41 : 7V-((4-Aminophenyl)sulfonyl)-2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l- methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carboxamide

[00464] Prepared according to general method VI from compound 40 (25 mg, 50%). ^l NMR (400 MHz, MeOD) δ 8.25 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 6.73 - 6.65 (m, 2H), 4.73 (d, J = 7.4 Hz, 1H), 4.47 (d, J = 10.3 Hz, 1H), 3.87 (dd, J = 9.8, 7.2 Hz, 1H), 3.72 (s, 1H), 3.59 - 3.40 (m, 4H), 3.21 (s, 3H), 3.00 (s, 1H), 2.75 (d, J = 4.0 Hz, 3H), 2.34 - 2.07 (m, 2H), 2.07 - 1.88 (m, 5H), 1.81 (d, J = 12.3 Hz, 2H), 1.65 (s, 2H), 1.23 (t, J = 7.0 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 0.97 (dd, J= 8.5, 6.8 Hz, 6H), 0.89 (d, J= 6.5 Hz, 3H). Mass calculated for ^HsoNeOeSz+H^ 679.3, found 679.4.

Intermediate 6-ii: tert-Butyl ((S)-l-(((S)-l-((4-(7V-(2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)- l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carbonyl)sulfamoyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methyl-l-oxobutan- 2-yl) carbamate

[00465] Prepared according to general method VIII from compound 41 (33 mg, 75%). Mass calculated for (C₄₈H₇₈NioOi i S2+H)⁺ 1035.5, found 1035.9.

Compound 42: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-((14S,17S)-l-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)phenyl)sulfonyl)thiazole-4-carboxamide

[00466] Prepared according to general methods III and IX from intermediate 6-ii (2.3 mg, 15%). Mass calculated for (C56H₈₇Ni iOi₅S2+H)⁺ 1218.6, found 1218.9.

EXAMPLE 2.7

Intermediate 7-i: tert-Butyl ((2S,3S)-l-(((LR,3i?)-l-ethoxy-4-methyl-l-(4-(((4-((2,2,2- trifluoroacetamido)methyl)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)pentan-3- yl)(methyl)amino)-3-methyl-l-oxopentan-2-yl)carbamate

[00467] Prepared according to general method XI from intermediate 2 and 2,2,2-trifluoro-N-(4- (sulfamoylmethyl)benzyl)acetamide (50 mg, quant.). Ή NMR (400 MHz, MeOD) δ 8.43 (s, 1H), 7.47 - 7.39 (m, 2H), 7.33 (dd, J = 12.5, 8.0 Hz, 2H), 4.82 (s, 2H), 4.45 (s, 2H), 4.38 (d, J = 10.9 Hz, 2H), 3.56 (q, J = 6.9 Hz, 2H), 3.12 (s, 3H), 2.16 - 1.92 (m, 3H), 1.87 (dd, J = 13.3, 3.9 Hz, 3H), 1.64 (s, lH), 1.43 (s, 9H), 1.24 (t, J = 6.9 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H), 0.93 (t, J = 7.4 Hz, 3H), 0.85 (d, J= 6.6 Hz, 3H). Mass calculated for (Cs^oFsNsOsSz+H^ 778.3, found 778.7.

Compound 43: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-((2,2,2- trifluoroacetamido)methyl)benzyl)sulfonyl)thiazole-4-carboxamide

[00468] Prepared according to general methods III and V from intermediate 7-i (48 mg, 90%). ¾ NMR (400 MHz, MeOD) δ 8.05 (s, IH), 7.42 (d, J= 8.1 Hz, 2H), 7.25 (d, J= 8.2 Hz, 2H), 4.78 (d, J = 7.4 Hz, IH), 4.62 (d, J = 2.6 Hz, 2H), 4.44 (s, 3H), 3.59 - 3.43 (m, 2H), 3.18 (s, 4H), 3.00 (s, IH), 2.26 (s, 4H), 1.98 (t, J= 11.8 Hz, 3H), 1.80 (d, J= 13.8 Hz, 3H), 1.73 - 1.52 (m, 5H), 1.37 (d, J = 43.6 Hz, 2H), 1.23 (t, J= 7.0 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.02 - 0.91 (m, 6H), 0.84 (d, J = 6.3 Hz, 3H). Mass calculated for (C₃₆H₅₃F₃N60₇S2+H)⁺ 803.3, found 803.7.

Compound 44: 7V-((4-(Aminomethyl)benzyl)sulfonyl)-2-((LR,3i?)-3-((2S,3S)-7V,3-dimethyl-2- ((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carboxamide

[00469] Prepared according to general method VI from compound 43 (17 mg, 50%). ^l NMR (400 MHz, MeOD) δ 8.08 (s, IH), 7.45 (s, 2H), 7.29 (s, 2H), 4.80 - 4.53 (m, 4H), 4.38 (d, J = 10.4 Hz, IH), 4.00 (s, 2H), 3.52 (s, 3H), 3.19 (s, 4H), 2.97 (s, 2H), 2.38 (s, 3H), 2.08 (s, IH), 1.90 (d, J = 44.1 Hz, 5H), 1.67 (dd, J = 28.9, 16.5 Hz, 4H), 1.41 (d, J = 13.7 Hz, IH), 1.21 (dt, J = 10.0, 7.1 Hz, 3H), 1.05 (d, J = 6.8 Hz, 3H), 0.95 (t, J = 7.4 Hz, 6H), 0.85 (d, J = 6.5 Hz, 3H). Mass calculated for

707.4, found 707.7.

Intermediate 7-ii: tert-Butyl ((S)-l-(((S)-l-((4-((^-(2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)- l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carbonyl)sulfamoyl)methyl)benzyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methyl-l- oxobutan-2-yl)carbamate

[00470] Prepared according to general method VIII from compound 44 (19 mg, 85%). Mass calculated for (C₅oH₈₂NioOi i S2+H)⁺ 1063.6, found 1064.0.

Compound 45: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-((4S,7S)-20-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-7-isopropyl-3,6,9-trioxo-4-(3-ureidopropyl)-12,15,18-trioxa-2,5,8- triazaicosyl)benzyl)sulfonyl)thiazole-4-carboxamide

[00471] Prepared according to general methods III and IX from intermediate 7-iiv (13 mg, 57%). Mass calculated for (C₅₈H₉iNi iOi5S2+H)⁺ 1246.6, found 1246.9.

EXAMPLE 2.8

Intermediate 8-i: tert-Butyi ((2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-(((4-(2,2,2- trifluoroacetamido)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3- methyl-l-oxopentan-2-yl)carbamate

[00472] Prepared according to general method XI from intermediate 2 and sulfonamide 2,2,2- trifluoro-N-(4-sulfamoylphenyl)acetamide (28 mg, 70%). ¾ NMR (400 MHz, MeOD) δ 8.42 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.48 - 7.40 (m, 2H), 4.81 (s, 2H), 4.47 - 4.32 (m, 2H), 3.55 (q, J = 6.9 Hz, 2H), 3.10 (s, 3H), 2.14 - 1.93 (m, 3H), 1.83 (d, J = 8.6 Hz, 3H), 1.74 - 1.56 (m, 1H), 1.43 (s, 9H), 1.24 (t, J = 7.0 Hz, 3H), 0.98 (d, J = 6.7 Hz, 6H), 0.93 (t, J = 7.5 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). Mass calculated for (CssH^FsNsO^+H^ 764.3, found 764.6.

Compound 46: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-(2,2,2- trifluoroacetamido)benzyl)sulfonyl)thiazole-4-carboxamide

[00473] Prepared according to general methods III and V from intermediate 8-i (26 mg, 93%). ¾ NMR (400 MHz, MeOD) δ 8.17 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 4.74 - 4.65 (m, 2H), 4.59 (t, J= 13.0 Hz, 2H), 4.49 (s, 2H), 3.50 (d, J = 7.1 Hz, 4H), 3.22 (s, 3H), 2.67 (s, 3H), 1.93 (ddd, J = 71.8, 42.4, 13.2 Hz, 9H), 1.70 - 1.52 (m, 3H), 1.42 - 1.36 (m, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.00 - 0.86 (m, 6H). Mass calculated for

789.3, found 789.7.

Compound 47: 7V-((4-Aminobenzyl)sulfonyl)-2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l- methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carboxamide

[00474] Prepared according to general method VI from compound 46 (6.0 mg, 30%). ^l NMR (400 MHz, MeOD) δ 8.34 (s, 1H), 7.11 (d, J = 8.3 Hz, 2H), 6.70 - 6.61 (m, 2H), 4.74 - 4.54 (m, 4H), 4.47 (d, J = 10.5 Hz, 1H), 3.55 (dd, J = 8.0, 5.5 Hz, 2H), 3.20 (s, 3H), 2.98 (s, 2H), 2.72 (s, 3H), 2.14 (d, J = 13.9 Hz, 3H), 1.94 (t, J = 17.2 Hz, 5H), 1.80 (t, J = 12.0 Hz, 2H), 1.63 (s, 3H), 1.24 (t, J = 7.0 Hz, 3H), 1.05 (dd, J = 7.0, 2.2 Hz, 3H), 1.01 - 0.84 (m, 9H). Mass calculated for (C₃₃H₅₂N₆0₆S₂+H)⁺ 693.3, found 693.3. Intermediate 8-ii: tert-Butyl ((S)-l-(((S)-l-((4-((^-(2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)- l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carbonyl)sulfamoyl)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methyl-l- oxobutan-2-yl)carbamate

[00475] Prepared according to general method VIII from compound 47 (28 mg, 46%). Mass calculated for (C₄9H₈₀Ni₀Oi i S₂+H)⁺ 1049.5, found 1049.8.

Compound 48: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((4-((14S,17S)-l-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)benzyl)sulfonyl)thiazole-4-carboxamide

[00476] Prepared according to general methods VIII, III and IX from compound 47 (3.2 mg, 10% over 3 steps). Major rotamer: ¾ NMR (400 MHz, MeOD) δ 8.07 (s, 1H), 7.58 (d, J= 8.6 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 5.7 Hz, 2H), 4.84 (d, J = 7.8 Hz, 1H), 4.62 (s, 2H), 4.54 (dd, J = 8.9, 5.1 Hz, 1H), 4.48 - 4.38 (m, 1H), 4.25 (d, J = 6.8 Hz, 1H), 3.78 - 3.52 (m, 21H), 3.27 - 3.14 (m, 5H), 3.00 - 2.92 (m, 1H), 2.63 - 2.53 (m, 3H), 2.17 - 1.96 (m, 6H), 1.86 - 1.55 (m, 1 1H), 1.26 (t, J = 7.0 Hz, 3H), 1.09 (d, J = 6.8 Hz, 3H), 1.03 - 1.00 (m, 9H), 0.97 (d, J = 7.5 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). Mass calculated for (C57H₈₉NiiOi₅S2)⁺ 1232.60, found 1232.9.

EXAMPLE 2.9

Intermediate 9-i: tert-Butyi ((2S,3S)-l-(((li?,3i?)-l-ethoxy-4-methyl-l-(4-(((3-(2,2,2- trifluoroacetamido)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)pentan-3-yl)(methyl)amino)-3- methyl-l-oxopentan-2-yl)carbamate

[00477] Prepared according to general method XI from intermediates 2 and 7 (31 mg, 62%). ¾ NMR (400 MHz, MeOD) δ 8.40 (s, 1H), 7.71 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 7.9 Hz, lH), 4.81 (s, 2H), 4.42 (t, J = 7.1 Hz, 2H), 3.57 - 3.51 (m, 2H), 3.10 (s, 3H), 2.18 - 1.90 (m, 3H), 1.83 (s, 3H), 1.64 (s, lH), 1.43 (s, 9H), 1.22 (dt, J = 13.9, 7.0 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H), 0.93 (t, J = 7.5 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). Mass calculated for (C₃₃H₄₈F₃N₅0₈S₂+H)⁺ 764.3, found 764.5.

Compound 49: 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((3-(2,2,2- trifluoroacetamido)benzyl)sulfonyl)thiazole-4-carboxamide

[00478] Prepared according to general methods III and V from intermediate 9-i (32 mg, qu ¾ NMR (400 MHz, MeOD) δ 8.16 (s, 1H), 7.71 (s, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.35 (t, J Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 4.69 (d, J = 8.0 Hz, 2H), 4.62 (s, 2H), 4.48 (s, 2H), 3.51 (q, J = 7.1 Hz, 4H), 3.23 (s, 3H), 2.70 (d, J= 17.7 Hz, 3H), 2.09 (d, J= 14.1 Hz, 2H), 2.01 - 1.71 (m, 7H), 1.64 (dd, J = 12.7, 6.8 Hz, 3H), 1.42 - 1.37 (m, 3H), 1.21 (dt, J= 8.0, 7.0 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 0.99 - 0.86 (m, 6H). Mass calculated for (CssHsiFsNeOySz+H 789.3, found 789.7. Compound 50: 7V-((3-Aminobenzyl)sulfonyl)-2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)-l- methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carboxamide

[00479] Prepared according to general method VI from compound 49 (12 mg, 50%). ¾ NMR (400 MHz, MeOD) δ 8.21 (s, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.77 (s, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 4.70 - 4.46 (m, 5H), 3.59 - 3.42 (m, 2H), 3.25 (s, 3H), 2.89 (s, 2H), 2.70 (s, 3H), 2.07 (s, 2H), 2.02 - 1.72 (m, 8H), 1.61 (d, J = 8.4 Hz, 3H), 1.25 - 1.18 (m, 3H), 1.05 (d, J = 6.9 Hz, 3H), 1.00 - 0.91 (m, 9H). Mass calculated for (C₃₃H₅₂N₆0₆S₂+H)⁺ 693.3, found 693.7.

Intermediate 9-ii: tert-Butyl ((S)-l-(((S)-l-((3-((^-(2-((li?,3i?)-3-((2S,3S)-7V,3-dimethyl-2-((i?)- l-methylpiperidine-2-carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)thiazole-4- carbonyl)sulfamoyl)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methyl-l- oxobutan-2-yl)carbamate

[00480] Prepared according to general method VIII from compound 50 (14 mg, 97%). Mass calculated for (C₄9H₈₀Ni₀Oi i S₂+H)⁺ 1049.5, found 1049.8. Compound 51 : 2-((li?,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-ethoxy-4-methylpentyl)-_JV-((3-((14S,17S)-l-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)benzyl)sulfonyl)thiazole-4-carboxamide

[00481] Prepared according to general methods III and IX from intermediate 9-ii (6.0 mg, 35%). Mass calculated for (C₅₇H₈₉Ni iOi₅S₂+H)⁺ 1232.6, found 1232.9.

[00482] Compound 52 ((l₁S",2i?)-2-(((25^,,3₁S)-l -((( l^,3i?)-l-(4-(((3-((14,S^,, 17₁S)-l-(2,5-Dioxo-2,5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12, 15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13, 16- diazaoctadecan- 18 -amido)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)- 1 -ethoxy-4-methylpentan-3 - yl)(methyl)amino)-3 -methyl- 1 -oxopentan-2-yl)carbamoyl)- 1 -methylpiperidine 1 -oxide) was isolated as a byproduct from the chromatography purification of compound 51 (2.1 mg, 12%). Mass calculated for (C₅₇H₈₉Ni iOi₆S₂+H)⁺ 1248.6, found 1248.9

[00483] EXAMPLES 2.10 AND 2.11 describe the preparation of certain compounds of general Formula I and drug-linkers comprising these compounds. The synthetic scheme is provided as Scheme 4 in FIG. 4 and employs the following general intermediates.

Intermediate 8: Methyl 2-((li?,3i?)-3-((2S,3S)-2-azido-yY,3-dimethylpentanamido)-4-methyl-l- ((triethylsilyl)oxy)pentyl)thiazole-4-carboxylate

[00484] The title compound was synthesized according to the procedures in J. Org. Chem. 2008, 73, 4362-4369 and J. Am. Chem. Soc. 2006, 128, 16018-16019 in comparable yield. Mass calculated for (CzAsNsC SSi+H^ 526.3, found 526.1. Intermediate 9: 2-((li?,3i?)-3-((2S,3S)-2-Azido-7V,3-dimethylpentanamido)-l-hydroxy-4- methylpentyl)thiazole-4-carboxylic acid

[00485] To a stirring solution of intermediate 8 (212 mg, 0.40 mmol) in 1,4-dioxane (4 mL) was added a 0.5M aqueous solution of LiOH (4 mL, 2.0 mmol). After stirring for 64h, the reaction mixture was concentrated in vacuo. The aqueous residue was acidified to pH 3-4 with 1M citric acid (1.5 mL) and extracted with EtOAc (3x). The combined organics was washed successively with H2O (2x) and brine (lx), dried (MgS04), filtered and concentrated in vacuo to yield the title compound (159 mg, 0.40 mmol, 99%) as a white solid. Ή NMR (400 MHz, CDCI3) δ 8.27 (s, 1H), 5.00 (br s, 1H), 4.68 (dd, J= 11.2, 2.5 Hz, 1H), 4.45 (t, 1H), 3.65 (d, J = 9.8 Hz, lH), 2.99 (s, 3H), 2.48 - 2.29 (m, 1H), 2.25 - 2.13 (m, 2H), 2.10 - 1.95 (m, 2H), 1.95 - 1.75 (m, 2H), 1.38 - 1.23 (m, 2H), 1.09 - 0.91 (m, 9H). Mass calculated for (Ci₇H₂7N₅04S+H)⁺ 398.2, found 398.0.

Intermediate 10: 2-((li?,3i?)-l-Acetoxy-3-((2S,3S)-2-azido-yY,3-dimethylpentanamido)-4- methylpentyl)thiazole-4-carboxylic acid

[00486] Prepared according to general method XII from acid intermediate 9 (0.20 g, 54%). ¾ NMR (400 MHz, MeOD) δ 8.15 (s, 1H), 5.73 (dd, J = 11.4, 2.6 Hz, 1H), 4.52 (t, J = 10.9 Hz, 1H), 3.87 (d, J = 9.0 Hz, 1H), 3.03 (s, 3H), 2.39 (ddd, J = 14.7, 11.3, 3.1 Hz, 1H), 2.31 - 2.19 (m, 1H), 2.17 (s, 3H), 2.04 (tdd, J= 9.1, 6.3, 3.0 Hz, 1H), 1.85 (dt, J= 10.3, 6.6 Hz, 1H), 1.76 (dtd, J= 15.1, 7.6, 4.5 Hz, 1H), 1.29 (ddt, J= 16.4, 14.3, 7.2 Hz, 1H), 1.06 (d, J= 6.5 Hz, 3H), 0.99 (t, J= 7.4 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H). Mass calculated for

440.2, found 440.1.

EXAMPLE 2.10 Intermediate 10-i: tert-Butyi (4-sulfamoylphenyl)carbamate

[00487] Prepared according to general method XIII from 4 -aminobenzene sulfonamide (2.72 g, quant.). NMR analysis shows the solid to be a 10: 1 mixture of carbamate to aniline. Ή NMR (400 MHz, DMSO-t/e) δ 9.73 (s, 1H), 7.74 - 7.67 (m, 2H), 7.63 - 7.57 (m, 2H), 7.18 (s, 2H), 1.49 (s, 9H). Mass calculated for (G ₁H₁₆N₂O₄S-H)- 271.1, found 271.6.

Intermediate 10-ii: (li?,3i?)-3-((2S,3S)-2-Azido-7V,3-dimethylpentanamido)-l-(4-(((4-((tert- butoxycarbonyl)amino)phenyl)sulfonyl)carbamoyl)thiazol-2-yl)-4-methylpentyl acetate

[00488] Prepared according to general method XI from acid intermediates 10 andlO-i (39 mg, 50%). ¾ NMR (400 MHz, MeOD) δ 8.20 (s, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 5.71 (dd, J = 11.0, 2.8 Hz, 1H), 4.56 (s, 1H), 4.48 (d, J = 11.0 Hz, 1H), 3.85 (d, J = 9.0 Hz, 1H), 3.01 (s, 3H), 2.43 - 2.22 (m, 3H), 2.16 (d, J = 6.3 Hz, 3H), 2.13 - 1.93 (m, 2H), 1.54 (s, 9H), 1.06 (d, J = 6.5 Hz, 3H), 0.99 (t, J= 7.5 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.90 (d, J= 6.6 Hz, 3H). Mass calculated for (C₃oH₄₃N₇0₈S2+H)⁺ 694.3, found 694.3.

Compound 53: (LR,3i?)-l-(4-(((4-((teri-

Butoxycarbonyl)amino)phenyl)sulfonyl)carbamoyl)thiazol-2-yl)-3-((2S,3S)-_JV,3-dimethyl-2- ((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl acetate

[00489] Prepared according to general methods XIV and V from intermediate 10-ii (20 mg, 46%). ¾ NMR (400 MHz, MeOD) δ 8.13 (s, 1H), 7.96 - 7.88 (m, 2H), 7.54 (d, J = 8.6 Hz, 2H), 5.77 (d, J = 11.4 Hz, 1H), 4.74 (d, J = 8.4 Hz, 1H), 4.57 (s, 1H), 3.75 (p, J = 6.6 Hz, 1H), 3.25 (q, J = 7.4 Hz, 1H), 3.18 (s, 3H), 2.72 (s, 3H), 2.14 (s, 3H), 1.89 (t, J = 8.5 Hz, 4H), 1.75 (s, 5H), 1.54 (s, 9H), 1.42 - 1.35 (m, 3H), 1.31 (s, 2H), 1.26 - 1.13 (m, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.98 - 0.86 (m, 6H). Mass calculated for (CsyHseNeOgSz+H 793.4, found 793.4.

Compound 54: (LR,3i?)-l-(4-(((4-Aminophenyl)sulfonyl)carbamoyl)thiazol-2-yl)-3-((2S,3S)-

_JV,3-dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl acetate

[00490] Prepared according to general method III from compound 53 (15 mg, 68%). ¾ NMR (400 MHz, MeOD) δ 8.19 (s, 1H), 7.73 (d, J = 8.6 Hz, 2H), 6.73 - 6.65 (m, 2H), 5.78 (s, 1H), 4.72 (d, J= 8.3 Hz, 1H), 4.57 (s, 1H), 3.80 - 3.69 (m, 1H), 3.24 (t, J= 7.4 Hz, 1H), 3.16 (s, 3H), 2.15 (s, 3H), 2.08 - 1.78 (m, 4H), 1.78 - 1.51 (m, 5H), 1.39 (d, J = 6.5 Hz, 3H), 1.31 (s, 2H), 1.26 - 1.11 (m, 2H), 1.01 (dd, J = 6.7, 2.8 Hz, 6H), 0.95 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H). Mass calculated for (C₃₂H₄₈N₆0₇S2+H)⁺ 693.3, found 693.8.

Intermediate 10-iii: (li?,3i?)-l-(4-(((4-((S)-2-((S)-2-((teri-Butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)phenyl)sulfonyl)carbamoyl)thiazol-2-yl)-3- ((2S,3S)-_JV,3-dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4- methylpentyl acetate

[00491] Prepared according to general method VIII from compound 54 (19 mg, quant.). Mass calculated for (C₄₈H₇6NioOi2S2+H)⁺ 1049.5, found 1049.8.

Compound 55: (LR,3i?)-3-((2S,3S)-yV,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-(4-(((4-((14S,17S)-l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14- isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16-diazaoctadecan-18- amido)phenyl)sulfonyl)carbamoyl)thiazol-2-yl)-4-methylpentyl acetate

[00492] Prepared according to general methods III and IX from intermediate 10-iii (6.1 mg, 19% over 4 steps from 376548-iii). Mass calculated for (C₅6H₈₅Ni iOi6S2+H)⁺ 1232.6, found 1232.9.

EXAMPLE 2.11 Intermediate 11-i: tert-Butyl (3-(sulfamoylmethyl)phenyl)carbamate

[00493] Prepared according to general method XIII from (3-aminophenyl)methanesulfonamide (0.23 g, quant.). ¾ NMR (400 MHz, DMSO-t/₆) δ 9.37 (s, 1H), 7.53 (t, J= 1.9 Hz, 1H), 7.38 (dd, J = 8.2, 1.9 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 6.96 (dt, J = 7.6, 1.3 Hz, 1H), 6.81 (s, 2H), 4.19 (s, 2H), 1.48 (s, 9H). Mass calculated for (Ci₂Hi₈N₂0₄S-H)- 285.1, found 285.1.

Intermediate 11-ii: (li?,3i?)-3-((2S,3S)-2-Azido-7V,3-dimethylpentanamido)-l-(4-(((3-((tert- butoxycarbonyl)amino)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)-4-methylpentyl acetate

[00494] Prepared according to general method XI from acid intermediates 10 and 11-i (49 mg, 62%). Ή ΝΜΡν (400 MHz, MeOD) δ 8.24 (s, lH), 7.50 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 5.71 (dd, J = 11.3, 2.6 Hz, 1H), 4.58 (d, J = 10.3 Hz, 1H), 4.50 (t,J = 10.8 Hz, 1H), 3.86 (d,J=9.0 Hz, 1H), 3.01 (s, 3H), 2.43 - 2.21 (m, 4H), 2.16 (s, 3H), 2.10-1.97 (m, 3H), 1.51 (s, 9H), 1.06 (d,J=6.5 Hz, 3H), 0.99 (t, J= 7.4 Hz, 3H), 0.96 (d,J=6.7 Hz, 3H), 0.91 (d, J= 6.6 Hz, 3H). Mass calculated for (CsAsNyOsSz+H 708.3, found 708.3.

Compound 56: (LR,3i?)-l-(4-(((3-((tert- Butoxycarbonyl)amino)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)-3-((2S,3S)-_JV,3-dimethyl-2- ((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl acetate

[00495] Prepared according to general methods XIV and V from intermediate 11-ii (36 mg, 65%). Ή NMR (400 MHz, MeOD) δ 8.12 (s, 1H), 7.44 (s, 1H), 7.39 (d, J= 7.7 Hz, 1H), 7.20 (t, J= 7.9 Hz, 1H), 7.06 (d,J= 7.5 Hz, 1H), 5.77 (d,J= 11.3 Hz, 1H), 4.75 (d,J= 8.4 Hz, 1H), 4.57 (d,J = 5.2 Hz, 3H), 3.75 (p,J=6.6Hz, 1H), 3.25 (q,J=7.3Hz, 1H), 3.18 (s, 3H), 2.61 (s, 3H), 2.15 (d,J = 2.1 Hz, 3H), 1.97- 1.77 (m, 4H), 1.66 (d, J =47.5 Hz, 5H), 1.51 (d,J=2.3 Hz, 9H), 1.39 (dd, J = 7.0, 1.7 Hz, 3H), 1.31 (s, 2H), 1.19 (s, 2H), 1.05 - 0.97 (m, 3H), 0.97 - 0.87 (m, 6H). Mass calculated for (C3₈H5₈N₆0₉S2+H)⁺ 807.4, found 807.5. Compound 57: (LR,3i?)-l-(4-(((3-Aminobenzyl)sulfonyl)carbamoyl)thiazol-2-yl)-3-((2S,3S)- _JV,3-dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl acetate

[00496] Prepared according to general method III from compound 56 (29 mg, 71%). ¾ NMR (400 MHz, MeOD) δ 8.16 (s, 1H), 7.03 (t,J= 7.8 Hz, 1H), 6.80 (t,J= 1.9 Hz, 1H), 6.74 (d,J= 7.7 Hz, 1H), 6.70 - 6.62 (m, 1H), 5.78 (d, J= 11.0 Hz, 1H), 4.73 (d, J= 8.3 Hz, 1H), 4.45 (t, J= 9.6 Hz, 3H), 3.75 (p, J = 6.7 Hz, 1H), 3.25 (q, J = 7.4 Hz, 1H), 3.16 (s, 3H), 2.16 (s, 3H), 1.92 (d, J = 7.9 Hz, 2H), 1.81 (s, 2H), 1.71 (d, J= 13.1 Hz, 3H), 1.65 - 1.49 (m, 3H), 1.39 (dd, J = 7.0, 1.9 Hz, 3H), 1.36 - 1.27 (m, 3H), 1.01 (dt, J = 7.0, 3.5 Hz, 6H), 0.98 - 0.85 (m, 6H). Mass calculated for (C₃₃H₅oN₆0₇S₂+H)⁺ 707.3, found 707.3. Intermediate 11-iii: (li?,3i?)-l-(4-(((3-((S)-2-((S)-2-((teri-Butoxycarbonyl)amino)-3- methylbutanamido)-5-ureidopentanamido)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)-3- ((2S,3S)-_JV,3-dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4- methylpentyl acetate

[00497] Prepared according to general method VIII from compound 57 (31 mg, quant.). Mass calculated for (C₃₃H₅oN₆0₇S₂+H)⁺ 1063.5, found 1063.7.

Compound 58: (LR,3i?)-3-((2S,3S)-yV,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-l-(4-(((3-((14S,17S)-l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14- isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16-diazaoctadecan-18- amido)benzyl)sulfonyl)carbamoyl)thiazol-2-yl)-4-methylpentyl acetate

[00498] Prepared according to general methods III, IX, and X from intermediate 11-iii (10 mg, 25% over 4 steps from 376551-iii). Mass calculated for (C₅7H₈₇NiiOi6S2+H)⁺ 1246.6, found 1246.9.

[00499] EXAMPLE 2.12 describes the preparation of a compound of general Formula I and a drug-linker comprising the compound. The synthetic scheme for these compounds is provided as Scheme 5 in FIG. 5.

Intermediate 12-i: Ethyl 2-((6S,9i?,lli?)-6-sec-Butyl-9-isopropyl-2,2,8-trimethyl-4,7,13-trioxo- 3,12-dioxa-5,8-diazatetradecan-ll-yl)thiazole-4-carboxylate

[00500] Prepared according to general procedure XII from ethyl 2-((li?,3i?)-3-((2,S^,,3₁S)-2-((tert- butoxycarbonyl)amino)-N,3-dimethylpentanamido)-l-hydroxy-4-methylpentyl)thiazole-4- carboxylate (35.5 mg, 89%). ¾ ΝΜΡν (400 MHz, Chloroform- ) δ 8.13 (s, 1H), 5.73 (dd, J = 11.5, 2.6 Hz, 1H), 5.16 (d, J = 9.9 Hz, 1H), 4.55 (t, J = 11.2 Hz, 1H), 4.49 - 4.36 (m, 1H), 4.43 (q, J = 7.2 Hz, 2H), 3.02 (s, 3H), 2.43 - 2.32 (m, 1H), 2.27 - 2.15 (m, 1H), 2.19 (s, 3H), 1.82 - 1.54 (m, 3H), 1.49 - 1.36 (m, 12H), 1.20 - 1.04 (m, 1H), 1.02 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.6 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H), 0.83 (d, J = 6.6 Hz, 3H). Mass calculated for (C26H₄3N30₇S+Na)⁺ 564.29, found 564.7.

Intermediate 12-ii: 2-((6S,9i?,lli?)-6-sec-Butyl-9-isopropyl-2,2,8-trimethyl-4,7,13-trioxo-3,12- dioxa-5,8-diazatetradecan-ll-yl)thiazole-4-carboxylic acid

[00501] Intermediate 12-i (35.5 mg, 0.066 mmol) was taken up in 1,2-dichloroethane (3 mL), and trimethyltin hydroxide (59 mg, 0.33 mmol, 5 eq) was added and the mixture heated to 80°C for 4 h. Upon cooling, the mixture was concentrated and purified by flash chromatography 10-100% (2% AcOH/EtOAc)/Hex. The residue was taken up in EtOAc, washed three times with dilute NaHSC>4, once with NaCl(_sat), dried over Na2S04(_S), filtered and concentrated to give 24.9 mg (74%). ¾ NMR (400 MHz, Chloroform-t δ 8.24 (s, 1H), 5.75 (dd, J = 11.0, 3.1 Hz, 1H), 5.39 (d, J = 9.8 Hz, 1H), 4.59 (t, J = 10.6 Hz, 1H), 4.45 (dd, J = 9.8, 7.6 Hz, lH), 3.05 (s, 3H), 2.38 (ddd, J = 14.5, 11.0, 3.3 Hz, 1H), 2.27 - 2.12 (m, 1H), 2.19 (s, 3H), 1.88 - 1.69 (m, 2H), 1.71 - 1.57 (m, 1H), 1.41 (s, 9H), 1.23 - 1.09 (m, 1H), 1.03 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H), 0.84 (d, J= 6.6 Hz, 3H). Mass calculated for (C_Z^N_SO_TS+H^ 514.26, found 514.7.

Intermediate 12-iii: (li?,3i?)-3-((2S,3S)-2-(teri-Butoxycarbonylamino)-7V,3- dimethylpentanamido)-4-methyl-l-(4-(4-nitrobenzylsulfonylcarbamoyl)thiazol-2-yl)pentyl acetate

[00502] Prepared according to general method XI from intermediate 12-ii and 4- nitrobenzylsulfonamide (26.9 mg, 78%). Ή NMR (400 MHz, Chloroform-d) δ 9.57 (s, 1H), 8.32 (s, 1H), 8.26 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 5.68 (dd, J = 10.8, 3.4 Hz, 1H), 5.17 (d, J= 9.8 Hz, 1H), 4.92 (s, 2H), 4.58 (t, J = 10.9 Hz, 1H), 4.44 (dd, J = 9.3, 7.7 Hz, 1H), 3.01 (s, 3H), 2.27 (dd, J = 17.9, 7.0 Hz, 2H), 2.18 (s, 3H), 2.16 - 1.94 (m, 3H), 1.43 (s, 9H), 1.21 - 1.05 (m, 1H), 1.02 (d, J= 6.5 Hz, 3H), 0.95 (d, J= 6.7 Hz, 3H), 0.90 (t, J= 7.3 Hz, 3H), 0.83 (d, J= 6.6 Hz, 3H). Mass calculated for (C3iH4₅N₅OioS2+Na)⁺ 734.26, found 734.7.

Compound 59: (LR,3i?)-3-((2S,3S)-7V,3-Dimethyl-2-((i?)-l-methylpiperidine-2- carboxamido)pentanamido)-4-methyl-l-(4-(4-nitrobenzylsulfonylcarbamoyl)thiazol-2- yl)pentyl acetate

[00503] Prepared according to general methods III and V from intermediate 12-iii (21 mg, 77%). Ή NMR (400 MHz, Methanol-^) δ 8.19 (d, J= 8.8 Hz, 2H), 8.12 (s, lH), 7.68 (d, J= 8.7 Hz, 2H), 5.76 (d, J = 11.3 Hz, 1H), 4.77 (s, 2H), 4.74 (d, J = 8.0 Hz, 1H), 3.51 - 3.38 (m, 1H), 3.17 (s, 3H), 3.1 1 - 2.95 (m, 2H), 2.76 (s, 3H), 2.36 - 2.21 (m, 1H), 2.16 (s, 3H), 1.98 - 1.50 (m, 10H), 1.27 - 1.09 (m, 2H), 1.06 - 0.98 (m, 6H), 0.95 (t, J = 7.4 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H). Mass calculated for

737.30, found 737.7.

Compound 60: (li?,3i?)-l-(4-(4-Aminobenzylsulfonylcarbamoyl)thiazol-2-yl)-3-((2S,3S)-7V,3- dimethyl-2-((i?)-l-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl acetate

[00504] Compound 59 (20 mg, 0.027 mmol) was treated with sufficient EtOAc to fully dissolve it (~5 mL), then the flask was purged with N2. 10% Pt/C (5.3 mg, 0.003 mmol, 0.10 eq) was added, and the flask was purged with ¾ with a balloon. After 22 h, the mixture was filtered through celite, washing with EtOAc and MeOH and concentrated to 12.1 mg (63%) of clear film. Ή NMR (400 MHz, Methanol-^) δ 8.10 (s, 1H), 7.14 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 8.0 Hz, 2H), 5.76 (d, J = 11.3 Hz, 1H), 4.75 (d, J= 8.3 Hz, 1H), 4.60 - 4.43 (m, 3H), 3.63 - 3.55 (m, 1H), 3.17 (s, 3H), 3.25 - 3.10 (m, 2H), 2.55 (s, 3H), 2.54 - 2.48 (m, 1H), 2.31 - 2.05 (m, 2H), 2.16 (s, 3H), 2.04 - 1.52 (m, 7H), 1.42 - 1.30 (m, 1H), 1.24 - 1.14 (m, 1H), 1.05 - 0.98 (m, 6H), 0.95 (t, J= 7.4 Hz, 3H), 0.92 - 0.83 (m, 3H). Mass calculated for (C33H5oN₆0₇S2+H)⁺ 707.33, found 707.8. Compound 61 : (lR,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-l-methylpiperidine-2- carboxamido)pentanamido)-l-(4-(4-((14S,17S)-l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-14- isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecanamido)benzylsulfonylcarbamoyl)thiazol-2-yl)-4-methylpentyl acetate

[00505] Prepared according to general methods VIII, III, and IX (4.5 mg, 20%) from compound 60. Ή NMR (400 MHz, Methanol-^) δ 8.15 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 6.83 (s, 2H), 5.77 (d, J = 1 1.1 Hz, 1H), 4.74 (d, J = 8.3 Hz, 1H), 4.66 - 4.44 (m, 3H), 4.21 (d, J = 6.9 Hz, 1H), 3.79 - 3.72 (m, 3H), 3.70 (t, J = 5.4 Hz, 2H), 3.63 (t, J = 5.2 Hz, 2H), 3.58 (d, J = 1.5 Hz, 8H), 3.16 (s, 3H), 3.24 - 3.08 (m, 3H), 2.82 - 2.68 (m, 1H), 2.61 (s, 3H), 2.57 (t, J = 6.1 Hz, 2H), 2.38 - 2.23 (m, 1H), 2.16 (s, 3H), 1.99 (s, 17H), 1.05 - 0.97 (m, 12H), 0.94 (t, J = 7.5 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H). Mass calculated for (C₅₇H₈₇Ni iOi₆S2+H)⁺ 1246.58, found 1247.1.

EXAMPLE 3: SYNTHESIS OF COMPOUNDS OF GENERAL FORMULA I AND CONJUGATES THEREOF

[00506] Compounds 1-9, 10 and 12 referred to in this Example were prepared as described in Example 1. Intermediate 1: Methyl 2-((lR,3R)-3-((2S,3S)-2-addo-N,3-dimethylpentanamido)-4-methyl-l- (triethylsilyloxy)pentyl)thiazole-4-carboxylate

[00507] The title compound was synthesized from compound 7 (see Example 1) and azido-L-isoleucine according to the procedures in J. Org. Chem. 2008, 73, 4362-4369 and J. Am. Chem. Soc. 2006, 128, 16018-16019 in comparable yield. Intermediate 2: 2-((lR,3R)-3-((2S,3S)-2-azido-N,3-dimethylpentanamido)-l-hydroxy-4- methylpentyl) thiazole-4-carboxylic acid

[00508] To a stirring solution of intermediate 1 (212 mg, 0.40 mmol) in 1,4-dioxane (4 mL) was added a 0.5M aqueous solution of LiOH (4 mL, 2.0 mmol). After stirring for 64h, the reaction mixture was concentrated in vacuo. The aqueous residue was acidified to pH 3-4 with \M citric acid (1.5 mL) and extracted with EtOAc (3x). The combined organics was washed successively with H₂0 (2x) and brine (lx), dried (MgS0₄), filtered and concentrated in vacuo to yield the title compound (159 mg, 0.40 mmol, 99%) as a white solid. ¾ NMR (400 MHz, CDC1₃) δ 8.27 (s, 1H), 5.00 (br s, 1H), 4.68 (dd, J = 1 1.2, 2.5 Hz, 1H), 4.45 (t, 1H), 3.65 (d, J = 9.8 Hz, 1H), 2.99 (s, 3H), 2.48 - 2.29 (m, 1H), 2.25 - 2.13 (m, 2H), 2.10 - 1.95 (m, 2H), 1.95 - 1.75 (m, 2H), 1.38 - 1.23 (m, 2H), 1.09 - 0.91 (m, 9H). m/z calc'd for Ci7H₂₇N₅0₄S = 397.2 Found [M+H]⁺ = 398.0.

Intermediate 3: 2-((lR,3R)-3-((2S,3S)-2-azido-N,3-dimethylpentanamido)-4-methyl-l- (triethylsilyloxy)pentyl)thiazole-4-carboxylic acid

[00509] To a stirring solution of intermediate 2 (159 mg, 0.40 mmol) in CH2CI2 (12 mL) was added imidazole (85 mg, 1.24 mmol). Once a solution was obtained, chlorotriethylsilane (0.21 mL, 1.25 mmol) was added. The resulting white suspension was stirred at ambient temperature for 1 h, passed through a bed of celite and concentrated in vacuo. The resulting white residue was purified by flash chromatography, eluted with 0-100% (2% AcOH/EtOAc) in hexanes, to yield the titled compound (159 mg, 78%) as a pale yellow viscous oil. Ή NMR (400 MHz, CDCI3) δ 8.21 (s, 1H), 4.96 (t, J = 5.7 Hz, 1H), 4.75-4.60 (m, 1H), 3.52 (d, J = 9.8 Hz, 1H), 2.94 (s, 3H), 2.28 - 2.04 (m, 3H), 1.90 - 1.70 (m, 2H), 1.35 - 1.20 (m, 1H), 1.08 (dd, J = 16.6, 6.6 Hz, 3H), 1.04 - 0.89 (m, 15H), 0.82 (d, J = 6.6 Hz, 3H), 0.70 (q, J = 8.0 Hz, 6H). m/z calc'd for C₂₃H₄₁N₅O₄SS1 = 511.3 Found [M+H]⁺ = 512.0. Intermediate 4: 2-((lR,3R)-3-((2S,3S)-2-azido-N,3-dimethylpentanamido)-4-methyl-l-

(triethylsilyloxy)pentyl)-N-(4-(l-(2,2,2-trifiuoroacetamido)cyclopropyl)phenylsulfonyl)M carboxamide

[00510] The title compound was prepared from intermediate 3 by following general method XI. Ή NMR (400 MHz, Methanol-^) δ 8.31 (s, 1H), 8.13 - 8.05 (m, 2H), 7.47 - 7.39 (m, 2H), 4.98 - 4.93 (m, 1H), 4.48 (d, J = 9.5 Hz, 1H), 3.88 (d, J = 8.9 Hz, 1H), 3.53 - 3.45 (m, 1H), 3.00 (s, 3H), 2.29 - 2.07 (m, 2H), 1.92 - 1.58 (m, 4H), 1.47 - 1.41 (m, 1H), 1.25 - 1.11 (m, 1H), 1.04 (d, J = 6.5 Hz, 4H), 1.01 - 0.93 (m, 12H), 0.91 (td, J = 6.3, 2.1 Hz, 6H), 0.77 - 0.64 (m, 6H). m/z calc'd for C34H50N7O6S2S1F3 = 801.3 Found [M+H]⁺ = 802.0. Intermediate 5: 2-((3S,6R,8R)-3-sec-butyl-10,10-diethyl-6-isopropyl-5-methyl-l-((R)-l- methylpiperidin-2-yl)-l,4-dioxo-9-oxa-2,5-diaza-10-siladodecan-8-yl)-N-(4-(l-(2,2,2- trifluoroacetamido)cyclopropyl)phenylsulfonyl)thiazole-4-carboxamide

[00511] To a stirring mixture of intermediate 4 (126 mg, 0.15 mmol) in EtOAc (1 mL) was added a solution of intermediate 18 (Mep-OPFP) (139 mg, 0.45 mmol) in EtOAc (1.5 mL) followed by 10 wt % Pd/C (67 mg, 0.063 mmol). The resulting black suspension was purged with ¾ and stirred at ambient temperature and pressure for 15 h and then at 50 °C for 24 h. The mixture was cooled to ambient temperature, passed through a bed of celite and concentrated in vacuo to afford a pale yellow oil. The crude material was carried to the next synthetic step without further purification. m/z calc'd for C41H63N6O7S2S1F3 = 900.4 Found [M+H]⁺ = 901.3.

Compound 68: N-(4-(l-aminocyclopropyl)phenylsulfonyl)-2-((lR,3R)-3-((2S,3S)-N,3-dimethy 2-((R)-l-methylpiperidine-2-carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)M

carboxamide

[00512] The crude yellow oil of intermediate 5 was dissolved in 1,4-dioxane (1.4 mL). To the stirring solution was added a 0.5 M aqueous solution of LiOH (1.4 mL, 0.70 mmol). The resulting suspension was stirred at ambient temperature for 2 h and then at 40 °C for 16 h. The mixture was concentrated in vacuo, diluted with a 0.25M phosphate buffer solution (pH 6.2) and saturated NH4CI, and extracted with CH2CI2 (5x). The combined organics was washed with brine (lx), dried (MgS04), filtered and concentrated in vacuo to afford an off-white solid. The crude solid was purified by preparative TLC, eluted with 15% (5% NFLOH/MeOH) in CH₂C1₂, to yield the title compound (21 mg, 20%) as an off-white solid. ¾ NMR (400 MHz, Methanol-^) δ 8.01 (s, 1H),

7.97 (dd, J = 8.4, 1.6 Hz, 2H), 7.47 - 7.41 (m, 2H), 4.77 (dd, J = 16.6, 8.8 Hz, 1H), 4.68 - 4.56 (m, 2H), 3.17 (d, J = 9.2 Hz, 3H), 2.98 (d, J = 11.0 Hz, 1H), 2.87 (d, J = 2.6 Hz, 1H), 2.62 (d, J = 10.6 Hz, 1H), 2.32 - 2.12 (m, 4H), 2.02 - 1.73 (m, 4H), 1.72 - 1.45 (m, 4H), 1.38 - 1.20 (m, 7.2 Hz, 2H), 1.19 - 1.05 (m, 4H), 1.05 - 0.96 (m, 6H), 0.93 (t, J = 7.4 Hz, 3H), 0.82 (t, J = 7.7 Hz, 3H). m/z calc'd for CssHsoNeOeSz = 690.3 Found [M+H]⁺ = 691.1 Compound 69: 2-((lR,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-l-methylpiperidine-2- carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)-N-((4-(l-((14S,17S)-l-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)cyclopropyl)phenyl)sulfonyl)thiazole-4-carboxamide

[00513] A solution of MT-VC-OH (37 mg, 0.067 mmol), EDC.HC1 (12 mg, 0.065 mmol) and HO At (9 mg, 0.067 mmol) in DMF/CH₂C1₂ (1 :8, v/v, 1 mL) was stirred for 3 min. This solution was then added to a vial containing compound 68 (14 mg, 0.021 mmol). To the resulting light brown/orange solution was added CuCh (10 mg, 0.078 mmol). The resulting olive green paste was stirred at ambient temperature for 40 min, passed through a bed of celite and rinsed with ample amounts of CH2CI2. The filtrate was concentrated in vacuo and diluted with DMSO (1.7 mL) and purified by preparative HPLC (25-45% MeCN/H₂0 with 0.1% formic acid) to yield the title compound (3.8 mg, 0.003 mmol, 15%) as a white solid, m/z calc'd for C57H87N11O15S2 1229.6 Found [M+H]⁺ = 1230.6, [(M+2H)/2]²⁺ = 615.9. Intermediate 6: 2-((lR,3R)-3-((2S,3S)-2-azido-N,3-dimethylpentanamido)-4-methyl-l- (triethylsilyloxy)pentyl)-N-(4-(2,2,2-trifluoroacetamido)benzylsulfonyl)thia^

[00514] The title compound was prepared from intermediate 3 by following general procedure XI. ¾ NMR (400 MHz, Methanol-^) δ 8.44 (s, 1H), 7.66 (dd, J = 8.4, 6.2 Hz, 2H), 7.45 (dd, J = 8.5, 6.5 Hz, 2H), 4.94 - 4.90 (s, 1H), 4.83 (s, 2H), 4.53 - 4.34 (m, 2H), 3.85 (d, J = 8.9 Hz, 1H), 2.95 (s, 3H), 2.16 - 2.02 (m, 2H), 1.91 - 1.69 (m, 2H), 1.49 - 1.29 (m, 1H), 1.23 - 1.10 (m, 1H), 1.07 - 0.90 (m, 17H), 0.88 (dd, J = 6.5, 4.4 Hz, 3H), 0.77 - 0.63 (m, 6H). m/z calc'd for C32H4₈N70₆S2SiF3 = 775.3 Found [M+H]⁺ = 776.0.

Intermediate 7: ( R) -per flu oroph enyl l-methylpiperidine-2-carboxylate, Mep-OPFP

[00515] The title compound was synthesized according to the procedures in J. Am. Chem. Soc. 2006, 128, 16018-16019 in comparable yield.

Intermediate 8: 2-((3S,6R,8R)-3-sec-butyl-10,10-diethyl-6-isopropyl-5-methyl-l-((R)-l- methylpiperidin-2-yl)-l,4-dioxo-9-oxa-2,5-diaza-10-siladodecan-8-yl)-N-(4-(2,2,2- trifluoroacetamido)benzylsulfonyl)thiazole-4-carboxamide

[00516] To a stirring mixture of intermediate 6 (113 mg, 0.15 mmol) in EtOAc (1 mL) was added a solution of Mep-OPFP (131 mg, 0.42 mmol) in EtOAc (1.5 mL) followed by 10 wt % Pd/C (60 mg, 0.056 mmol). The resulting black suspension was purged with ¾ and stirred at ambient temperature and pressure for 15 h and then at 50 °C for 24 h. The mixture was cooled to ambient temperature, passed through a bed of celite and concentrated in vacuo to afford a pale yellow oil. The crude material was carried to the next synthetic step without further purification, m/z calc'd for C39H61N6O7S2S1F3 calcd m/z = 874.4 Found [M+H]⁺ = 875.2.

Compound 65: N-(4-aminobenzylsulfonyl)-2-((lR,3R)-3-((2S,3S)-N,3-dimethyl-2-((R)-l- methylpiperidine-2-carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)thiazole-4- carboxamide

[00517] Intermediate 8 was dissolved in 1,4-dioxane (1.4 mL). To the stirring solution was added a 0.5 M aqueous solution of LiOH (1.4 mL, 0.70 mmol). The resulting suspension was stirred at ambient temperature for 15 h. The mixture was concentrated in vacuo, diluted with a 0.25M phosphate buffer solution (pH 6.2) and saturated NH4CI, and extracted with CH2CI2 (5x). The combined organics was washed with brine (lx), dried (MgS04), filtered and concentrated in vacuo to afford a yellow solid. The crude solid was purified by preparative TLC, eluted with 15% (5% LOH/MeOH) in CH₂C1₂, to yield the title compound (21 mg, 22%) as an off-white solid. Ή NMR (400 MHz, Methanol-^) δ 8.01 (s, 1H), 7.20 - 7.12 (m, 2H), 6.69 - 6.62 (m, 2H), 4.79 (dd, J = 18.6, 8.8 Hz, 1H), 4.71 - 4.56 (m, 3H), 4.50 (s, 2H), 3.37 (s, 1H), 3.20 (d, J = 11.0 Hz, 3H), 2.99 (d, J = 11.9 Hz, 1H), 2.89 (d, J = 3.4 Hz, 1H), 2.69 - 2.56 (m, 1H), 2.35 - 2.15 (m, 4H), 2.07 - 1.73 (m, 4H), 1.71 - 1.47 (m, 2H), 1.41 - 1.17 (m, 2H), 1.10 (d, J = 6.5 Hz, 1H), 1.06 - 0.97 (m, 6H), 0.94 (t, J = 7.4 Hz, 3H), 0.84 (t, J = 7.5 Hz, 3H). m/z calc'd for CsAsNeOeSz = 664.3 Found [M+H]⁺ = 665.2. Compound 70: 2-((lR,3R)-3-((2S,3R)-N,3-dimethyl-2-((R)-l-methylpiperidine-2- carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)-N-((4-( ( 14R, 17R)-l-(2, 5-dioxo-2, 5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)benzyl)sulfonyl)thiazole-4-carboxamide

[00518] The title compound was prepared from compound 65 by application of general method VII. m/z calcd. for C55H85N11O15S2 = 1203.6 found [M+H]⁺ = 1204.5. Compound 71: N-( ( 4-aminophenyl)sulfonyl)-2-( ( lR,3R)-3-((2S,3R)-N,3-dimethyl-2-( (R)-l- methylpiperidine-2-carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)thiazole-4- carboxamide

[00519] The title compound was prepared from intermediate 24 by application of general method VI. ¾ NMR (400 MHz, Methanol-^) δ 8.15 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 4.81 - 4.60 (m, 2H), 4.12 - 4.03 (m, 1H), 3.23 (s, 3H), 2.82 - 2.71 (m, 1H), 2.58 (s, 3H), 2.50 - 2.15 (m, 2H), 2.01 - 1.80 (m, 5H), 1.80 - 1.68 (m, 2H), 1.68 - 1.46 (m, 3H), 1.14 - 0.77 (m, 12H). m/z calcd. for Csot^eNeOeSz = 650.3 found [M+H]⁺ = 651.8. Compound 72: 2-((lR,3R)-3-((2S,3R)-N,3-dimethyl-2-((R)-l-methylpiperidine-2- carboxamido)pentanamido)-l-hydroxy-4-methylpentyl)-N-((4-( ( 14R, 17R)-l-(2, 5-dioxo-2, 5- dihydro-lH-pyrrol-l-yl)-14-isopropyl-12,15-dioxo-17-(3-ureidopropyl)-3,6,9-trioxa-13,16- diazaoctadecan-18-amido)phenyl)sulfonyl)thiazole-4-carboxamide

[00520] The title compound was prepared from compound 71 by application of general method VII. m/z calcd. for C54H83N11O15S2 = 1189.6 found [M+H]⁺ = 1190.9. EXAMPLE 4: CYTOTOXICITY OF COMPOUNDS OF FORMULA I IN JURKAT, HCC1954, NCI-N87, SKOV-3 AND DU-145 CELLS LINES

[00521] Cell lines: Human T-cell leukemia cell line Jurkat (ATCC: TIB-152); HCC 1954 (ATCC: CRL. 2338); Human gastric carcinoma cell line NCI-N87 (ATCC: CRL. 5822); Human ovarian SKOV-3 (ATCC: HTB-77) and Human prostate DU-145 (ATCC HTB-81).

[00522] Compounds were tested on Jurkat, HCC1954, NCI-N87, SKOV-3 and DU-145 cell lines to assess their cytotoxicity. Each cell line was grown in its respective growth medium until seeding day. Cells were removed from their culture vessels and the resulting cell suspension were counted using the ViCell. Cells were then diluted in their growth medium to 25000/mL such that 100 μΕ/well = 2500 cells/well. Each cell line was seeded in the inner 60 wells of 96-well black walled TC plate, outer wells were filled with water. HCC 1954, NCI-N87, SKOV-3 and DU-145 were seeded one day prior to compound plating to allow cell adhesion to the plate. Compounds were diluted to a 5 ^χ dose-response of each compound using RPMI + 10% FBS in a deep-well 96-well plate. This "master" dilution plate was used for each cell line. 25

of the 5 ^χ dose responses were spiked into each cell line in triplicate. Compounds were then titrated 1 :3 starting at various concentrations (between 30 nM to 1000 nM). A control plate with no test article present (growth medium and cells alone) was included in sextuplicate. The plates were then incubated for 5 nights for HCC 1954, NCI-N87, SKOV-3 and DU-145 cells and for 3 nights for Jurkat cells. Cell viability was quantified using 30 μΕ/well of l x CellTiter-glo reagent. After at least 10 minutes of incubation, the luminescence was measured using the SpectraMax (500 ms integration).

[00523] The results are shown in Table 2. Compounds 65, 68 and 71 containing a free hydroxyl group showed minimal cytotoxicity when tested against HCC 1954 and Jurkat cells.

EXAMPLE 5: PREPARATION OF ANTIBODY-DRUG CONJUGATES

Preparation of Antibody-Drug Conjugates from MTvc-Toxins, General Methods

[00524] To a solution of antibody (Trastuzumab, 1 -10 mg/mL) in 25 mM sodium borate, 25 mM sodium chloride, 1 mM DTPA (pH 8.0) wa added TCEP from a freshly prepared stock (1-10 mM) in the same buffer (2.0-3.0 molar equivalents). The solution was mixed thoroughly and incubated at 37 °C for 2 h before cooling on ice. In some instances, the reduced antibody solution was further diluted with either ice-cold phosphate buffered saline containing 1 mM DTPA (final protein concentration 2.0 mg/mL) or ice-cold 25 mM sodium borate, 25 mM sodium chloride, 1 mM DTPA (pH 8.0), to obtain a solution with a final protein concentration of between 1 and 4 mg/mL. To the reduced protein solution stored on ice was added the maleimide functionalized toxin (10-12 molar equivalents) from a 10 mM DMSO stock solution. The conjugation reaction was immediately mixed thoroughly by inversion and conjugation was allowed to proceed on ice for a period of approximately 1 hour before purification by passage over Zeba™ Spin Desalting Columns (40 KDa MWCO; Pierce) pre-equilibrated with phosphate buffered saline or 10 mM sodium citrate, 150 mM sodium chloride, pH 5.5. The eluate was pooled, filter sterilized (Steriflip®, Millipore), and stored at 4 °C. The purified ADCs were analyzed for total protein content (bicinchonic acid assay, Pierce microBCA protocol, catalogue #23225). The ADC product was characterized by reducing and non-reducing PAGE, HPLC-HIC, SEC, and RP-UPLC-MS.

Assay of Selective In Vitro Cytotoxicity of Antigen-positive Cells by Trastuzumab-based Antibody Drug Conjugates [00525] Selective killing of an antigen positive cell line (including HCC1954, NCI-N87, and SKOV3 cell lines) over antigen-negative Jurkat cells was demonstrated for each ADC prepared. Briefly, each cell line was grown in its respective growth medium until seeding day. Cells were removed from their culture vessels and the resulting cell suspension was counted using the ViCell. Cells were then diluted in their growth medium to 25000/mL such that 100 μΕ/well = 2500 cells/well. Each cell line was seeded in the inner 60 wells of 96-well black walled TC plates, outer wells filled with water. HCC1954, NCI-N87, SKOV-3 and DU-145 were seeded a day prior to compound plating to allow cell adhesion to the plate. Compounds were diluted to a 5 ^χ dose- response of each compound using RPMI + 10% FBS in a deep-well 96-well plate. This "master" dilution plate was used for each cell line. 25

of the 5 ^χ dose responses were spiked into each cell line in triplicate. Compounds were then titrated 1 :3 starting at various concentrations (30 nM to 1000 nM). A control plate with no test article present (growth medium and cells alone) was included in sextuplicate. The plates were then incubated for 5 nights for HCC 1954, and SKOV-3 cells and for 3 nights for Jurkat cells. Cell viability was quantified using 30 μΕ/well of l x CellTiter-glo reagent. After at least 10 minutes of incubation, the luminescence was measured using the SpectraMax (500 ms integration). [00526] The results are shown in Table 3. None of the ADCs tested showed any cytotoxicity towards Jurkat cells over the concentration range tested.

Estimation of Drug to Antibody Ratio (PAR)

[00527] The average degree of conjugation of toxin-linker to antibody was assessed by hydrophobic interaction chromatography and/or high performance liquid chromatography-mass spectrometry. These techniques are described in Antibody Drug Conjugates, Methods in Molecular Biology vol. 1045, 2013. pp 275-284. L. Ducry, Ed., and in Chakraborty, et al , Characterization of an IgGl Monoclonal Antibody and related Sub-structures by LC/ESI-TOF/MS: Application note, Waters Corporation. March 2007. 720002107EN. [00528] Average DAR estimates for the ADCs in Table 3 were in the range of 3.5-4.5.

Method 1. Hydrophobic Interaction Chromatography

[00529] Antibody drug conjugates were subjected to hydrophobic interaction chromatography (HIC) on a TSKgel® Butyl-NPR column (Tosoh Bioscience; 4.6 mm ^χ 35 mm i.d.; 2.5 μιη particle size) connected to an Agilent 1100 series HPLC. Samples were injected (5

at or above 1 mg/mL. Where necessary, ADCs are concentrated prior to injection using PALL Nanosep Omega centrifugal concentration devices (part # OD010C34). A linear gradient elution was employed starting at 95% mobile phase A/5% mobile phase B, transitioning to 5% mobile phase A/95% mobile phase B over a period of 12 min (mobile phase A: 1.5 M ammonium sulfate + 25 mM sodium phosphate at pH 6.95 and mobile phase B: 25% isopropanol, 75% 25 mM sodium phosphate at pH 6.95). Injection of unmodified antibody provided a means of identifying the peak with DAR = 0. Antibodies and ADCs were detected on the basis of absorbance at 280 nm.

Method 2. Ultra Performance Liquid Chromatography-Mass Spectrometry for DAR estimation

[00530] Reversed phase ultra performance liquid-chromatography tandem ESI-QToF-mass spectrometry (UPLC-ESI-QToF-MS) was used to characterize antibody drug conjugates for extent of drug conjugation following ADC reduction with dithiothreitol. The characterization was performed using Acquity-UPLC® (H-class) Bio coupled to a Quattro-Premier™ QToF mass spectrometer with an electrospray ion source (WATERS Corporation). UPLC analysis of the reduced ADC sample is performed at 70 °C with a PolymerX™ 5u PR-1 100A, 50 2.0 mm column (Phenomenex, Inc.) and with a mobile phase composed of solvent A: acetonitrile/water/trifluoroacetic acid/formic acid (10/90/0.1/0.1, v/v%), and solvent B: acetonitrile/formic acid (100/0.1, v/v). Components of the reduced ADC sample are eluted with a linear gradient starting at solvent A/solvent B (80/20 v/v and a flow rate of 0.3 mL/min to solvent A/solvent B (40/60, v/v) over 25 min, and then to solvent A/solvent B( 10/90, v/v%) over 2 min before equilibrating back to initial conditions. The total run time is 30 min. The ESI-ToF MS total ion current (TIC) data is acquired over 500-4,500 m/z range using MassLynx™ data acquisition software (Waters Corporation). Sample component mass data is acquired in the positive ion V- mode, and the ESI source is operated at source temperature: 150 °C, desolvation temperature: 350 °C, desolvation gas: 800 L/h, sample cone voltage: 60 V, capillary voltage: 3.0 kV, desolvation gas: nitrogen, and collision gas: argon. The summed TIC mass spectra for each peak is deconvoluted by the Maximum Entropy™ 1 (Max-Entl) algorithm to generate the neutral mass data of the peak component.

Preparation of Reduced ADC samples for UPLC/ESI-ToF MS analysis [00531] Reduction of the disulfide bonds in the antibody of the ADC ( ~ 1 solution) to generate the light and heavy chains is performed using 20 mM DTT at 60 °C for 20 min. An injection volume of 5-10 of the reduced ADC sample was employed for UPLC/ESI-ToF-MS analysis.

EXAMPLE 6: OTHER EXAMPLES OF ANTIBODY-DRUG CONJUGATES Exemplary Linkers

[00532] Compounds of general Formula I having, or modified to have, a thiol moiety may be used for conjugate formation using, for example, the commercially available cleavable linker sulfosuccinimidyl 6-[3 ^'(2-pyridyldithio)-propionamido] hexanoate (sulfo-LC-SPDP: Thermo Pierce Cat# 21650) or non-cleavable linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-l- carboxylate (SMCC: Thermo Pierce Cat# 22360). The coupling procedure is performed in two major steps: 1) incorporation of the linkers onto the antibody via reaction with antibody primary amine groups (lysine residues) and the N-hydroxysuccinimide (NHS) ester moiety of the linkers; and 2) reaction of the incorporated maleimide group (SMCC) or 2-pyridyldithio group (LC-SPDP) with thiol-containing compounds. Activation of Antibody with Cleavable (LC-SPDP) or Non-Cleavable (SMCC) Linkers

[00533] Antibody (Herceptin®) is diluted into either potassium phosphate pH 8 (sulfo-LC-SPDP) or D-PBS (Invitrogen) pH 7.4 (SMCC) to 5 mg/mL. To the diluted antibody, freshly dissolved linker is added, using ultra-pure water for sulfo-LC-SPDP or anhydrous N,N-Dimethylacetamide (DMA) for SMCC. 10-14 fold molar-excesses of SMCC:antibody or sulfo-LC-SPDP: antibody result in incorporation of 5-7 linkers/antibody. The linker-antibody "activation" reaction is incubated at 28 °C for 2 hours. Following the incubation, the unreacted linker is removed from each antibody sample using 40 kda Zeba™ size-exclusion chromatography/de salting columns (Thermo Pierce Cat# 87771, or 87772 depending on the scale). During the same chromatography step the buffer is exchanged in preparation for the next reaction: either phosphate buffer/EDTA pH 6.5 (LC-SPDP), or citrate buffer/EDTA pH 5 (SMCC). The purified preparations are then assayed for total protein content versus an antibody standard curve using the microplate adapted BCA assay (Thermo Pierce Cat# 23225). To estimate the extent of linker incorporation a small scale reaction with excess (~ 10-fold compared to protein concentration) cysteine is performed. Following a 10 min incubation the unreacted cysteine is detected using 5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent, Thermo Pierce Cat# 22582). By interpolating the concentration from a cysteine standard curve the linker concentration is determined by subtracting the determined value from the known concentration of cysteine used.

Reaction of Thiol-Containing Compounds to Linker-Activated Antibody [00534] In the second step of the coupling reaction, the activated-antibody is utilized by first diluting the preparation to 2 mg/mL using either phosphate buffer/EDTA pH 6.5 (LC-SPDP), or citrate buffer/EDTA pH 5 (SMCC). Prior to use, the thiol containing N-acyl sulfonamide compounds are reduced using TCEP-agarose beads to ensure the thiol group is available to react to the incorporated linkers. In brief, compounds are diluted to 5 mM using phosphate buffer/EDTA pH 6.5. In instances where aqueous solubility is an issue, a small volume of 37% HC1 (1 :300) is added and this is sufficient to solubilize the compounds at 5 mM. TCEP-agarose beads (Thermo Pierce Cat# 77712), are equilibrated with phosphate buffer/EDTA/ 10% DMA prior to use. The compound dilutions are rotated with TCEP-agarose beads for at least 0.5 hours, or up to 3 hours. The reduced compounds are collected by centrifugation over a filter which excludes the TCEP- agarose. The extent of reduction and thiol concentration is measured using Ellman's reagent (compared to a cysteine standard curve). The reduced thiol-containing compounds are then added to the activated antibody samples at a molar excess of ~2-fold compared to the previously determined linker concentrations. In order to monitor the coupling reaction effectiveness an "overnight" conjugation control is prepared by diluting each compound into phosphate buffer/EDTA pH 6.5 or citrate buffer/EDTA pH 5 at the same dilution factor that is used in the conjugation reaction. The remaining compound stocks are frozen at -80 °C. The reactions and overnight controls are incubated at ambient temperature overnight. The next morning the frozen compound stocks are thawed and another control is prepared for each compound exactly like the "overnight" control - this is the "fresh" control. A small volume of each conjugation reaction is compared to the overnight and fresh compound controls using Ellman's reagent. Non-reacted compound is purified away from the ADCs using 40 kda Zeba™ Size-exclusion/desalting columns; during the same step the buffer is exchanged to D-PBS pH 7.4 (Invitrogen). The purified ADCs are then analyzed for: total protein content (BCA assay, Pierce microBCA protocol), relative affinity for antigen binding (equilibrium native binding), and selective cytotoxic killing of HER2 -positive cells (HCC1954) compared HER2 -negative cells (Jurkat).

Analysis of Antibody-Drug Conjugate (ADC) by EsiToF Mass Spectrometry.

[00535] An electrospary ionization time of flight (EsiToF) mass spectrometer (MS) instrument (QStar XL Hybrid quadrupole-TOF LC/MSMS; AB Sciex) is used to determine the molecular weight of the ADCs and to evaluate the drug-to-antibody ratio (DAR). The EsiToF MS instrument is equipped with an electrospray ionization turbo spray source. Data acquisition is performed in the positive ion mode, and the sample's total ion current is acquired over the mass range 2,000 m/z to 4,000 m/z using Analyst QS 1.1 software. The ion source is operated with an ion spray needle voltage of 5.2 KV, a nebulization (gas 1) at 25 (arbitrary units), a curtain gas of 30 (arbitrary units), a declustering potential of 150 V and at a temperature of 150 °C. The ADC test sample solutions is introduced at 5 μΕ/πύη into the ion source by direct infusion via a fused silica capillary with the help of syringe and syringe pump. Typically, the DAR ranges from 0 to 4.

Preparation of the ADC Sample for ESI-ToF MS Analysis

[00536] All ADC samples are deglycosylated using EndoS(IgGZERO)™ endoglycosidase and buffer exchanged with water prior to EsiToF-MS analysis. Briefly, the original ADC sample is run through a 100 K MWCO Amicon concentrator for buffer exchange in sodium phosphate buffer. The buffer exchanged sample is then treated with IgGZERO™ (1 unit/1 μg of antibody) in sodium phosphate cleavage buffer, containing 150 mM NaCl, and incubated for 30 min at 37 °C. The resulting deglycosylated ADC is again buffer-exchanged with water using a 100 K MWCO Amicon concentrator, and diluted with 0.1% formic acid in acetonitrile/water (50/50 v/v%) to a concentration of 3.0 μg/μL prior to analysis.

EXAMPLE 7: EFFICACY STUDY OF TOXINS IN PC-3 TUMOR-BEARING MICE

[00537] Test articles are administered IV. Dosage is near the maximum tolerated dosage. One injection of test article is delivered every seven days for four repeats/injections or one injection every seven days for three repeats/injections. Vehicle: 6.3% trehalose, 0.05% Tween® 20, 20 mM citrate buffer, pH 5.0, 4 °C .

[00538] Procedure Overview: Female athymic nude mice, purchased from Harlan Laboratories at 7-8 weeks of age, are inoculated subcutaneously in the back with 5 ^χ 10⁶ PC-3 tumor cells on experimental day 0. Tumors are measured every Monday, Wednesday, and Friday. Once tumors reach 150-200 mm³ in size, animals are assigned to one of 4 treatment groups by counterbalancing the average tumor size across groups. Animals are treated with their respective compound, and tumor measures continue every Monday, Wednesday, and Friday.

[00539] PC-3 Cells: Cell Preparation— Tissue Culture: The PC-3 human prostate adenocarcinoma cell line is obtained from ATCC (Cat # CRL-1435). Cells are started from a frozen vial of lab stock which are frozen down from the original ATCC vial, tested for mycoplasma negative and kept in lab liquid nitrogen tanks. Cell cultures with passage #3 to #10 and a confluence of 80-90% are harvested for in vivo studies. Cells are grown in Ham's F12 medium supplemented with 2 mM L-glutamine and 10% FBS at 37 °C/5% CO2 environment. Cells are sub- cultured once a week with split ratio 1 :3 to 1 :6 and expanded. The medium is renewed once a week. [00540] Cell preparation— Harvesting for Implantation: Cells are rinsed briefly one time with 2 mL of fresh trypsin/EDTA solution (0.25% trypsin with EDTA 4Na), then the extra trypsin/EDTA is aspirated. Then 1.5 mL of trypsin/EDTA is added, and the flask is laid horizontally to ensure the cells are covered by trypsin/EDTA. The cells are then incubated at 37 °C for a few minutes. The cells are observed under an inverted microscope to ensure the cell layer is dispersed, then fresh medium is added, and 50 of cell suspension is sampled and mixed with trypan blue (1 : 1) and the cells are counted and cell viability assessed using the Cellometer® Auto T4. The cells are centrifuged at 1,000 rpm for 7 min and the supernatant aspirated. The cells are then re-suspended in growth medium to the appropriate concentration for inoculation. Injection volume is 100 per animal.

[00541] Tumor Cell Implantation— SC Back: On Day 0, 5.0 ^χ 10⁶ tumor cells are implanted subcutaneously into the back of mice in a volume of 100 using a 27/28-gauge needle under isoflurane anesthesia.

[00542] Animal Housing: Animals are housed in ventilated cages, 2 to 5 animals per cage, in a 12-hour light/dark cycle. Animals receive sterile food and water ad libitum and housing and use of animals is performed in accordance with Canadian Council on Animal Care guidelines. Animals are handled aseptically, and cages changed once every 10-14 days.

[00543] Data Collection (Tumor size): Mice are monitored every Monday, Wednesday and Friday for tumor development. Dimensions of established tumors are measured with calipers. Tumor volumes are calculated according to the equation [L ^χ W²] ÷ 2 with the length (L) being the longer axis of the tumor. Animals are also weighed at the time of tumor measurement. Tumors are allowed to grow to a maximum of 800 mm³.

[00544] Analysis Methods (Tumor Volume X Experimental Day Growth Curves): Tumor volumes of each group across the treatment days are plotted. Growth curves are cutoff for each group at the time point when the first animal reaches the tumor-size experimental endpoint (800 mm³), or at the last day of the study. Any animal that is withdrawn from the study prior to the group growth curve cutoff is removed entirely from the study.

[00545] Animal Exclusions: Any animal with ulcerating tumors, necessitating euthanasia of the animal, with tumor volume of 700 mm³ or smaller are removed from the study and do not contribute to the data analysis (except for Days to Recurrence if the final tumor volume is > 2.0 fold higher than on the treatment day). EXAMPLE 8: EFFICACY DOSE RANGE FINDING OF ANTIBODY DRUG CONJUGATES IN THE NCI-N87 TUMOR MODEL USING NOD SCID GAMMA MICE

[00546] Test articles are administered IV, one treatment only. Dosages tested are 3, 7, and 12 mg/kg. Vehicle: 20mM sodium citrate, 6.3% trehalose, 0.02% Tween® 20, pH 5, 4 °C. [00547] Procedure Overview: Seventy-six (76) female NOD/SCID Gamma mice (NSG), purchased from The Jackson Laboratory (JAX® Mice) at 7-8 weeks of age, are inoculated subcutaneously in the lower back with 5 ^χ 10⁶ NCI-N87 tumor cells in matrigel on experimental day 0. Tumors are measured every Monday, Wednesday, and Friday. Once tumors reach 150-200 mm³ in size, animals are assigned to one of 10 treatment groups by counterbalancing the average tumor size across groups. Animals are treated with their respective compound, and tumor measures continue every Monday, Wednesday, and Friday.

[00548] Cell preparation— Tissue Culture: NCI-N87 Cells: NCI-N87 human gastric carcinoma cells are derived from a liver metastasis of a well differentiated carcinoma of the stomach taken prior to cytotoxic therapy. The tumor is passaged as a xenograft in athymic nude mice for three passages before the cell line is established. NCI-N87 cells are obtained from the ATCC (Cat # CRL-5822) and are tested negative at RADIL for Mycoplasma and mouse pathogens.

[00549] Cells are started from a frozen vial of lab stock which is frozen down from the original ATCC vial and kept in lab liquid nitrogen tanks. Cell cultures with passage #3 to #10 and a confluence of 80-90% are harvested for in vivo studies. NCI-N87 cells are grown in RPMI 1640 medium with 1.0 mM L-glutamine and 10% FBS at 37 °C/5% CO2 environment. Cells are subcultured once or twice a week with the split ratio 1 :3 or 1 :4 and expanded. The medium is renewed once a week. Cell are frozen with 5% DMSO.

[00550] Cell preparation— Harvesting for Implantation: Cells are rinsed briefly one time with Hank's Balanced Salt Solution without Ca, Mg. Fresh trypsin/EDTA solution (0.25% trypsin with EDTA 4Na) is added, and the flask is laid horizontally to ensure the cells are covered by trypsin/EDA, and then the extra trypsin/EDTA is aspirated. The cells are incubated at 37 °C for a few minutes. Cells are observed under an inverted microscope until the cell layer is dispersed, and fresh medium is then added. Then, 50 of cell suspension is collected and mixed with trypan blue (1: 1), and the cells are counted and assessed for viability on a haemocytometer. Viability should be > 90%. The cells are centrifuged at 125 RCF (1,000 rpm) for 7 min and the supernatant is aspirated off. The cells are resuspended in cold growth medium to 2 times the desired final concentration (100 ^χ 10⁶/mL). The suspension is mixed (on ice) with matrigel (1 : 1). The resulting cell suspensions (50 ^χ 10⁶ cells/mL) is used to deliver 5 ^χ 10⁶ cells in an injection volume of 100 per animal. All equipment coming into contact with matrigel (needles, syringes, pipette tips) is chilled prior to injection.

[00551] Tumor Cell Implantation— SC (NCI-N87): Prior to inoculation, an area, approximately 2 x 2 cm, is shaved in the lower back region of each mouse and cleaned with alcohol. On Day 0, 5.0 x 10⁶ tumor cells are implanted subcutaneously into the back of mice in a volume of 100 using a 27/28-gauge needle under isoflurane anesthesia.

[00552] Animal Housing, Data Collection (Tumor size), Analysis Methods (Tumor Volume X Experimental Day Growth Curves) and Animal Exclusions are carried out as described in Example 7.

EXAMPLE 9: EFFICACY COMPARISON OF ANTIBODY DRUG CONJUGATES IN THE NCI-N87 TUMOR MODEL USING NOD SCID GAMMA MICE

[00553] Test articles are administered IV, with one administration of 3 mg/kg. Vehicle: 20 mM sodium citrate, 6.3% trehalose, 0.02% Tween® 20, pH 5, 4 °C.

[00554] Procedure Overview: Twenty-four (24) female NOD/SCID Gamma mice (NSG), purchased from The Jackson Laboratory (JAX® Mice) at 7-8 weeks of age, are inoculated subcutaneously in the lower back with 5 ^χ 10⁶ NCI-N87 tumor cells in matrigel on experimental day 0. Tumors are measured every Monday, Wednesday, and Friday. Once tumors reach 150-200 mm³ in size, animals are assigned to one of 3 treatment groups by counterbalancing the average tumor size across groups. Animals are treated with their respective compound, and tumor measures continued every Monday, Wednesday, and Friday. [00555] Cell preparation (Tissue Culture: NCI-N87 Cells and Harvesting for Implantation), Tumor Cell Implantation (subcutaneous (NCI-N87)), Animal Housing, Data Collection (Tumor size), Analysis Methods (Tumor Volume X Experimental Day Growth Curves) and Animal Exclusions are carried out as described in Example 8. [00556] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their entirety to the extent not inconsistent with the present description. From the foregoing it will be appreciated that, although specific embodiments described herein have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope described herein. Accordingly, the disclosure is not limited except as by the appended claims.

Table 2: EC50 Values (nM) for Exemplary Compounds of General Formula I

* nd: not determined

Table 3: EC50 Values (nM) for Antibody-Drug Conjugates Comprising Compounds of General Formula I

* nd: not determined

Claims

WHAT IS CLAIMED IS:

1. A compound of general Formula I:

or a pharmaceutically acceptable salt thereof, or a precursor thereof that comprises one or more protecting groups, wherein:

R is selected from: amino-G-C6 alkyl, amino-aryl, amino-G-G cycloalkyl, amino- heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from aryl, aryl-Ci-C6 alkyl, G-C6 alkyl, G-C6 alkylthio, carboxyl, carboxamide, C3-C7 cycloalkyl, C3-C7 cycloalkyl -G-G alkyl, guanidino, halo, G-C6 haloalkyl, heterocyclyl, heterocyclyl-G-C6 alkyl, hydroxyl, and thio; or

R is R^NCHtR³)-;

R¹ is selected from: H and G-C6 alkyl;

R² is G-C6 alkyl; and

R³ is selected from: H, R¹⁵ and R⁷-CH(CH₃)₂-; or

R⁴ is selected from: C1-C4 alkyl and C3-C6 cycloalkyl;

R⁵ is selected from: H and C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1 -C4 acyloxy;

R⁶ is selected from: G-C6 alkyl, aryl, aryl-G-C6 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, each optionally substituted with one or more substituents selected from: G - Ce alkyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkyl, C1-C6 alkylamino, amino, amino-Ci-C6 alkyl, amino-aryl, amino-C3-C? cycloalkyl, amino-heteroaryl, amino-heterocyclyl, aryl, carboxamide, carboxyl, C3-C7 cycloalkyl, cyano, G-C6 haloacyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, halo, hydroxyl, nitro, thio, and thio-G-C6 alkyl;

R⁷ is selected from: H, aryl, C3-C7 cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: C1-C4 acylthio, C2-C4 alkenyl, C1-C4 alkyl, C1-C4 alkylamino, C1 -C4 alkyloxy, amino, amino-Ci-C4 alkyl, halo, C1-C4 haloalkyl, hydroxyl, hydroxy-Ci-C4 alkyl, and thio, wherein C2-C4 alkenyl, C1-C4 alkylamino and C1-C4 alkyloxy are further optionally substituted with one substituent selected from C1-C4 alkylaryl, hydroxyl, and thio;

R⁹ and R¹⁰ are each independently selected from: H, C1-C4 alkyl, aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl-Ci-C4 alkyl, and hydroxy-G-C4 alkyl;

R¹⁵ is Ci-C₆ alkyl;

X is selected from C2-C3 alkenyldiyl and C2 alkyldiyl, wherein C2 alkyldiyl is substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹;

Y is heteroaryldiyl, or Y is absent; and

Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-, or Z is absent; with the proviso that when R is R^NCHiR³)-; R¹ is H; R² is methyl; R³ is PhCH(CH₃)₂-; R⁵ is methyl; X is -CH=CH(CH₃)-; Y is absent and Z is absent, then R⁴ is other than fert-butyl.

2. The compound according to claim 1, wherein: X is C2 alkyldiyl substituted with one substituent selected from: G-C6 acyloxy, G-C6 alkyloxy, hydroxyl, oxo, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹, and

Y is heteroaryldiyl.

The compound according to claim 1, wherein the compound is a compound of Formula la:

la or a pharmaceutically acceptable salt thereof, wherein:

R¹, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, X, Y and Z are as defined in claim 1, and n is 0, 1, or 2.

The compound according to claim 1, wherein the compound is a compound of Formula lb:

lb or a pharmaceutically acceptable salt thereof, wherein:

R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, X, Y and Z are as defined in claim 1, and

R² is G-C6 alkyl.

The compound according to claim 1, wherein the compound is a compound of Formula Ie:

le or a pharmaceutically acceptable salt thereof, wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵ and Z are as defined in claim 1 ;

R⁹ and R¹⁰ are each independently H or C1-C4 alkyl, and

R¹¹ is selected from: H, G-C₆ acyl, Ci-C₆ alkyl, and -C(0)NR⁹R¹⁰, with the proviso that when R¹ is H; R² and R³ taken together with the atoms to which they are each bonded form piperidin-2-yl; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; R¹¹ is acetyl; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl.

6. The compound according to claim 5, wherein:

R¹ and R² are each independently G-C6 alkyl; and

R³ is H or R¹⁵.

7. The compound according to claim 5, wherein R² and R³ taken together with the atoms to which they are each bonded form a heterocyclyldiyl.

8 The compound according to claim 7, wherein the heterocyclyldiyl is selected from pyrrolidinyldiyl, piperidinyldiyl, and azepanyldiyl.

9. The compound according to any one of claims 5, 7 or 8, wherein R¹ is H or C1-C4 alkyl.

10. The compound according to any one of claims 5 to 9, wherein R⁴ is C1-C4 alkyl or cyclopropyl.

11. The compound according to any one of claims 5 to 10, wherein R⁵ is C1-C4 alkyl optionally substituted with one substituent selected from: C1-C4 alkyloxy and C1-C4 acyloxy.

12. The compound according to any one of claims 5 to 11, wherein R⁶ is selected from: C1-C4 alkyl, aryl, aryl-Ci-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the G- alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-G alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G- G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio-G-G alkyl.

13. The compound according to any one of claims 5 to 11, wherein R⁶ is aryl or aryl-G-G alkyl, each optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino- heterocyclyl, carboxamide, carboxyl, cyano, G-G haloacyl, G-G haloalkyl, G-G haloalkoxy, hydroxyl, nitro, thio, and thio-G-G alkyl.

14. The compound according to any one of claims 5 to 12, wherein R¹¹ is selected from: G-G acyl, G-G alkyl, and -C(0)NR⁹R¹⁰.

15. The compound according to any one of claims 5 to 12, wherein R¹¹ is G-G acyl or G-G alkyl.

16. The compound according to any one of claims 5 to 15, wherein Z is absent.

17. The compound according to any one of claims 5 to 15, wherein Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

18. The compound according to claim 17, wherein R⁸ is aryl optionally substituted with one substituent selected from amino and hydroxyl.

19. The compound according to claim 1, wherein the compound is a compound of Formula Ix:

Ix or a pharmaceutically acceptable salt thereof, wherein: R¹, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰ and Z are as defined in claim 1;

R¹⁴ is selected from: C1-C6 acyloxy, C1-C6 alkyloxy, hydroxyl, -OC(0)NR⁹R¹⁰, -OC(S)NR⁹R¹⁰, -OC(0)OR⁹, and -OC(S)OR⁹, and n is 0, 1, or 2, with the proviso that when n is 1; R¹ is H; R⁴ is fert-butyl or sec-butyl; R⁵ is methyl; R¹⁴ is acetoxy; Z is -C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)- and R⁸ is phenyl, then R⁶ is other than cyclopropyl.

20. The compound according to claim 19, wherein R¹ is selected from: H and C1-C4 alkyl.

21. The compound according to claim 19 or 20, wherein R⁴ is C1-C4 alkyl or cyclopropyl.

22. The compound according to any one of claims 19 to 21, wherein R⁵ is C1-C4 alkyl optionally substituted with C1-C4 acyloxy.

23. The compound according to any one of claims 19 to 22, wherein R⁶ is selected from: C1-C4 alkyl, aryl, aryl-G-C4 alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl, wherein the G- C4 alkyl is substituted with a substituent selected from: amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl or amino-heterocyclyl, and the aryl, aryl-G-G alkyl, C3-C7 cycloalkyl, heteroaryl, and heterocyclyl are optionally substituted with one or more substituents selected from: amino, amino-G-G alkyl, amino-aryl, amino-G-G cycloalkyl, amino-heteroaryl, amino-heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, G- C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-Ci-C6 alkyl.

24. The compound according to any one of claims 19 to 22, wherein R⁶ is aryl or aryl-G-C4 alkyl, each optionally substituted with one or more substituents selected from: amino, amino-Ci-C4 alkyl, amino-aryl, amino-C3-C7 cycloalkyl, amino-heteroaryl, amino- heterocyclyl, carboxamide, carboxyl, cyano, C1-C4 haloacyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, hydroxyl, nitro, thio, and thio-G-C6 alkyl.

25. The compound according to any one of claims 19 to 24, wherein R⁹ and R¹⁰ are each independently H or G-C4 alkyl.

26. The compound according to any one of claims 19 to 25, wherein R¹⁴ is selected from: G- G acyloxy, G-G alkyloxy, hydroxyl and -OC(0)NR⁹R¹⁰.

27. The compound according to any one of claims 19 to 25, wherein R¹⁴ is G-G acyloxy or G-G alkyloxy.

28. The compound according to any one of claims 19 to 25, wherein R¹⁴ is G-G acyloxy or G-G alkyloxy.

29. The compound according to any one of claims 19 to 28, wherein Z is absent.

30. The compound according to any one of claims 19 to 28, wherein Z is

C(0)NHCH(CH₂R⁸)CH₂CH(CH₃)-.

31. The compound according to claim 30, wherein R⁸ is aryl optionally substituted with one substituent selected from amino and hydroxyl.

32. The compound according to claim 1, wherein the compound is selected from:

Compound 25

Compound 26

33. A pharmaceutical composition comprising the compound according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier.

A conjugate of general Formula VI:

T-(L-(D)_m),

VI wherein:

T is a targeting moiety; L is an optional linker;

D is a monovalent radical of a compound of any one of claims 1 to 32; m is an integer between 1 and about 10, and n is an integer between 1 and about 20.

35. The conjugate composition according to claim 34, wherein m is 1.

36. The conjugate according to claim 34 or 35, wherein n is between 2 and 8.

37. The conjugate according to any one of claims 34 to 36, wherein the targeting moiety is an antibody or antibody fragment.

38. The conjugate according to claim 37, wherein the antibody binds to a cancer antigen.

39. The conjugate according to any one of claims 34 to 38, wherein the linker is a non- cleavable linker.

40. The conjugate according to any one of claims 34 to 38, wherein the linker is a cleavable linker.

41. The conjugate according to claim 40, wherein the linker is a peptide -containing linker.

42. The conjugate according to claim 41, wherein the linker has general Formula VII:

VII wherein:

Z is a functional group capable of reacting with the target group on (T); Str is a stretcher;

X is a self-immolative group;

43. The conjugate according to claim 42, wherein:

The conjugate according to claim 42 or 43, wherein:

O O

r_is — (CH2)p— C— . — (CH₂CH₂0)_q-C- . — (CH₂)_p— (CH₂CH₂0)_q-

O O R O

-(CH₂CH₂0)_q— (CH₂)_p— C— — (CH2)p— C-N-(CH2)p— C— or

O R 0

-(CH₂)_p— C-N-(CH₂CH₂0)_q— C— wherein:

R is H or optionally substituted G-C6 alkyl; p is an integer between 2 and 10, and q is an integer between 1 and 10.

45. The conjugate according to claim 42 or 43, wherein:

O O O

Str _is— (CH₂)_P -C- _s— (CH₂CH₂0)_q-C- _Qr— (CH₂CH₂0)_q— (CH₂)_p -C-

46. The conjugate according to any one of claims 42 to 45, wherein:

AAi-[AA2]m is selected from Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile- Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, lie-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me₃Lys- Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Met-Cit-Val, Gly-Cit-Val, (D)Phe -Phe-Lys, (D)Ala-Phe-Lys, Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu.

47. The conjugate according to any one of claims 42 to 45, wherein m is 1.

48. The conjugate according to claim 47, wherein AAi-[AA2]_m is selected from Val-Lys, Ala- Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit and Trp-Cit.

49. The conjugate according to any one of claims 42 to 48, n is 1.

50. The conjugate according to any one of claims 42 to 49, wherein each X is independently selected from /j>-aminobenzyloxycarbonyl (PABC), /j>-aminobenzyl ether (PABE) and methylated ethylene diamine (MED).

51. The conjugate according to any one of claims 42 to 49, wherein o is 0.

52. The conjugate according to claim 34, wherein L-D is a monovalent radical of a compound selected from:

53. A pharmaceutical composition comprising the conjugate according to any one of claims 34 to 52 and a pharmaceutically acceptable carrier.

54. A method of inhibiting proliferation of cancer cells comprising contacting the cancer cells with an effective amount of a compound according to any one of claims 1 to 32.

55. A method of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 32.

56. A method of treating cancer in a subject comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 32.

57. A method of inhibiting proliferation of cancer cells comprising contacting the cancer cells with an effective amount of a conjugate according to any one of claims 34 to 52.

58. A method of inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a conjugate according to any one of claims 34 to 52.

59. A method of treating cancer in a subject comprising administering to the subject an effective amount of a conjugate according to any one of claims 34 to 52.